JP6940558B2 - Ejecting a selected group of micro-objects from a micro-fluid device - Google Patents

Description

Background In bioscience and related fields, it can be useful to remove selected elements from microfluidic devices. Some aspects of the invention are for selecting a group of micro-objects in a particular holding pen in a micro-fluid device and for carrying out a selected group from a micro-fluid device. Includes equipment and processing.

Overview In some embodiments, the present invention provides a process of ejecting a microobject from a microfluidic device. The process may include selecting a group of micro-objects that are placed in a holding enclosure that is placed inside the enclosure of the microfluidic device. The holding enclosure can be one of a plurality of retaining enclosures placed inside the enclosure. The process moves a group of selected micro-objects to a staging area inside the enclosure, and moves the selected group of micro-objects from the staging area through a passage through the enclosure to the enclosure. It may further include carrying out to a location outside the body. In some embodiments, each retaining enclosure in the enclosure comprises an isolated region configured to retain a plurality of microobjects. In some embodiments, moving a swarm of micro-objects comprises isolating the swarm of micro-objects from all other micro-objects located within the micro-fluid device.

In some embodiments, one or more (or all) of the microobjects in the selected group are living cells. In some embodiments, the group of microobjects selected is a single living cell, such as an immune cell, a cancer cell, a transformed cell, a stem cell or a homologous one. In another embodiment, the group of microobjects selected is a plurality of cells, such as a clonal population of living cells.

In some embodiments, selecting a group of micro-objects comprises determining that the group of micro-objects has a particular activity or physical characteristic.

In some embodiments, selecting a group of microobjects is such that a pattern of light activates a DEP force that surrounds the microobjects of the selected group and captures the microobjects of the selected group. Includes directing a pattern of light in. In one related aspect, moving a group of selected micro-objects captures the selected group of micro-objects by the DEP force triggered by the light pattern, even as the light pattern moves to the staging area. It involves moving the pattern of light surrounding the group to the staging area so that it remains as it is.

In some embodiments, the staging area is located in a channel defined by the enclosure of the microfluidic device. For example, the staging area may be close to the opening into the channel from the holding enclosure containing the selected group of microobjects. In another embodiment, the staging area is placed within a holding enclosure containing a group of selected microobjects. In certain related embodiments, moving a group of selected micro-objects to a staging area involves attracting the group of micro-objects to the staging area by gravity.

In some embodiments, unloading a group of selected micro-objects comprises pulling the selected group of micro-objects out of the staging area through passages in the enclosure. In some embodiments, the unloading device is used to pull a selected group of microobjects through the proximal end of the unloading device, and thereby through a passage in the enclosure. In some embodiments, pulling a selected group of micro-objects through a passage in the enclosure comprises creating a pressure difference that pulls the group of micro-objects into an opening at the proximal end of the unloading device. In some embodiments, the export device is inserted into an export interface located on a passageway in the enclosure before the selected group of microobjects is exported. In some embodiments, by inserting the unloading device into the unloading interface, an opening at the proximal end of the unloading device is placed close to the passage in the enclosure.

In some embodiments, the microfluidic device comprises a carry-out interface having a self-closing cover that is placed and overlying a passage in the enclosure. In some embodiments, the unloading device presses the proximal end of the unloading device against a separation in the self-closing cover, moving the proximal end of the unloading device closer to the passage in the enclosure. , Can be inserted into such a carry-out interface.

In some embodiments, the microfluidic device comprises a carry-out interface with a self-healing cover that is placed and covered over a passage in the enclosure. In some embodiments, the unloading device can be inserted into such a unloading interface by penetrating the self-healing cover at the proximal end of the unloading device so that the proximal end of the unloading device is close to the passage in the enclosure. Can be positioned in.

In some embodiments, the selected group of microobjects is carried out in a medium having a volume of 1 μL or less or in another solution. In another embodiment, the selected group of microobjects is carried out in a medium or other solution having a volume of 5 μL, 10 μL, 25 μL, 50 μL or more.

In some embodiments, after a group of selected micro-objects has been unloaded from the microfluidic device, the staging area, unloading interface, unloading device, or any combination thereof, for the selected group of tiny objects that have failed to be unloaded. Can be investigated. In some embodiments, missed tiny objects are removed from the staging area, unloading device and / or unloading interface. Missing unloading micro-objects can be removed, for example, by flushing and / or neutralizing the staging area, unloading device and / or unloading interface.

In some embodiments, the enclosure of the microfluidic device includes a first passage and a second passage. In some embodiments, the first passage is placed close to the first end of the staging area and the second passage is placed close to the second end of the staging area. In some embodiments, the staging area has an elongated shape. In certain related embodiments, moving a group of selected micro-objects is in a staging area that is placed at the first end, the second end, or between the first and second ends of the staging area. Includes moving the group to a position. In certain relevant embodiments, unloading a group of selected microobjects causes a liquid to flow from a first unloading device through a first passage in the enclosure towards the first end of the staging area, thereby staging. This includes the occurrence of a flow from the first end of the area to the second end of the staging area. In another related aspect, carrying out a group of selected micro-objects takes the selected group of micro-objects from the second end of the staging area through a second passage in the enclosure to a second passage. Includes pulling into an opening at the proximal end of a second unloading device placed in close proximity.

In some embodiments, the process of removing a microobject from a microfluidic device may include creating a flow of liquid medium directly into the enclosure of the microfluidic device to the outlet for the medium from the enclosure. .. In some embodiments, the flow of the liquid medium is in a channel located within the enclosure. The process also includes selecting a group of micro-objects in the retention enclosure inside the enclosure and moving the group selected from the retention enclosure into the flow path of the liquid medium. obtain. In some embodiments, the flow of the liquid medium is sufficiently maintained for the flow to clear the selected group to and from the outlet and out of the enclosure through the outlet. In some embodiments, the flow of the liquid medium is slowed or stopped while the selected group is moved from the holding enclosure into the flow path.

In some embodiments, the present invention provides a microfluidic device. The microfluidic apparatus may include a siege configured to contain a liquid medium and a passage through the siege. The microfluidic device may further include a channel located inside the enclosure and an open off of holding enclosure. In some embodiments, the microfluidic device comprises a plurality of retaining enclosures that are open through the channel. Each holding enclosure may be configured to hold a group of microscopic objects. In some embodiments, each retention enclosure is configured to isolate a group of microobjects from the microobjects in the other retention enclosure in the microfluidic device. In some embodiments, each retaining enclosure comprises an isolated area.

In some embodiments, the microfluidic device comprises a unloading interface. For example, the unloading interface may be placed on the enclosure in close proximity to the passage through the enclosure. In some embodiments, the unloading interface includes a cover that entirely covers the passage through the enclosure. In some embodiments, the unloading interface is configured to connect with the unloading device such that the opening in the first (or proximal) end of the unloading device is placed close to the passage through the enclosure.

In some embodiments, the cover of the carry-out interface comprises a crevice that divides the cover into a series of valves. In some embodiments, the valves are biased to contact each other, thereby covering the entire passage through the enclosure. In some embodiments, the valve of the cover separates when the first end of the carry-out device is pressed against the crevice that divides the cover into a series of valves, forming an opening that can accommodate the first end of the carry-out device. It is flexible enough. In another aspect, the cover of the unloading interface comprises a self-healing material that can be penetrated by the unloading device. In some embodiments, such penetration creates a hole for receiving the unloading device, which can self-heal, thereby closing when the unloading device is removed from the cover.

In some embodiments, the enclosure of the microfluidic device comprises at least one staging area. In some embodiments, at least one staging area is located in close proximity to the passageway through the enclosure. In some embodiments, the staging area is located in the channel. In some embodiments, the enclosure comprises a plurality of staging areas. In some embodiments, each retaining enclosure in the microfluidic device includes a staging area.

In some embodiments, the present invention provides a system. The system may include a microfluidic device as well as a means for capturing and moving a swarm of microobjects located within the microfluidic device. The microfluidic device can be any of the devices described herein. In some embodiments, the means for catching and moving a swarm of micro-objects is for catching a swarm of micro-objects that are placed in a holding enclosure within a microfluidic device, and of the captured micro-objects. Suitable for moving the swarm to the staging area.

These and other aspects and advantages of the methods, systems and devices of the present invention will be specified or more fully demonstrated in subsequent statements and in the accompanying claims. Features and benefits may be recognized and obtained using the equipment and combinations specifically pointed out in the accompanying examples and claims. Moreover, aspects and advantages of the described systems, devices and methods may be familiar with the practice or will be apparent from the description, as set forth herein below.

Brief description of drawings
FIG. 1A is a perspective view of a microfluidic device.

FIG. 1B is a horizontal cross-sectional view of the microfluidic device of FIG. 1A.

FIG. 1C is a vertical cross-sectional view of the microfluidic device of FIG. 1A.

FIG. 1D is a partial vertical cross-sectional view of the microfluidic device of FIG. 1A illustrating insertion of the unloading device into the unloading interface.

FIG. 2A is a partial vertical cross-sectional view of the microfluidic device of FIGS. 1A-1C, without a holding enclosure in which the selector is configured as a dielectrophoresis (DEP) device (for ease of explanation).

FIG. 2B is a partial horizontal cross-sectional view of FIG. 2A.

FIG. 3 is an example of a process for carrying out a group of minute objects from a minute fluid device.

FIG. 4 shows an example of a group of micro-objects in a holding enclosure in the micro-fluid device of FIGS. 1A-1C.

FIG. 5 illustrates an example of selecting and capturing a group of micro-objects in one of the holding enclosures.

FIG. 6 shows an example of moving a group of captured micro-objects from a holding enclosure in a micro-fluid device towards a staging area.

FIG. 7 shows a group of microscopic objects in the staging area.

FIG. 8 describes an example of a unloading device inserted into the unloading interface of the microfluidic device of FIGS. 1A-1C.

FIG. 9 shows an example of a micro object being pulled out of the staging area into the unloading device.

FIG. 10 describes an example of processing for carrying out a group of minute objects from the staging area.

FIG. 11 is an example of a process for excluding minute objects that have not been unloaded from the staging area and the unloading interface.

