JP7037475B2 - Sheet cell culture recovery system and method - Google Patents

Description

The present invention relates to a method for recovering a sheet-like cell culture and a system for using the method.

Despite recent breakthroughs in the treatment of heart disease, a treatment system for severe heart failure has not yet been established. As a treatment method for heart failure, medical treatment with β-blockers and ACE inhibitors is performed, but for heart failure that has become so severe that these treatments do not work, replacement treatment such as ventricular assist device or heart transplantation, that is, surgical treatment Is done.

Severe heart failure targeted for such surgical treatment includes those caused by advanced valvular disease and severe myocardial ischemia, acute myocardial infarction and its complications, acute myocarditis, ischemic cardiomyopathy (ICM), and dilated cardiomyopathy. There are various causes such as chronic heart failure due to type cardiomyopathy (DCM) and its acute exacerbation.
Depending on the cause and severity of these, valvuloplasty, replacement surgery, coronary artery bypass grafting, left ventricular plasty, mechanical assisted circulation, etc. are applied.

Among these, for those who have suffered heart failure due to severe left ventricular dysfunction due to ICM or DCM, only heart transplantation or replacement-type treatment with an artificial heart has been regarded as an effective treatment method. However, replacement-type treatment for these patients with severe heart failure has many problems to be solved such as chronic donor shortage, the need for continuous immunosuppression, and the onset of complications, and it is a universal treatment for all severe heart failure. Is hard to say.

On the other hand, recently, it is considered indispensable to develop new regenerative medicine as a solution for the treatment of severe heart failure.
In severe myocardial infarction or the like, cardiomyocytes become dysfunctional, and further, fibroblast proliferation and interstitial fibrosis progress, resulting in heart failure. As heart failure progresses, cardiomyocytes are damaged and undergo apoptosis, but since cardiomyocytes hardly undergo cell division, the number of cardiomyocytes decreases and cardiac function further declines.
Cell transplantation is useful for recovering cardiac function in such patients with severe heart failure, and clinical application using autologous skeletal myoblasts has already begun.

In recent years, as an example, by using a temperature-responsive culture dish to which tissue engineering has been applied, a three-dimensionally constructed cell culture that can be applied to the heart, including cells derived from parts other than the adult myocardium, and its cells. A manufacturing method has been provided (Patent Document 1).

Although the temperature-responsive culture dish described in Patent Document 1 facilitates the peeling of the cell culture from the culture dish, it cannot be completely peeled off, and artificial separation such as pipetting is required for complete peeling. Since it requires an operation, it causes contamination, wrinkles, tears, breakage, etc., and it is necessary to be careful especially when exfoliating fragile cell cultures.

Special Table 2007-528755 Japanese Unexamined Patent Publication No. 2011-1158080 Japanese Unexamined Patent Publication No. 2011-155869

An object of the present invention is to provide a method for recovering a sheet-shaped cell culture from a culture surface easily, quickly and without damaging it, and a system for carrying out such a method.

A system for automating the peeling operation has been provided for the purpose of reducing the above-mentioned human operation which becomes a problem at the time of peeling (Patent Documents 2 and 3). However, these systems also took a long time to exfoliate, especially when dealing with fragile sheet-like cell cultures, and there was still the possibility of wrinkles, tears, breakage, etc. during exfoliation.

Therefore, in further research on a method for exfoliating a sheet-shaped cell culture, the present inventors apply reciprocating vibration when vibrating the sheet-shaped cell culture in a liquid, which is faster than applying rotational vibration. It was found that the risk of damage due to vibration can be reduced because it can be peeled off and the time for applying vibration can be shortened. Furthermore, if wrinkles or wrinkles occur in the sheet-shaped cell culture during peeling, the sheet is stretched and the wrinkles or wrinkles are improved by applying rotational vibration around the sheet-shaped cell culture in the liquid. As a result of diligent research, we have completed the present invention.

That is, the present invention relates to the following.
[1] A method of peeling a sheet-shaped cell culture from an adhesive culture surface, in which the sheet-shaped cell culture adhered to the adhesive culture surface is immersed in a liquid in a container, and the liquid is vibrated (1). The above method comprising vibrating the container by rotating or circularly vibrating it).
[2] The method of [1], wherein the sheet-shaped cell culture is a fragile sheet-shaped cell culture.