FIG. 12A is a partial perspective view of the microfluidic device of FIGS. 1A-1C having an aspect of a carry-out interface including a self-closing cover.

FIG. 12B is a partial vertical cross-sectional view of FIG. 12A.

FIG. 12C shows a unloading device inserted into the unloading interface of FIGS. 12A and 12B.

FIG. 13A is a partial vertical cross-sectional view of the microfluidic device of FIGS. 1A-1C having a unloading interface aspect including a self-healing cover.

FIG. 13B illustrates a unloading device in the form of a hypodermic needle inserted into the unloading interface of FIG. 13A.

FIG. 13C shows a hypodermic needle inserted into a different portion of the carry-out interface of FIG. 13A.

FIG. 14 is a partial vertical cross-sectional view of the microfluidic device of FIGS. 1A-1C having an aspect of a unloading interface that includes an attached tubular unloading device.

FIG. 15A is a partial perspective view of a microfluidic device including a plurality of unloading interfaces.

FIG. 15B is a horizontal cross-sectional view of FIG. 15A illustrating a staging area including an intermediate portion between the opposite ends.

FIG. 15C is a vertical cross-sectional view of FIG. 15A.

FIG. 16 is a vertical cross-sectional view of the microfluidic device of FIG. 15A illustrating a unloading device inserted into the unloading interface.

FIG. 17 is a horizontal cross-sectional view of a microfluidic device including a holding enclosure in the flow path in the device and a large number of staging areas.

FIG. 18 is a horizontal cross-sectional view of a microfluidic device including a retention enclosure and staging area, in which the retention enclosure opens from a channel separated by a continuous barrier from the flow path in which the staging area is located. doing.

FIG. 19A is a horizontal cross-sectional view of a microfluidic device including a holding enclosure with a staging area located in a channel (where the holding enclosure opens) in close proximity to an opening to each holding enclosure.

FIG. 19B is a vertical cross-sectional view of the microfluidic device of FIG. 19A.

FIG. 20 describes an example of processing for carrying out a minute object from the devices of FIGS. 19A to 19B.

FIG. 21A is a partial vertical cross-sectional view of a microfluidic device in which the unloading interface may be placed in direct proximity to the holding enclosure.

FIG. 21B is a partial horizontal cross-sectional view of a microfluidic device in which the holding enclosure includes an isolated area, a portion of which is a staging area.

FIG. 21C is a partial horizontal cross-sectional view of the retention enclosure in a microfluidic device in which the retention enclosure includes an isolated area and a connected staging area.

Detailed Description of Illustrated Embodiments This specification describes exemplary embodiments and applications of the present invention. However, the invention is not limited to these exemplary embodiments and applications, or to the manner in which the exemplary embodiments and applications work or are described herein. The figure may also show a simplified or partial view, the dimensions of the elements in the figure may appear larger for clarity, or otherwise balanced. It does not have to be. In addition, when the terms "on", "attached to" or "coupled to" are used herein, one. Elements (eg, materials, layers, substrates, etc.) are one or more elements, whether or not one element is directly attached to or coupled to it on top of another. Whether the intervening element is between one element and the other, it is "on", "attached to", or "coupled" with another element. It may be "has been". If provided, directions (eg above, below, top, bottom, side, up, down, down) Also (under), up (over), up (upper), down (lower), horizontal (horizontal), vertical (vertical), "x", "y", "z", etc.) It is relative and is provided merely by way of example for ease of explanation and review and has no purpose of limitation. In addition, when referring to a list of elements (eg, elements a, b, c), such reference consists of any one of the loaded elements itself, less than all of the loaded elements. It is intended to include combinations and / or combinations consisting of all of the mounted elements.

As used herein, "substantially" means sufficient to work as intended. Thus, the term "substantially" may be in perfect or perfect condition, size, size, insignificant small variation from the results, or, as would be expected of one of ordinary skill in the art, for example, as a whole. Allows similar performances that do not perceptibly affect performance. The term "one" means more than one.

The term "suction" used for a small object means to move the small object using a pressure difference, and "aspirator" is a device that generates a pressure difference.

As used herein, the term "microobject" may include one or more of: microparticles, microbeads (eg, polystyrene beads, Luminex ™ beads or the like), magnetic beads, microrods. Inanimate microobjects such as (microrod), microwire, quantum dots; from cells (eg embryos, egg mother cells, sperm, cells dissociated from tissue, blood cells, hybridomas, cultured cells, cell lines) Biological microscopic objects such as cells, cancer cells, infected cells, transgenic and / or transformed cells, reporter cells, liposomes (eg from synthetic or membrane preparations), lipid nanorafts (lipid nanoraft), etc .; or a combination of inanimate microobjects and biological microobjects (eg, microbeads attached to cells, microbeads coated with liposomes, magnetic beads coated with liposomes or The same kind). Lipid nanorafts are described, for example, in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods Enzymol., 464: 211-231.

The term "flow" as used herein in accordance with a liquid refers primarily to the bulk movement of a liquid due to any mechanism other than diffusion. For example, the flow of the medium can be accompanied by the movement of the fluid medium from one point to another due to the pressure difference between the points. Such a flow may include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or a combination thereof. When one fluid medium flows into another, turbulence and mixing of the medium can occur.

The phrase "substantially no flow" refers to the rate of flow of a liquid that is less than the rate of diffusion of the components of the material into or within the liquid (eg, the analysts of interest). The rate of diffusion of the components of such a material can depend, for example, on the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein in accordance with a fluid medium, "diffuse" and "diffuse" refer to the thermodynamic movement of components of a fluid medium below a concentration gradient.

As used herein in accordance with different regions within a microfluidic device, the phrase "fluidically connected" refers to a region when the different regions are substantially filled with fluid, such as a fluid medium. It means that the fluids in each are connected so as to form a single body of the fluid. This does not mean that the composition of the fluid (or fluid medium) in the different regions is necessarily the same. Rather, the fluids in the different regions of the microfluidic device that are fluidly connected are fluid so that the solute moves down and / or the fluid flows through the device. It may have different compositions (eg, different concentrations of solutes such as proteins, carbohydrates, ions or other molecules).

The microfluidic devices or devices of the present invention may include "cleaned" and "non-cleaned" regions. Areas that are not cleared are areas and fluids that are cleared, provided that the fluid connection allows diffusion, but is constructed so that there is virtually no flow of medium between the areas that are cleared and the areas that are not cleared. Can be connected. Thus, the microfluidic device substantially isolates the non-cleaned region from the flow of media in the cleared region, but is substantially the only diffusible region between the cleared and non-cleaned regions. It can be constructed to allow fluid communication.

"Sterilizing" a living cell means making the cell incapable of reproducing.

The "group" of micro-objects used herein can be a single micro-object or multiple micro-objects. The group of microobjects can be a group of clones of living cells (eg, one cell, multiple cells or all cells from a clonal colony). A colony of living cells is a "clonal" if all of the living cells in the colony that are capable of replication are daughter cells derived from a single parent cell. A "cloned cell" is a cell of the same clonal colony.

In some embodiments of the invention, a swarm of micro-objects can be captured from a selected holding enclosure in a microfluidic device and moved from the retaining enclosure to a staging area, where the swarm of micro-objects. Can be unloaded from the microfluidic device. Microobjects can be living cells. Each retention enclosure can isolate living cells in the retention enclosure from living cells in other retention enclosures (eg, by placing the living cells in the isolated region of the retention enclosure). The micro-objects captured from the selected retention enclosure may be clonal cells, and aspects of the invention may select a particular group of clonal cells in a microfluidic device and migrate the clonal cells to a staging area. And the clonal cells can be removed from the microfluidic device while maintaining the clonality of the group.

1A-1D illustrate an example of a microfluidic device 100 including a holding enclosure 156, a selector 122 and a unloading interface 162. As will be appreciated, selector 122 may select micro-objects (not shown) and move them from any of the enclosures 156 to the staging area 172 inside the enclosure 102 of device 100 and carry them out. The interface 162 provides an interface within the enclosure 102 for an external unloading device 182 capable of unloading micro-objects (not shown) from the staging area 172.

As shown in FIGS. 1A-1D, the microfluidic device 100 may include an enclosure 102, a selector 122, a flow controller 124 and an export interface 162 for an external export device 182. As shown similarly, the microfluidic device 100 may include auxiliary elements such as a control module 130, a source of electromagnetic radiation 136 (hereinafter EM source 136), a detector 138 and / or the like.

The enclosure 102 may define a flow region 140 and may hold the liquid medium 144. The enclosure 102 may include, for example, a microfluidic structure 104 disposed on a base (eg, a substrate) 106. The microfluidic structure 104 is a gas permeable, flexible material such as rubber, plastic, elastomer, silicone (eg, patterned silicone), polydimethylsiloxane (“PDMS”) or the like. Can include. Other examples of materials that may constitute the microfluidic structure 104 include cast glass, etchable materials such as silicon, photoresists (eg SU8) or the like. In some embodiments, the material-and thus the microfluidic structure 104-may be rigid and / or substantially impermeable to gas. Whatever the microfluidic structure 104 may be located on the base 106. The base 106 may include one or more substrates. Although described as a single structure, the base 106 may include a number of interconnected structures such as a number of substrates. The microfluidic structure 104 may similarly include a number of structures that can be interconnected. For example, the microfluidic structure 104 also includes a cover (not shown) made of the same or different material as the other materials in the structure.

The microfluidic structure 104 and the base 106 may define a microfluidic flow region 140. Although one flow region 140 is shown in FIGS. 1A-1C, the microfluidic structure 104 and the base 106 can define multiple flow regions for the medium 144. The flow region 140 may include channels (152, 153 in FIG. 1B) and chambers that can be interconnected to form a microfluidic circuit. 1B and 1C describe the inner surface 142 of the flow region 140 in which the medium 144 can be placed as flat (eg, flat) and featureless. However, the inner surface 142 may instead be non-flat (eg, non-planar) and may include features (not shown) such as electrical terminals.

As shown, the enclosure 102 may include one or more inlets 108 through which the medium 144 may be charged into the flow region 140. The inlet 108 can be, for example, an inlet, an opening, a valve, another channel, a fluid connector, a tubing, a fluid pump (eg, a capacitive syringe pump) or the like. The enclosure 102 may also include one or more outlets 110 through which the medium 144 can be removed from the flow region 140. The outlet 110 can be, for example, an outlet, an opening, a valve, a channel, a fluid connector, a pipe, a pump or the like. As another example, Exit 110 ejects droplets, such as any ejection mechanism disclosed in US Patent Application No. 13 / 856,781 (attorney docket no. BL1-US) filed April 4, 2013. May include a mechanism. All or part of the enclosure 102 may be gas permeable to allow gas (eg, ambient air) to enter and exit the flow region 140. In an enclosure containing more than one flow region 140, each flow region 140 has one or more inlets 108 and one or more for charging the medium 144 and removing it from the flow region 140, respectively. Can be associated with exit 110 of.

As shown in FIGS. 1B and 1C, the holding enclosure 156 may be located in the flow area 140. For example, each holding enclosure 156 may include a barrier 154 disposed on the inner surface 142 of the enclosure 102. Many such holding enclosures 156 can be in the flow area 140 arranged in any pattern, and the holding enclosure 156 can be of any of many different sizes and shapes. For example, the retaining enclosure 156 may include the structures described in U.S. Provisional Application 62 / 058,658 filed October 1, 2014. Thus, for example, the retention enclosure 156 may include a contiguous zone (not shown) and an isolated zone (not shown). As shown in FIG. 1B, the openings of the holding enclosure 156 may be placed in close proximity to channels 152, 153, which are conduits for the medium 144 flowing past the openings of more than one enclosure 156. obtain. The opening of each retention enclosure 156 may allow the natural replacement of the liquid medium 144 flowing through channels 152, 153, but otherwise each retention enclosure 156 is a living body in any one enclosure 156. Micro-objects such as cells (not shown) can be sufficiently closed to prevent mixing with micro-objects in any other enclosure 156. Eight enclosures 156 and two channels 152, 153 are shown, but may be more or less. For example, there may be a single channel that is fluidly connected to a holding enclosure of 100, 250, 500, 1000, 2500, 5000, 10000, 25000, 50000, 100,000 or more.

The medium 144 can flow past the openings in the holding enclosure 156 into channels 152, 153. The flow of medium 144 in channels 152, 153 can provide nutrients, for example, to biological micro-objects (not shown) in retention enclosure 156. The flow of medium 144 in channels 152, 153 can also provide the removal of waste from the retention enclosure 156. In the retention enclosure 156 containing the isolated region, the exchange of nutrients and waste between the isolated region and channels 152, 153 can occur substantially only by diffusion.

As also shown, there may be a staging area 172 in the flow area 140 and thus inside the enclosure 102. The staging area 172 may simply be an area on the inner surface 142 of the enclosure 102, which may or may not be marked. Instead, the staging area 172 is a recess into the surface 142 (as shown in FIGS. 1B-1D), a platform extending from the surface 142 (not shown), a barrier (not shown) (similar to barrier 154 as an example). ), Chambers or similar structures may be included in the flow region 140. Whatever, as shown in FIG. 1D, the staging area 172 is located in close proximity to the passage 174 via the enclosure 102 (eg, across the flow region 140 and opposite the passage 174) and into the flow region 140. Can be done. The passage 174 can be from the outside of the enclosure 102 to the inside of the enclosure 102. The passage 174 can be, for example, a hole in the enclosure 102.

The unloading interface 162 may provide a contact point to the passage 174 into the siege 102 for an external unloading device 182 that may be an external device to the siege 102. As shown in FIG. 1C, the unloading interface 162 may be located on the enclosure 102 in close proximity to the aisle 174 (eg, on and / or within the aisle 174). The unloading interface 162 may include an opening 164 to aisle 174. The unloading interface 162 may further include a cover 166 above the opening 164. The cover 166 may be removable or otherwise breachable by the unloading device 182 so that the unloading device 182 pulls the passage 174 into the enclosure 102. , Can be inserted into the opening 164 in the carry-out interface 162. Although shown as separate entities, the unloading interface 162 and the microfluidic structure 104 can be integrally formed from the same material and thus be part of one physical entity.

The unloading interface 162 may include either a flexible material, such as polystyrene, or any of the materials described above with respect to the microfluidic structure 104. Alternatively, the unloading interface 162 may include a combination of a hard or stiff material or a flexible material and a hard or hard material. Examples of the cover 166 include a cover that can be attached to and removed from the enclosure 102, a cork-like structure that can be inserted into the opening 164 but smaller than the passage 174, or the like. As seen in FIGS. 12A-13B, other examples of the cover 166 include a self-closing cover 1206 that can be pushed and opened by the unloading device 182 and a unloading interface 1302 in the form of a penetrating self-healing structure.

As shown in FIG. 1D, the unloading device 182 can be a hollow tube-like structure. For example, the unloading device 182 may include a tubular housing 184 that defines an internal passage 186 from the first end 188 to the opposite second end 192. When the cover 166 of the unloading interface 162 is removed, the unloading device 182 can be inserted into the opening 164 in the interface 162 such that the first end 188 is near or in contact with the enclosure passage 174. Thus, the opening 190 at the first end 188 of the unloading device 182 can be installed in the enclosure 102 in close proximity to the passage 174.

In some embodiments, the unloading device 182 can be a device that produces a pressure difference (eg, an aspirator, a capacitive syringe pump, an exhaust pump, a peristaltic pump or the like). For example, the unloading device 182 can be connected to a source (not shown) capable of generating a pressure difference, which produces a pressure difference from the first end 188 to the second end 192 of the unloading device 182. I can let you. Thus, the unloading device 182 pulls a small object (not shown) from the staging area 172 through the staging area 172 through the passage 174 into the opening 190 in the first end 188, through the internal passage 186, and into the second end 192. It can be constructed, where micro-objects (not shown) can be recovered or otherwise discarded. Examples of unloading devices 182 include hollow needles such as pipettes, tubes, hypodermic needles, combinations thereof or the like. The internal passage 186 of the unloading device has a diameter of about 5 μm to 300 μm (eg, about 25 to 300 μm, about 50 to 300 μm, about 75 to 300 μm, about 100 to 300 μm, about 100 to 250 μm, about 100 to 200 μm, about 100 to It can have 150 μm, about 150-200 μm or any other diameter or range defined by one or more of the endpoints described above).

The unloading device 182 may further include a sensor (not shown) capable of detecting micro-objects passing through the unloading device. The sensor may be located proximal to the unloading interface 162. Alternatively, the sensor may be placed distal to the unloading interface 162 (eg, at the distal end of the unloading device 182). The sensor can be, for example, an imaging device such as a camera (eg, a digital camera) or an optical sensor (eg, a charge-coupled device or a complementary metal oxide semiconductor imaging device). Instead, the sensor can be an electrical device, such as a device that detects changes in complex impedance as a small object passes by.

The selector 122 may be configured to selectively generate an electrokinetic force on a micro object (not shown) in the medium 144. For example, the selector 122 may be configured to selectively activate (eg, turn on) and start and stop (eg, turn off) the electrodes on the inner surface 142 of the flow region 140. The electrodes may generate a force in the medium 144 that attracts or moves away micro-objects (not shown) in the medium 144, so the selector 122 may select and move one or more micro-objects in the medium 144. .. The electrode can be, for example, a dielectrophoresis (DEP) electrode.

For example, selector 122 is one or more dielectrophoretic electrode devices, optical (eg, laser) tweezers devices, and / or one or more photoelectric tweezers (OET) devices (eg, US Pat. No. 7,612,355, which is by reference). (Whole incorporated herein) or as disclosed in US Patent Application No. 14 / 051,004 (attorney docket no. BL9-US) (also incorporated herein by reference in its entirety). Can include. As yet another example, the selector 122 may include one or more devices (not shown) for moving droplets of medium 144 in which one or more of the microobjects are suspended. Such devices (not shown) may include electrowetting devices such as photoelectric wetting (OEW) devices (eg, as disclosed in U.S. Pat. No. 6,958,132) or other electrowetting devices. Thus the selector 122 can be characterized as a DEP device in some embodiments.

2A and 2B illustrate an example in which the selector 122 includes a DEP device 200. As shown, the DEP device 200 is the same as the first electrode 204, the second electrode 210, the electrode activation substrate 208, the power supply 212 (eg, alternating current (AC) power supply) and the light source 220 (which is the same as the EM source 136). May also be different). The medium 144 and the electrode activation substrate 208 in the flow region 140 can separate the electrodes 204 and 210. Changing the pattern 222 of the light from the light source 220 may selectively start and stop the changing pattern of the DEP electrode in the region 214 of the inner surface 142 of the flow region 140. (Hereinafter, region 214 is also referred to as "electrode region".)

In the example described in FIG. 2B, the light pattern 222'directed onto the inner surface 142 illuminates the obliquely parallel electrode regions 214a in the square pattern shown. No light is applied to the other electrode region 214, which is sometimes referred to as the "dark" electrode region 214. The relative electrical impedance from each dark electrode region 214 to the second electrode 210 beyond the electrode activation substrate 208 is from the relative impedance from the first electrode 204 across the medium 144 in the flow region 140 to the dark electrode region 214. big. However, shining light on the electrode region 214a increases the relative impedance from the lighted electrode region 214a to the second electrode 210 beyond the electrode activation substrate 208 from the first electrode 204 in the flow region 140. It is reduced beyond the medium 144 to less than the relative impedance to the illuminated electrode region 214a.

When the power supply 212 is activated, the aforementioned causes an electric field gradient in the medium 144 between the lighted electrode region 214a and the adjacent dark electrode region 214, which in turn near the medium 144. Generates a local DEP force that attracts or moves away small objects (not shown). Thus, the DEP electrode that attracts or moves microscopic objects in the medium 144 by varying the light pattern 222 projected from the light source 220 (eg, a laser source or other type of light source) into the microfluidic device 200. At many different such electrode areas 214 on the inner surface 142 of the flow area 140, it can be selectively started and stopped. Whether the DEP force attracts or moves nearby micro-objects may depend on parameters such as the frequency of the power supply 212 and the dielectric properties of the medium 144 and / or micro-objects (not shown).