[3] The vibration of the liquid is caused by the reciprocating vibration of the container, the frequency of the reciprocating vibration is constant, and the amplitude increases as the adhesion area between the sheet-like cell culture and the adhesion culture surface decreases. The method of [1] or [2].
[4] The method of [3], wherein the frequency is 200 rpm or less.
[5] The reciprocating vibration of the liquid is caused by the reciprocating vibration of the container, the amplitude of the vibration is constant, and the frequency increases as the adhesion area between the sheet-like cell culture and the adhesion culture surface decreases. The method of [1] or [2].
[6] The methods of [1] to [5], wherein the liquid is an amount of liquid that the sheet-shaped cell culture is just immersed in.
[7] The method of [6], wherein the liquid is a Hanks balanced salt solution.
[8] The methods of [1] to [7], wherein the properties of the adhesive culture surface change depending on the surrounding environment.

[9] A method for extending a sheet-shaped cell culture, which comprises rotating the liquid in the container while the sheet-shaped cell culture is immersed in the liquid in the container.
[10] The method of [9], wherein the sheet-shaped cell culture is a fragile sheet-shaped cell culture.

[11] The method of [9] or [10], wherein the rotation of the liquid in the container is caused by rotationally vibrating the container.
[12] The methods of [9] to [11], wherein the liquid is a Hanks equilibrium salt solution.

According to the present invention, it is possible to exfoliate a sheet-like cell culture from the adherent culture surface more easily, quickly, and with a reduced risk of breakage than in the past. INDUSTRIAL APPLICABILITY According to the present invention, the peeling step of the sheet-shaped cell culture, which is a rate-determining step and a step having a risk of quality deterioration in the production of the sheet-shaped cell culture, is improved. Further, even if wrinkles or wrinkles occur, it is possible to quickly perform the stretching work following the peeling work, so that the quality can be further improved in combination with the reduction of the risk of breakage. Therefore, it can contribute to the improvement of production efficiency and quality of sheet-shaped cell culture.

FIG. 1 is a photographic diagram showing the results of a vibration peeling test due to horizontal vibration. FIG. 2 is a photographic diagram showing the results of a vibration peeling test by various vibration methods. FIG. 3 is a photographic diagram showing the results of the rotational extension test.

The present invention relates to a simple, rapid and method for exfoliating a sheet-like cell culture with a reduced risk of breakage. Further, the present invention relates to a method for stretching a sheet-shaped cell culture in which wrinkles and wrinkles are formed, and for eliminating wrinkles and wrinkles. Further, the present invention relates to a method for recovering a sheet-shaped cell culture, which comprises a peeling step using the peeling method and a stretching step using the stretching method, and a system for realizing the method.
Hereinafter, the present invention will be described based on a preferred embodiment of the present invention.
(1) Peeling Method The method for recovering a sheet-shaped cell culture of the present invention includes a step of peeling the sheet-shaped cell culture from the adherent culture surface. Therefore, in the present invention, in one aspect, the sheet-like cell culture adhered to the adherent culture surface is immersed in the liquid in the container, and the liquid is subjected to a single-axis reciprocating vibration in parallel with the adherent culture surface. The present invention relates to a method for exfoliating a sheet-like cell culture from an adhesive culture surface.

In the present invention, the "sheet-shaped cell culture" is typically used for the treatment of diseases, injuries and illnesses of mammals such as humans and livestock, but is not limited thereto. A cell culture is typically composed of cultured cells and / or products thereof, but various materials (supplementary materials and / or supplement materials) for supplementing and / or supporting a predetermined part of a living body (for example, an affected part). Support material) etc. are also included.

The cells constituting the sheet-shaped cell culture are not particularly limited as long as they can form the sheet-shaped cell culture, and include, for example, adherent cells (adherent cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Cells, synovial cells, cartilage cells, etc.) and stem cells (eg, myoblasts, tissue stem cells such as heart stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) And so on. Somatic cells may be differentiated from stem cells, particularly iPS cells. Examples of cells constituting the sheet-like cell culture include, but are not limited to, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots, etc.). Muscle tissue, endometrial membrane, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, heart stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells) , Retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), renal cells, adrenal cells , Root membrane cells, gingival cells, bone membrane cells, skin cells and the like. The animals from which the cells constituting the sheet-shaped cell culture are derived are not particularly limited, and include, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like.

In the present invention, the "adhesive culture surface" (sometimes simply referred to as "culture surface") means the surface to which the cells forming the sheet adhere. Usually, a sheet-like cell culture is formed so as to adhere to the adhesive culture surface. The adhesive culture surface typically includes, but is not limited to, the culture surface of the cell culture container, the surface of the cell culture carrier, and the like. The adhesive culture surface does not necessarily have to be fixed to the container containing the liquid, but is preferably fixed.