The square pattern 222'of the illuminated electrode region 214a, described in FIG. 2B, is only an example. Any pattern of the electrode region 214 can be illuminated by the pattern 222 of the light projected into the device 100, and the pattern of the electrode region 222'to which the light is applied is repeated by changing the light pattern 222. Can be changed.

In some embodiments, the electrode activation substrate 208 may be a photoconductive material and the inner surface 142 may be featureless. In such an embodiment, the DEP electrode 214 can be generated anywhere and in any pattern on the inner surface 142 of the flow region 140 according to the light pattern 222 (see FIG. 2A). Therefore, the number and pattern of the electrode regions 214 are not fixed, but correspond to the optical pattern 222. An example is described in U.S. Pat. No. 7,612,355 above. Here, the non-doped amorphous silicon material 24 shown in the drawings of the patent described above can be an example of a photoconductive material that can constitute the electrode activation substrate 208.

In another aspect, the electrode activation substrate 208 may include a circuit board such as a semiconductor material including a plurality of dope layers, an electrically insulating layer and a conductive layer that form a semiconductor integrated circuit as is known in the semiconductor field. In such an embodiment, the electrical circuit element forms an electrical connection between the electrode region 214 on the inner surface 142 of the flow region 140 and the second electrode 210 which can be selectively started and stopped by the light pattern 222. obtain. Each electrical connection is high so that when not activated, the relative impedance from the corresponding electrode region 214 to the second electrode 210 is greater than the relative impedance from the first electrode 204 through the medium 144 to the corresponding electrode region 214. Can have impedance. However, when activated by the light in the light pattern 222, the relative impedance from the corresponding electrode region 214 to the second electrode 210 is smaller than the relative impedance from the first electrode 204 to the corresponding electrode region 214 via the medium 144. As such, each electrical connection can have a low impedance, which activates the DEP electrode in the corresponding electrode region 214, as described above. Thus, the DEP electrode that attracts or moves away microscopic objects (not shown) in the medium 144 is selectively activated or stopped by the light pattern 222 at many different electrode regions 214 on the inner surface 142 of the flow region 140. Can be done. Non-limiting examples of such configurations of the electrode activation substrate 208 are the optical transistor-based devices described in FIGS. 21 and 22 of US Pat. No. 7,956,339 and the US patent application filed on October 10, 2013. Includes devices 200, 400, 500 and 600 described throughout the drawings in No. 14 / 051,004.

In some embodiments, the first electrode 204 may be part of the first wall 202 (or cover) of the enclosure 102, the electrode activation substrate 208 and the second electrode 210, as generally described in FIG. 2A. , Can be part of the second wall 206 (or base) of the enclosure 102. As shown, the flow area 140 can be between the first wall 202 and the second wall 206. However, the above is nothing but an example. In another embodiment, the first electrode 204 may be part of the second wall 206 and one or both of the electrode activation substrate 208 and / or the second electrode 210 may be part of the first wall 202. As another example, the first electrode 204 may be part of the same wall 202 or 206 as the electrode activation substrate 208 and the second electrode 210. For example, the electrode activation substrate 208 may include a first electrode 204 and / or a second electrode 210. Also, the light source 220 may instead be placed under the enclosure 102.

Therefore, when configured as the DEP device 200 of FIGS. 2A and 2B, the light pattern 222 surrounds and captures a minute object in order to activate one or more DEP electrodes in the electrode region 214 of the inner surface 142 of the flow region 140. By projecting into the device 200 in a pattern, the selector 122 may select micro-objects (not shown) in the medium 144 in the flow region 140. The selector 122 can then move the captured micro-object by moving the light pattern 222 relative to the device 200. Instead, the device 200 can be moved relative to the light pattern 222.

The barrier 154 defining the retention enclosure 156 is described in FIGS. 1B and 1C and is described above as a physical barrier, but the barrier 154 is instead a virtual barrier containing the DEP force activated by the light pattern 222. obtain.

The flow controller 124 may be configured to control the flow of the medium 144 in the flow area 140. For example, the flow controller 124 may control the direction and / or speed of the flow. Non-limiting examples of the flow controller 124 include one or more pumps or fluid actuators. In some embodiments, the flow controller 124 may include additional elements, such as one or more sensors (not shown) for sensing the flow speed of the medium 144 in the flow area 140.

The control module 130 may be configured to receive and control signals from the selector 122, the EM source 136, the detector 138 and / or the flow controller 124. As shown, the control module 130 may include a controller 132 and a memory 134. In some embodiments, the controller 132 is a machine-readable instruction (eg, software, eg, stored as a non-transient signal in memory 134, which can be, for example, a digital electronic, optical or magnetic memory device. , Firmware, microcode or the like) can be a digital electronic controller (eg, a microprocessor, a microcontroller, a computer or the like) configured to operate according to. Instead, the controller 132 includes a combination of a real-wired digital and / or analog network or a digital electronic controller that operates according to machine-readable instructions and a real-wired digital and / or analog network. obtain. The control module 130 may be configured to perform all or any part of any of the processes, functions, steps or the like disclosed herein.

The EM source 136 may selectively direct electromagnetic radiation into the enclosure 102. Electromagnetic radiation can include wavelengths that are absorbed and / or excite it by a label such as a chromophore (eg, fluorophore). Electromagnetic radiation can include wavelengths in the visible or ultraviolet region of the electromagnetic spectrum. The EM source 136 can be, for example, a laser light source, a light emitting diode (LED), a high intensity discharge lamp or the like, which is a control module to direct light to a specific target area inside the flow area 140. It can be controlled by 130. The detector 138 can be a mechanism for capturing an image (eg, a digital image) of a flow region 140 including, for example, an enclosure 156 in a medium 144, a micro object (not shown) and a staging area 172. The detector 138 can also capture an image of the unloading interface 162. Examples of suitable imaging devices that the detector 138 may include include digital cameras or optical sensors such as charge-coupled devices and complementary metal oxide semiconductor imaging devices. Images can be captured and analyzed by such devices (eg by control module 130 and / or human operator).

In some embodiments, the enclosure 156 can be shielded from illumination (eg by selector 122, EM source and / or detector 138) or can be selectively illuminated for only a short period of time. Thus, the biological microobject 402 (see FIG. 4) can be protected from illumination, or the illumination of the biological microobject 402 can be seen in the biological microobject 502 (FIGS. 5 and 6). See) can be minimized before it is unloaded.

FIG. 3 illustrates an example of process 300 for carrying out a group of microobjects from a microfluidic device according to some embodiments. Process 300 can be performed by the microfluidic device 100 of FIGS. 1A-1D. The selector 122 may be configured as a DEP device as shown in FIGS. 2A and 2B. However, the process 300 can be performed by other microfluidic devices, but not so limited.

At step 302, process 300 may move a group of microobjects from the holding enclosure in the microfluidic device to the staging area. The group consists of one or more specific micro-objects in the holding enclosure. FIGS. 4 to 7 explain an example.

As shown in FIG. 4, there can be a group of micro objects 402 in the plurality of holding enclosures 156. For example, the microobject can be a living cell, and each group 402 in one of the retention enclosures 156 can be a clonal colony of cells. As shown similarly, the flow 404 of the medium 144 may be provided, for example, from the inlet 108 to the outlet 110.

As described in FIG. 5, one of the groups 402 in one of the holding enclosures 156 can be selected. The particular group 402 and / or retention enclosure 156 can be identified, for example, from an image of the flow region 140 captured by the detector 138 (see FIG. 1A). The choice may be based on the physical characteristics of the micro-objects in the retention enclosure 156 or their associated activity. Such physical features may include, for example, the number of micro-objects, their size, their morphology or the like. Activities associated with microobjects include, for example, the production or presentation of the antigen of interest (antibodies, cytokines, growth factors, metabolites, cancer cell markers or the like), the rate of growth, or a combination thereof. Can include. In FIG. 5, the selected group 402 is labeled 502. The selected group 502 is described as including all of the micro-objects in the enclosure 156, but instead the selected group 502 is more than all of the micro-objects in the group 402 of the enclosure 156. It may contain less (eg, one, two, three, four, five or so).

In the example shown in FIG. 5, the selected group 502 of microobjects is captured by an optical trap 504 that can be generated by a selector 122 configured as the DEP device 200 of FIGS. 2A and 2B. For example, the light trap 504 may include a continuous pattern of activated DEP electrodes 214 surrounding the selected group 502. As mentioned above, the DEP electrode 214 may be selectively activated by the pattern 222 of light directed onto the electrode activation substrate 208 (see FIGS. 2A and 2B).

As shown in FIG. 6, the light trap 504 can be moved from the holding enclosure 156 towards the staging area 172, which also moves the selected group 502 of microobjects. The light trap 504 can then be moved into the staging area 172. As described above, the light trap 504 can be moved by varying the light pattern 222 directed onto the electrode activation substrate 208. The image of the flow region 140 captured by the detector 138 may facilitate selecting, capturing, and moving the group 502 to the staging area 172.

The light trap 504 may be configured to keep small objects in the medium 144 away, for example. Thus, the optical trap 504 may continue to carry the micro-objects of the selected group 502 inside the trap 504 and all other micro-objects from any other group 402 outside the trap 504. obtain. Thus, the optical trap 504 can prevent micro-objects in the selected group 502 from mixing with any other group 402 of micro-objects in device 100 during step 302 of FIG. 3 and select. Any micro-objects not found in group 502 can be prevented from mixing with group 502.

As shown in FIG. 7, once the selected group 502 comes to the staging area 172, the light trap 504 can be turned off. As shown in FIG. 7, the flow 404 of the medium 144 (see FIG. 4) can be turned off so that the microobjects of the selected group 502 are not flushed from the staging area 172. Alternatively, the light trap 504 can be kept held in the staging area 172 to hold the group 502 in place in the staging area 172 with respect to the flow of the medium 144 in the flow area 140. As yet yet another alternative, the staging area 172 may include a physical barrier (not shown) that can hold the group 502 in place against the flow of the medium 144 in the flow area 140.

In step 304 of FIG. 3, the process 300 can carry the group 502 of microobjects out of the staging area 172. 8 and 9 illustrate an example.

As shown in FIG. 8, the cover 166 of the unloading interface 162 can be removed and the unloading device 182 has an opening 190 in the first end 188 of the unloading device 182 proximal to the passage 174 into the enclosure 102. Or it can be inserted into interface 162 to make contact with it. Thus, as shown in FIG. 8, the opening 190 in the first end 188 of the unloading device 182 may be in close proximity to the group 502 of microobjects in the staging area 172.

As shown in FIG. 9, a pressure difference can occur in the unloading device 182. Thus, the micro-objects in group 502 located in the staging area 172 can be pulled out through the passage 174 into the enclosure 102 and into the interior 186 of the unloading device 182. The microobjects of group 502 can then be recovered at the second end 192 of the unloading device 182 or otherwise discarded. Alternatively, when the unloading device 182 is not a device that produces a pressure difference or no pressure difference is produced, another mechanism pulls the micro-objects in group 502 into the unloading device 182. For example, a flow (not shown) of the medium 140 that pushes the micro-objects in group 502 from the staging area 172 into the unloading device 182 can be generated. Other forces that can be used to carry out micro-objects can include gravity and magnetic force. As yet another alternative, a combination of forces such as the flow of the medium 140 and the pressure difference generated by the unloading device 182 can be used to pull the micro-objects in group 502 into the unloading device 182.

FIG. 10 is an example of the process 1000 in which step 304 of FIG. 3 can be performed. As will be appreciated, the fluid flow can move the microobjects of group 502 from the staging area 172 into the unloading device 182. The flow of such fluid may include the flow of the medium 144 in the device 100, the pressure difference due to the unloading device 182, the capillary force due to the unloading device 182, and the like.

At step 1002, the flow 404 (see FIG. 4) of the medium 144 in the flow region 140 can be substantially stopped. For example, the control module 130 may cause the flow controller 124 to substantially stop the flow 404. Alternatively, the flow 404 can be slowed down in step 1002. At step 1004, the unloading device 182 can be inserted into the unloading interface 162, for example as shown in FIG. At step 1006, the outlet 110 of the microfluidic device 100 (see FIGS. 1A-1C) may be closed. Any other outlet from the enclosure 102 other than the carry-out interface 162 may also be closed.

In step 1008, the flow 404 of the medium 144 in the flow region 140 can be restarted. Since the unloading interface 162 is the only outlet opening from the enclosure 102, the direction of the flow 404 will be towards and into the unloading interface 162. Thus, the flow 404 can wipe out the micro-objects of group 502 from the staging area 172 towards the unloading device 182.

At step 1010, a pressure difference can be created in the unloading device 182, which can pull microobjects of group 502 into and towards the interior 186 of the second end 192 of the unloading device 182. Instead, when the pressure difference is not created in step 1010, the flow generated in step 1008 alone (optionally coupled with other forces such as gravity or magnetic force) is microscopic in group 502. The object is pulled out of the staging area 172 into the unloading device 182. Thus, some aspects of the unloading device 182 do not have a pressure differential capability.

Once the group 502 of microobjects has been pulled into the unloading device 182, the flow 404 of the medium 144 can again be substantially stopped or slowed down in step 1012. The unloading device 182 can optionally be removed from the unloading interface 162 in step 1014. The pressure difference due to the unloading device 182 can also be turned off in step 1012 or 1014. At step 1016, the outlet 110 of the microfluidic device 100 can be opened, and the flow 404 of the medium 144 in the flow region 140 can be restarted again at step 1018.

Process 100 is only an example and may vary. For example, the order of steps 1002-1018 may differ from that shown in some cases. In addition, not all steps need to be performed. For example, the unloading device 182 can be connected substantially permanently to the unloading interface 162, whereby steps 1004 and 1014 can be skipped. Similarly, in a series of unloading of microobjects from different holding enclosures 156, outlet 110 may be closed for the first unloading, but may remain closed for subsequent unloading steps thereafter. .. Thus, steps 1016 of process 1000 can be skipped, steps 1006 and 1016 of process 1000 can be skipped, steps 1014 and 1016 of process 1000 can be skipped, and steps 1004, 1006, 1014 and 1006 of process 1000 can be skipped. Obtaining or steps 1004, 1006 and 1014 of process 1000 may be skipped.

With reference to FIG. 3 again, in step 306, process 300 may exclude micro-objects that were not unloaded at the staging area and unloading interface. For various reasons, one or more of the microobjects in group 502 may not be carried out as part of step 304. For example, microobjects may unintentionally remain in the staging area 172. As another example, micro-objects may become clogged in the carry-out interface 162.

For various reasons, such micro-objects that were not unloaded at the staging area 172 and the unloading interface 162 may need to be excluded. For example, in some applications it may be important to prevent micro-objects in one of the groups 402 from mixing with micro-objects in the other group 402 (see FIG. 4). For example, as mentioned, each group 402 of microobjects in enclosure 156 can be a clonal colony of living cells, in this case from mixing live cells in one group 402 that can replicate with other groups. Prevention can be important. This is because colonies with mixed cells are no longer clones. If micro-objects from group 502 were left in the staging area 172 or the unloading interface 162, the un-unloaded micro-objects could be mixed with the next group 402 of micro-objects and removed from the holding enclosure 156. , Moved into the staging area 172 and carried out via the carry-out interface 162.

Step 306 can be performed in any of various ways. For example, the detector 138 may capture images of the staging area 172 and the unloading interface 162, which images may be examined for tiny objects that were not unloaded from group 502. The image can be examined by a human operator and / or by utilizing an image processing algorithm performed by the control module 130. Any of the detected micro-objects can be excluded in any of the various possible ways.

For example, micro-objects that were not unloaded at the staging area 172 and / or the unload interface 162 utilize a selector 122 (configured as the DEP device 200 of FIGS. 2A and 2B as an example) to unload the staging area 172 and / or unload. It can be removed by flushing micro-objects that have not been unloaded from interface 162 or the like. As another example, step 306 may be performed by repeating step 304 one or more times without first moving a group of new microobjects into the staging area 172 in step 302. ..

As another example, if the non-delivered microobject is a living cell, the non-carried living cell can be excluded by sterilizing or killing the non-carried cell. FIG. 11 is an example of process 1100 for carrying out step 306 of FIG. 3 when the micro-object is a biological micro-object such as a cell. At step 1102, the procedure can be applied to the staging area 172 and the unloading interface 162. Treatment can kill or sterilize biological microscopic objects. For example, the procedure may include flushing a chemical agent that is fatal to or sterilizes the biological micro-object via the staging area 172 and the unloading interface 162. As another example, the procedure may include directing electromagnetic radiation that is fatal to or sterilizes the biological micro-object onto the staging area 172 and the carry-out interface 162. For example, the EM source 136 may direct the laser beam over the staging area 172 and the unloading interface 162. In some embodiments, the detector 138 may capture an image of the staging area 172 and / or the export interface 162, and such a laser beam may be specifically directed at any biological microobject detected from the image. .. Other examples of killing or sterilizing cells that were not exported in the staging area 172 or export interface 162 are electrical, hot or cold, lysed (eg by mechanical cutting microdevices), ultrasonic vibration or the use of the like. including.

At step 1104, process 300 may flush the staging area 172 and the unloading interface 162 to wash away any biological micro-objects in the staging area 172 or the unloading interface 162. For example, the staging area 172 and the unloading interface 162 can be flushed by the medium 144. As another example, the staging area 172 and the unloading interface 162 can be flushed with deionized water.

At step 1106, the staging area 172 and the unloading interface 162 can be examined for biological microobjects that have not been unloaded. For example, the detector 138 can capture images of the staging area 172 and the unloading interface 162, but they can be examined for biological micro-objects that were not unloaded, as described above. If undelivered biological micro-objects are detected, step 1102 to apply the procedure and step 1104 to flush can be repeated until the non-delivered micro-objects are no longer detected in step 1106.

The process 1100 of FIG. 11 is only an example, and fluctuations are taken into consideration. For example, examining step 1106 may precede treatment step 1102 and flushing step 1104. Thus, in some embodiments, they are carried out only if undelivered micro-objects are detected. As another example, process 1100 can be performed without one or both of flushing step 1104 and examining step 1106. As yet another example, treatment 1100 can be performed without step 1102, such as by flushing undelivered biological micro-objects into a waste container.

In fact, the devices and processes described in FIGS. 1A-11 are examples and variations are considered. 12A-16 illustrate an example of such variation.

12A-12C illustrate another example of the unloading interface 1202. It is thus replaceable with the unloading interface 162 in any of the figures or discussions herein. As shown, the unloading interface 1202 may be located on the siege 102, above the aisle 174. The unloading interface 1202 may include an opening 1204 to passage 174, a cover 1206 including a crevice 1208 (eg, a slit) and a valve 1210, and an inflow opening 1222.

As shown in FIG. 12B, fissure 1208 can define valve 1210 and thus separate. For example, crevice 1208 may include one or more notches in cover 1206. Cover 1206 is described to include one fissure 1208 and two valves 1210, but cover 1206 may include more fissures 1208 and valves 1210. Whatever the cover 1206 may comprise a flexible elastic material, the cover 1206 may have an elastic internal force across or substantially of the passage 174 through which the valve 1210 and the cover 1206 pass through the enclosure 102. It can be constructed to be proximal or contact biased to cover the entire area. However, the flexibility of the valve 1210 may allow the valve 1210 to move away in response to the force of the first end 188 of the unloading device 182 being pressed against the cover 1206 near the crevice 1208. As described in FIG. 12C, the valve 1210 may allow the first end 188 of the unloading device 172 to be moved into the enclosure 102 proximally or in contact with the passage 174. .. Although not shown, the elasticity of the valve 1210 is such that when the unloading device 182 is removed from the unloading interface 1202, the cover 1206 covers the passage 174 shown in FIG. 12B entirely or substantially entirely. The valve 1210 can be moved in the opposite direction while making contact.

Thus, the cover 1206 is when the end 188 of the unloading device 182 is pressed against the cover 1206 through it (in this case, the cover 1206 is open to accept the unloading device 182). Except, it can be closed. When the unloading device 182 is removed from the cover 1206, the cover 1206 self-closes.

The inflow 1222 portion of the carry-out interface 1202 can function as a guide for facilitating the insertion of the carry-out device 182 into the carry-out interface 1202. As described, the inflow 1222 may include an inclined side wall 1224.

13A-13C illustrate another example of the unloading interface 1302, which can be replaced with the unloading interface 162 in any of the figures or discussions herein. As shown, the unloading interface 1302 may be located on the aisle 174 and on the enclosure 102. The unloading interface 1302 may include a mass of material that entirely covers (and thus covers 166) the passage 174 shown in FIG. 13A. However, the material can be sufficiently supple to be easily penetrated by the unloading device 1304 shown in FIG. 13B. In FIG. 13B, the unloading interface 1302 is penetrated through the unloading interface 1302 by pressing, for example, a subcutaneous injection needle or a first end 1388 of a unloading device 1304 which may be of the same type. As a result, a hole 1306 is formed in the carry-out interface 1302. The unloading device 1304 can be particularly thin, but otherwise it can generally be something like the unloading device 182. Thus, while inserted via the unloading interface 1302 shown in FIG. 13B, the first end 1388 of the unloading device 1304 may enter the enclosure 102 proximally or in contact with the passage 174 and be a micro object (FIG. 13B). (Not shown in 13B) can be pulled into the opening at the first end 1388 of the carry-out device 1304 generally described above with respect to the carry-out device 182. Therefore, the unloading device 1304 is an example of the unloading device 182 and can be replaced with the unloading device 182 in any of the figures or studies herein. The unloading interface 1302 may self-heal so that the holes 1306 made by the unloading device 182 are closed when the unloading device 1304 is removed from the unloading interface 1302. Examples of self-healing materials for unloading interface 1302 include silicone. As described in FIG. 13C, the unloading interface 1302 can be pierced many times in many places by the unloading device 1304. Rather than being a structure separated from the microfluidic structure 104, the unloading interface 1302 may instead be part of the microfluidic structure 104 in the immediate vicinity of the staging area 172. In such cases, the structure 104 does not need to include the passage 174.

FIG. 14 shows yet another example of a unloading interface 1406 that can replace the unloading interface 162 in any of the figures or studies herein. In FIG. 14, the unloading interface 1406 permanently secures the access tube 1402 to the enclosure 102 such that the first end 1488 of the tube 1402 is proximal to or in contact with the passage 174 into the enclosure 102. It can be a formula adhesion mechanism. The micro-object (not shown in FIG. 14) can be pulled into the opening in the first end 1488 of the access tube 1402, as generally described above for the unloading device 182. Therefore, the access tube 1402 is an example of the carry-out device 182, and can be replaced with the carry-out device 182 in any of the figures or studies herein. As shown, the access tube 1402 may include a valve 1408 that can open and close the internal passage from the first end 1488 to the second end 1492 in the tube 1402. The valve 1408 may be closed, for example, except during the removal of microobjects from the staging area 172. However, the access tube 1402 does not need to include the valve 1408, as adjusting the capillary force and the velocity of the fluid flow through the flow area 140 replaces the valve 1408.

FIG. 15A shows a partial perspective view of the microfluidic device 1500 which may be an example of variation of the device 100 of FIGS. 1A-1D. With a few exceptions, the microfluidic device 1500 can be of the same type as the device 100, and the similarly numbered elements in FIGS. 15A-15C can be the same as in FIGS. 1A-1D.

As shown in FIGS. 15A-15C, the unloading interface 162 may be the first unloading interface of the microfluidic device 1500, which is also located on the second passage 1574 into the flow region 140 via the enclosure 102. The second unloading interface 1562 may also be included. Otherwise, the second unloading interface 1562 may be similar to the first unloading interface 162. For example, the second unloading interface 1562 may include an opening 1564 and a cover 1566 to the passage 1574, but may be similar to the opening 164 and cover 166 of the first unloading interface 162 described above. As shown similarly, the passages 174, 1574 into the enclosure 102 and thus the unloading interfaces 162, 1562 can be on opposite sides of the enclosure 102 and do not need to be aligned with each other.

The microfluidic device 1500 may also have a staging area 1512 that is different from the staging area 172 of the device 100. For example, as shown in FIGS. 15B and 15C, the staging area 1512 is generally aligned with the passage 174 and thus the first unloading interface 162 with the first end 1518; the passage 1574 and thus the second unloading interface 1562. The second end 1514; and the intermediate portion 1516 between the ends 1514, 1518 may be included. Otherwise, the staging area 1512 may be of the same type as the staging area 172 of device 100 described above. Alternatively, the device 100 may have a staging area similar to the 1512, so that it can be replaced with the staging area 172 in FIGS. 1A-11 and the above discussion.

The process 300 of FIG. 3 can be performed by the microfluidic device 1500 as follows. Step 302 can generally be performed as described above. For example, although not visualized in the figures shown in FIGS. 15A-15C, device 1500 may include a holding enclosure 156, each of which holds a group of microobjects 402, eg, as shown in FIG. obtain. The holding enclosure 156 may include an isolation space in which a group of microobjects 402 are located. The group of micro-objects can be moved from the holding enclosure 156 to the staging area or a portion thereof (eg, intermediate portion 1516), as generally described in FIGS. 4-7.

Step 304 can be performed as described in FIG. Covers 166, 1566 of the unloading interfaces 162, 1562 can be removed, and the first unloading device 182 has a first unloading device 182 such that the opening 190 in the first end 188 is proximal to or in contact with the first passage 174 via the enclosure 102. It can be inserted into one interface 162. The first unloading device 182 can be the same as the unloading device 182 described above, and the one described above can be the same as the one described above.

The second unloading device 1682 can be similarly inserted into the second interface 1562. The second unloading device 1682 may be the same as or similar to the first unloading device 182. Thus, the second unloading device 1682 includes a hollow tube-like structure that includes a tubular housing 1684 that defines an internal passage 1686 from the first end 1688 to the opposite second end 1692. When the cover 1566 of the second unloading interface 1682 is removed, the second unloading device 1682 is evacuated so that the first end 1688 is proximal to or in contact with the second passage 1574 into the enclosure 102. It can be inserted into the opening 1564 in interface 1562.

As described in FIG. 16, when the unloading devices 182, 1682 are inserted into the unloading interfaces 162, 1562, the flow of liquid (eg medium 144) flows into the flow region 140 close to the staging area 1512. The flow of liquid through a portion of the can be generated between the openings 190 of the first unloading device 182 to the openings 1690 of the second unloading device 1582. To facilitate the flow of such liquid, a pressure difference can be generated between the opening 190 of the first unloading device 182 and the opening 1690 of the second unloading device 1682.

For example, as shown in FIG. 16, a flow of liquid (eg, medium 144) can be generated from the second end 192 to the first end 188 of the first unloading device 182. This can create a flow 1604 in the enclosure 102 from the first end 1518 to the second end 1514 of the staging area 1512. The flow 1604 can clear the selected group 502 of microobjects from the intermediate portion 1516 of the staging area 1512 towards the opening 1690 at the first end 1688 of the second end 1514 and thus the second unloading device 1682. At the same time, a pressure difference can be generated from the first end 1688 to the second end 1692 of the second unloading device 1682. Such a pressure difference can pull a selected group 502 of microobjects into the second unloading device 1682 via the second passage 1574 in the enclosure 102. Thus, the micro-objects of the selected group 502 can be recovered at the second end 1692 of the second unloading device 1682.

The selected group 502 of the microobjects moved to the staging area 1512 in step 302 of FIG. 3 can thus be carried out in step 304. Generally, as described above, thereafter in step 306, the staging area 1512, the first unloading interface 162 and / or the second unloading interface 1562 can be flushed and / or small objects that have not been unloaded from them. Can be excluded.

FIG. 17 illustrates another variation of the device 100 of FIGS. 1A-1D. As shown, device 1700 in FIG. 17 may include multiple staging areas 1772, each of which may be of the same type as staging area 172 as described above, where device 1700 may include multiple outlets 110a, 110b. (They may have the exit 110 as described above). In some embodiments, each staging area 1772 may be associated with a single retaining enclosure 156. Otherwise, device 1700 may be of the same type as device 100 of FIGS. 1A-1D, and similarly numbered elements may be of the same type.

As described in FIG. 17, the medium flow 1704 can be directed to the staging area 1772. For example, all outlets 110 except outlet 110a can be closed, bypassing fluid flow from channel 152, and generally a single flow path 1752 from inlet 108 to staging area 1772 and to the only open outlet 110a. Can occur. A micro object (not shown in FIG. 17) can be moved to a staging area 1772 by simply moving the micro object from one of the enclosures 156 into a flow 1704 (which can carry the micro object to the staging area 1772). There, micro-objects (not shown) can be removed via a close carry-out interface (not shown), as described above. Alternatively, in some embodiments, the micro-object (not shown in FIG. 17) can be moved from one of the enclosures 156 to the staging area 1772, and the flow 1704 moves the micro-object to and from exit 110a. Can be carried. Therefore, the outlet 110a can function as a carry-out interface. The outlet 110a can be of the same type as any of the examples of outlet 110 described above, including a mere hole through the enclosure 102.

FIG. 18 illustrates the variation of device 1700 of FIG. As shown, the device 1800 of FIG. 18 is generally the same as the device 1700, except that the device 1800 generally includes a single barrier 1854 that defines a holding enclosure 156 and separates the channel 152 from the flow path 1752. It is possible (and elements with similar numbers can be the same). In addition, device 1800 includes (or none at all) a single staging area 1772 located in flow path 1752. As described above for FIG. 17, the flow 1704 of the medium 144 can be generated in the flow path 1752 by closing all outlets 110 except the outlet 110a. The micro-object (not shown) wraps the micro-object from one of the enclosures 156 to the staging area 1772 (where the micro-object (not shown) goes through the proximity carry-out interface (not shown) as described above. It can be removed from the device 1800 by moving it to (can be removed). Instead, the micro-object (not shown in FIG. 18) is a device by moving the micro-object from one of the enclosures 156 into the staging area 1772 or into the flow 1704 (if the staging area does not exist). Can be unloaded from 1800. Thus, the flow 1704 can carry microobjects to and from outlet 110a. The outlet 110a can be of the same kind as any of the examples of the outlet 110 described above, including a mere hole through the enclosure 102.

19A-19B illustrate another variation of device 1700 of FIG. As shown, the device 1900 in FIGS. 19A-19B can generally be the same as (and similarly numbered) the device 1700, except that the device 1900 includes a channel 152 that also functions as a flow path 1752. The elements that were made can be the same). In the embodiments shown, each staging area 1772 is placed in channel 152 in close proximity to an opening to a holding enclosure 156. However, the number of staging areas in device 1900 can be less than the number of retention enclosures 156 and / or the staging areas need not have a well-defined location in channel 152 (ie, in channel 152). Both locations can be considered staging areas 1772). As shown in FIG. 19B (cross section of device 1900), the inlet 108 to and / or the outlet 110 from the flow region 140 may be placed on top of the flow region 140. The exit can serve as a carry-out interface with aisle 174. The outlet 110 may be connected to the unloading device (not shown) in the manner generally described above. Moreover, the holding enclosure 156 may have an isolated region 1960 and a connecting region 1958, which is fluidly connected to the channel 152 via the connecting region 1958. The micro-object (not shown in FIGS. 19A-19B) can be unloaded from device 1900 by moving the micro-object from one of the enclosures 156 to the staging area 1772 in channel 152. The flow 1904 can then carry the micro object to the outlet 110 and out of the unloading device.

FIG. 20 describes an example of process 2000 for carrying out a minute object from the device 1900 of FIGS. 19A-19B. As shown, in step 2002, process 2000 can generate a flow 1904 of the medium 144 in the enclosure 102 of the device 1900 directly to the outlet 110. For example, flow 1904 may flush media 144 out of outlet 110 via channel 152. The flush in step 2002 may remove tiny objects from the device 1900 that may be around the exit 110 in the channel 152 (ie, the unloading interface) and / or in the unloading device (not shown). In some embodiments, the amount of medium 144 flushed through the device 1900 in step 2002 is at least twice the combined volume of fluid that can be held by the device 1900 and the unloading device (eg, at least 3, 4, 5, 10, 15, 20, 25 times or more). The medium to be flushed is about 0.05 to 5.0 μL / sec (eg, about 0.1 to 2.0, 0.2 to 1.5, 0.5 to 1.0 μL / sec or about 1.0 to 1.0 to. It can flow into the channel at a rate of 2.0 μL / sec). In step 2002, the medium flow 1904 can be a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or a combination thereof.

In step 2004, process 2000 may include slowing down or substantially stopping the flow 1904 of medium 144 in channel 152. For example, the flow 1904 of medium 144 in channel 152 can be slowed down to a rate of about 0.05 μL / sec or less.

At step 2006, process 2000 may select a group of microobjects in one of the holding enclosures 156 and at step 2008 it is placed into channel 152 (eg, close to the opening of enclosure 156). The selected group can be moved (to the staging area 1772 or some other area of channel 152). Although not shown in FIGS. 19A-19B, the holding enclosure 156 may contain the group of microobjects 402 described in FIG. Steps 2006 and 2008 are described, for example, in FIGS. 5-7, except that the selected group 502 is moved into channel 152 in FIGS. 19A-19B rather than the staging area 172 in FIGS. 5-7. Can be carried out as it is. When a light trap (eg, light trap 504 in FIGS. 5-7) is used to move a group of micro-objects 502, the light trap is off once the micro-object 502 is located in channel 152. Can be done.

At step 2010, the flow 1904 of the medium 144 in the channel 152 can be restarted and the group 502 of selected microobjects can be carried by the flow 1904, through the outlet 110 and through the distal end of the unloading device. Can be unloaded out of the unloading device (not shown). Throughout step 2010, the flow 1904 of medium 144 in channel 152 is, for example, about 0.05-0.25 μL / sec (eg, about 0.1-0.2 μL / sec or about 0.14-0.15 μL). Can be restarted at a speed of (/ sec). The medium flow 1904 can be a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or a combination thereof.

In process 2000 (as well as processes 300 and 1000), the unloading device is configured to unload the medium 144 (or other solution) into a container such as a test tube, a well in a microtiter plate or the like. obtain. The distal end of the unloading device (eg, the second end 192 in FIG. 1D or the second end 1692 in FIG. 16) could come into contact with the surface of the liquid contained in the container, thereby being unloaded. The volume of the medium 144 comes into direct contact with the liquid in the container. Alternatively, the distal end of the unloading device may contact the bottom wall or side wall of the container. Such direct contact with either the container or the liquid contained therein can help reduce surface tension, which is otherwise carried out in association with the unloading device 144 ( Or it may tend to keep the volume of some fractions) constant.

Step 2010 of process 2000 may include disposing of the unwanted medium 144 before carrying out the group 502 of selected microobjects. Once the microobject group 502 is moved to the staging area 1772 (eg, the area of channel 140 where the selected microobject group 502 is located close to the opening of the retaining enclosure 156 from which it has been removed). (See FIG. 19A), the "leading volume" of the liquid medium 144 is (i) part of the channel 140 located between the staging area 1772 and the outlet 110, as well as (ii) the proximal end (ii). As an example, it will occupy the internal passage (not shown) of the unloading device between the first end 188 of the unloading device 182) and the distal end (eg the second end 192 of the unloading device 182). Since this leading volume of the liquid medium 144 cannot contain any microobjects, it may be desirable to dispose of it, for example, in a disposal container that is easily accessible to the second end of the unloading device. Therefore, step 2010 completes the flow of a volume of liquid medium 144 equivalent to the leading volume through the device 1900 so that the leading volume is disposed of, and then the removal of the group 502 of selected microobjects. May include doing. After disposing of the leading volume of the liquid medium 144, but before completing the unloading of the group 502 of micro-objects, the flow 1904 of the liquid medium 144 in the channel 140 is at the distal end of the unloading device. Can be delayed or substantially stopped for a sufficient period of time to move the octopus into a container, into which a group of microobjects 502 can be unloaded.

Once the group of selected micro-objects 502 has been unloaded, step 2010 further comprises excluding any unremoved micro-objects that could clog the channel 140, outlet 110 and / or the unloading device. Any of the above excluded techniques can be utilized. For example, the technique to exclude may include or consist of flushing the device 1900, including the unloading device. Such flushing can be a component of step 2002 of the new round of unloading.

In one variation relative to process 2000, in step 2002 for flushing the channel 140, outlet 110 and / or unloading device (not shown), and in step 2010 for unloading the group 502 of selected microobjects. The liquid medium 144 used is replaceable with an alternative solution. Alternative solutions may be suitable for treating the group 502 of microobjects after they have been carried out. The solution can be, for example, a solution that does not support cell growth, a solution that cells cannot tolerate well over a long period of time, or a solution that is suitable or useful for assaying biological activity. For example, the solution can be HEPES buffer, phosphate buffered saline (PBS), osmotically balanced sugar water or the like. In this variation relative to process 2000, step 2010 may include flushing with a liquid medium 144 such that the alternative solution is washed off the device 1900. Exposure to alternative solutions of groups of microobjects 402 (not shown) placed in the isolation region of retention enclosure 156 can be limited, provided that steps 2002-2010 are carried out rapidly. By this, any of the damaging effects on the group of microobjects 402 that the alternative solution may otherwise have is substantially reduced.

Any of the steps 2000 described above, such as disposing of the leading volume of the liquid medium 144, or flushing and carrying it out into a liquid different from the liquid medium 144, as well as specifics such as flow rate. Any of the conditions and the like can be applied in the same manner as in the process 300 or 1000, if necessary.

In any of treatments 300, 1000 or 2000, the group 502 of selected microobjects to be carried out can be carried out into a predetermined volume of medium 144 (or other solution). The volume of the medium 144 may depend on the number of micro-objects in the group and how the group 502 of micro-objects after export is to be processed. Thus, for example, group 502 of microobjects is the volume of medium 144 that is less than 5 μL (eg, 4 μL, 3 μL, 2 μL, 1 μL, 750 nL, 500 nL, 250 nL, 200 nL, 150 nL, 100 nL, 75 nL, 50 nL or less). Can be carried out inside. Instead, group 502 of microobjects is about 1-10 μL, 5-15 μL, 10-20 μL, 15-25 μL, 20-30 μL, 25-35 μL, 30-40 μL, 35-45 μL, 40-50 μL or the aforementioned 2 It can be carried into a volume that is in any range defined by one endpoint. In the application of single cell genomics or of the same species, the group 502 of microobjects can consist of a single cell, and the volume of the carried-out medium 144 containing the single cell is relatively small (eg,). It can be less than 5 μL). In the development of cell lines or clonal proliferation of cells, groups of microobjects can contain multiple cells and the volume of the exported medium 144 containing the multiple cells is relatively large (eg 5 μL, 10 μL, 25 μL). , 50 μL or more). In assaying activity associated with micro-objects, the micro-object group 502 may contain one or more micro-objects, and the volume of the unloading medium 144 containing the micro-object group 502 may be a particular assay. Can be relatively small or relatively large, depending on the needs of.

In order to prevent dilution and / or evaporation of the carried-out volume of the medium 144, especially when the carried-out medium 144 has a small volume (eg, 1 μL or less), the carried-out medium 144 volume (of microobjects). (Contains a group) can be carried out into a non-aqueous liquid such as an oil (eg, mineral oil). Instead, the volume of the exported medium 144 is contained within the volume of the medium (eg, fresh cell growth medium), assay medium (eg, containing the appropriate assay components), or the exported medium 144. It can be carried out into some other solution (eg PBS, cell lysis buffer, etc.) that is unsuitable for subsequent treatment of the microscopic object.

In any of process 300, 1000 or 2000, the group of selected micro-objects 502 can be carried out individually or continuously with other groups of selected micro-objects (ie, process). 300, 1000 and 2000 can be repeated). In some embodiments, the continuous removal of a group of microobjects involves repeating certain steps of the process, but not the other steps of the process. For example, in process 2000, steps 206 and 208 may be repeated multiple times without repeating steps 2002, 2004 and 2010 so that a plurality of selected groups of microobjects 502 are present in channel 140. Become. A predetermined volume of the liquid medium 144 can be continuously delivered into different containers throughout the delivery in step 2010. By adjusting the volume of the liquid medium 144 so that a given volume contains only a single group of selected micro-objects, mixing of the micro-objects among multiple selected groups can be avoided. ..

21A-21C illustrate additional variations of the devices disclosed herein (eg, device 100 in FIGS. 1A-1D or device 1900 in FIGS. 19A-19B). As shown, device 2100 of FIG. 21A allows a hypodermic needle 1304 or other such unloading device to penetrate interface 1302 and move microobjects (not shown in FIG. 21A) directly from enclosure 156. It may include a passage 174 through a microfluidic structure 104 and an unloading interface 1302 in the form of a penetrating cover in close proximity to each of the holding enclosures 156. Each passage 174, each unloading interface 1302 and the hypodermic needle 1304 could otherwise be the same numbered elements as in FIGS. 13A-13C. Also, device 2100 could otherwise be similar to device 100 in FIGS. 1A-1D or device 1900 in FIGS. 19A-19B, and similarly numbered elements could be the same. 21B and 21C show different ways in which the unloading interface 1302 may be in close proximity to the holding enclosure 156. In FIG. 21B, the retention enclosure 156 includes an isolation region 1960, a portion of which forms a staging area 2172 on which the unloading interface 1302 may be placed. Therefore, the micro-object 204 (not shown) placed in the staging area 2172 portion of the holding enclosure 156 is convenient for direct removal from the holding enclosure 156. In FIG. 21C, the retaining enclosure 156 includes an isolated area 1960 (in this case by a passage) connected to a staging area 2172, on which the carry-out interface 1302 may be placed. Cells will begin to migrate into the staging area as a group of microscopic objects 402 (not shown), such as a population of cells, grows in the isolation region 1960 of the retention enclosure of FIG. 21C. Such movement can be achieved, for example, by a force involving gravity. Again, once micro-objects are present in the staging area 2172, they are convenient for direct removal from the holding enclosure 156.

As shown in FIG. 21A, rather than the unloading interface 1302 in the form of a penetrating cover 1302 and the unloading device in the form of a hypodermic needle 1304, the device 2100 is the unloading interface and unloading device described herein. Any of these can be used. For example, the unloading interface 162 of FIGS. 1A-1D can be replaced with any or all of the unloading interface 1302 in FIG. 21A, and the unloading device 182 can be replaced with a hypodermic needle 1304. As another example of variation in device 2100, the unloading interface 1302, rather than being a structure separated from the microfluidic structure 104, is instead a part of the microfluidic structure 104 in the immediate vicinity of the enclosure. could be. In such cases, the structure 104 does not need to include the passage 174.

A method of unloading, including direct unloading from the holding enclosure 156, can be used to unload group 502 of selected microobjects in a very small volume of fluid medium 144 (or other solution). For example, a unloading device (such as a hypodermic needle 1304) is placed within a proximal end prior to being inserted through unloading interface 1302 (ie, first end 1388 inserted through / through unloading interface 1302). It can have a small volume of air. Upon insertion, the unloading device may suck up (eg, absorb upwards) a predetermined volume of the liquid medium 144 containing the group 502 of selected microobjects. As soon as the unloading device can be removed from the unloading interface 1302 and moved to a suitable container, the volume of the liquid medium 144 sucked up by the unloading device (together with the group of microobjects 502) is the container. It is released into. Thus, the air placed within the proximal end of the unloading device can prevent the unloaded volume of the liquid medium 144 from mixing with the fluid that would otherwise have been present in the unloading device. In addition, by avoiding the carry-out path through the channel 152, the carry-out interface 162 and / or the internal passage length of the carry-out device 182, the selected micro-object group 502 was carried out due to patrolling and scattering. Volume increase and / or loss of micro-objects in the middle of the carry-out route can be avoided. Also, the carry-out device in this embodiment (eg, a hypodermic needle 1304) can be easily removed, and / or micro-objects that have not been carried out, such as a cleaning or neutralizing solution. The solution may be prepared for further use by flushing the unloading device. Chemicals that can be harmful into microfluidic devices (eg, devices 100, 1700, 1800, 1900 or the like) by flushing the solution into an external waste container via the unloading device. The introduction of substances can be avoided.

All of the steps 300, 1000, 1100, 2000 of processes 300, 1000, 1100, 2000 of FIGS. 3, 10, 11 and 20 are in place for the unloading interfaces 1202, 1302, 1406, and unloading interfaces 162 in FIGS. It can be carried out by the unloading interface 1562 in 1406 or outlet 110a of FIG. 17 or 18 and / or in FIGS. 1A-11 and 15A-16. Similarly, any of the steps 300, 1000, 1100, 2000 of processes 300, 1000, 1100, 2000 of FIGS. It can be carried out by devices 1304 and 1402. Similarly, the processes 300, 1000, 1100, 2000 of FIGS. 3, 10, 11 and 20 are performed by the microfluidic devices 1500, 1700, 1800, 1900, 2100 of FIGS. 15A-19B and 21A-C, rather than device 100. Can be done.

Specific aspects and applications of the invention are described herein, but these aspects and applications are for purposes of illustration only and may vary in many ways. For example, the passage 174 is described in the drawing as passing through the top edge of the enclosure 102, but instead the passage 174 can pass through the side or bottom edge of the enclosure 102 and thus the unloading interface 162, 1202, 1302, 1406 (or outlets 110, 110a) can be on the side or bottom edge of the enclosure 102.

Claims (20)

It is a process to carry out a group of cells from a microfluidic device.
The microfluidic device
An enclosure comprising a substrate and a microfluidic structure disposed on the substrate, the enclosure defining a flow region configured to contain a liquid medium, the enclosure being an inlet and an outlet. Envelopment, each associated with the flow area for charging the liquid medium and removing it from the flow area, the outlet providing a passage through the enclosure. With the body
A flow area comprising a channel disposed inside the enclosure, wherein the channel has a plurality of holding enclosures open through the channel, and each of the plurality of holding enclosures is open. With the flow region, which comprises a contiguous zone and an isolated region.
A first electrode, a second electrode, and an electrode starting substrate having a DEP electrode on the inner surface of the flow region,
With
The isolated region
Holds a clonal population of cells
Clone populations of the cells were isolated from the cells in the other retention enclosures of the plurality of retention enclosures.
To limit the exchange of nutrients and waste between the holding enclosure and the channel to occur by diffusion.
It is composed and
The process is
Select a group of cells from a clonal population of cells located in the retention enclosure, which is one of the plurality of retention enclosures open through the channel inside the enclosure of the microfluidic device. To do and
To move the selected group of cells to the channel,
Carrying the selected group of cells through the passage in the enclosure into the unloading device
Including
Group of the selected cells, rather less than all of the clonal population of cells,
Selecting the group of cells comprises providing a DEP force to capture the group of selected cells by means of the DEP electrode.
Moving the group of selected cells comprises providing a DEP force with the DEP electrode to move the group of selected cells to the channel.
process. The process of claim 1, wherein the selected group is a single cell. The process of claim 1 or 2, wherein the selection comprises determining that the group of cells has a particular activity or physical characteristic. The process according to any one of claims 1 to 3 , wherein moving the selected group of cells further comprises moving the selected group to a staging area located in the channel. The unloading further comprises inserting an opening at the proximal end of the unloading device into an unloading interface located above the passage in the enclosure, thereby the unloading device. The process according to any one of claims 1 to 4 , wherein the opening at the proximal end is installed so as to retract the passage. The carry-out interface is located above the aisle and comprises a self-closing cover covering it.
The insertion opens the cover by pressing the proximal end of the unloading device against a crevice in the cover and pulls the proximal end of the unloading device into the passage. Including moving to,
The process according to claim 5. The unloading interface is located above the aisle and comprises a self-healing cover covering it.
The insertion comprises penetrating the cover at the proximal end of the unloading device and positioning the proximal end of the unloading device in close proximity to the aisle.
The process according to claim 5. The process according to any one of claims 1 to 7 , wherein the selected group of cells is carried out in a medium having a volume of 1 μL or less or in another solution. The process of claim 4 , further comprising examining the staging area and / or the unloading device for any cells in the selected group that were not unloading after the unloading. After said that unloading, for any cells that have not been unloaded from among the selected group, further comprising: examining the unloading interface process according to any one of claims 5 7. The process of claim 9 , further comprising removing any of the cells that have not been removed from the staging area and / or the removal device. The process of claim 10, further comprising removing all of the cells that were not carried out from the carry-out interface. The passage in the enclosure is the second passage in the enclosure and is arranged so as to be close to the second end of the staging area.
The movement comprises moving the selected group to an intermediate portion of the staging area between the second end of the staging area and the first end on the opposite side thereof.
To carry out
A liquid is flowed from the first unloading device through the first passage in the enclosure toward the first end of the staging area, thereby from the first end of the staging area to the first end of the staging area. To generate a flow to the two ends and
The cells of the selected group are placed in the vicinity of the second carry-out device arranged in the vicinity of the second passage from the second end of the staging area through the second passage in the enclosure. Pulling it into the opening at the end of the position
4. The process according to claim 4. The process according to any one of claims 1 to 13 , wherein the selected group of cells is delivered in a volume of liquid of about 1 microliter to about 5 microliters. The treatment according to any one of claims 1 to 14 , wherein the selected group of cells comprises immune cells. The treatment according to any one of claims 1 to 14 , wherein the selected group of cells comprises cancer cells or transformed cells. The treatment according to any one of claims 1 to 14 , wherein the selected group of cells comprises stem cells. The process according to any one of claims 1 to 17, wherein the DEP electrode is activated by directing the patterned light onto the inner surface of the flow region. The process according to any one of claims 5 to 7 , further comprising excluding any of the cells located in the enclosure, the unloading interface, and / or the unloading device that have not been unloaded. The process according to any one of claims 1 to 19 , wherein carrying out includes disposing of the leading volume of the liquid medium.

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