The peeling method of the present invention is carried out in a state where the sheet-shaped cell culture is immersed in a liquid. In the present invention, the state of being "immersed in the liquid" means a state in which the entire sheet is immersed in the liquid when it is allowed to stand. That is, if the sheet-like cell culture is covered with a liquid at the time of standing, it is in a state of being "immersed in a liquid", and when vibration is applied from that state, a part of the sheet is temporarily liquid. Even if it may be exposed to the outside, if it returns to the state of being covered with liquid when it is left to stand again, the state of being temporarily exposed to the outside is also the state of being "immersed in the liquid". I can say.

In the peeling method of the present invention, a frictional force is generated between the liquid and the sheet-shaped cell culture by vibrating the liquid in which the sheet-shaped cell culture is immersed, and the frictional force is applied to the sheet-shaped cell culture. It is considered that the force of peeling from the adherent culture surface makes peeling possible. Therefore, the most efficient peeling proceeds when the peeling force acts perpendicularly to the outer edge of the surface where the sheet-shaped cell culture and the adherent culture surface are adhered. On the contrary, it is the most inefficient that the peeling force acts in a direction close to parallel to the outer edge. Therefore, circular vibration or rotation in which the liquid in the container moves in the direction of rotation is not preferable.

The peeling method of the present invention comprises vibrating the sheet-like cell culture adhering to the adhesive culture surface in a state of being immersed in the liquid in the container. Such vibration may be any vibration as long as the frictional force generated between the liquid and the sheet-shaped cell culture is a force for peeling the sheet-shaped cell culture from the adhesive culture surface. Typically, for example, a single-axis reciprocating vibration parallel to the adhesive culture surface can be mentioned. Here, "to make a single axis reciprocating vibration parallel to the adhesive culture surface" means that the axis of the reciprocating vibration exists on a plane parallel to the adhesive culture surface and is on one axis (also referred to as "vibration axis"). It means going back and forth along. At this time, the vibration axis itself does not necessarily have to be fixed, and may be arbitrarily changed. However, as described above, from the viewpoint of the action efficiency of the peeling force, it is not desirable when the change of the vibration axis and the reciprocating vibration are combined to form a circular vibration as a whole. Therefore, preferably, the peeling method of the present invention does not include the case where the container is rotated or circularly vibrated so that the liquid rotates in the container.

As a method for vibrating the liquid, any method known in the art can be used as long as the sheet-like cell culture existing in the liquid is not damaged. Specific examples thereof include, but are not limited to, methods such as reciprocating a container containing a liquid to vibrate, and reciprocating a plate-shaped stirrer or the like in the liquid. From the viewpoint of less risk of directly damaging the sheet-like cell culture and less risk of contamination, a method of vibrating the liquid by moving the container without directly touching the liquid is preferable. Examples of the movement of the container include horizontal vibration, rocking, rotation, circular vibration, horizontal eccentric vibration, and the like, preferably horizontal vibration, rocking, horizontal eccentric vibration, etc. from the viewpoint of the action efficiency of the peeling force. Can be mentioned.

In the peeling method of the present invention, the frictional force between the liquid in which the sheet-shaped cell culture is immersed and the sheet-shaped cell culture is used as the peeling force, so that the peeling force becomes extremely strong or a biased load is applied. Does not take. Therefore, it can be suitably used particularly when the sheet-shaped cell culture is a fragile sheet-shaped cell culture. A "fragile" sheet is, for example, an evaluation of tensile properties by a normal tensile tester (for example, one described in JIS K 7161 etc.) in which the sheet is fixed to a gripper outside the liquid. Refers to a sheet that is difficult or virtually impossible for. Examples of such a sheet include a sample in which each numerical value of the tensile property is small and it is difficult to measure accurately with a normal tensile tester. Examples of such a fragile sheet include those showing a breaking load of less than 10N (Newton), less than 5N, less than 2N, less than 1N, less than 0.5N, less than 0.1N, and less than 0.05N in a tensile test. Be done. Further, since the measurement limit of a normal tensile test is generally about 1 N as a breaking load, those showing a breaking load lower than this (for example, less than 0.5 N) can be mentioned.

If the sheet-like cell culture to be exfoliated is a fragile sheet, it is difficult to pinch it with an instrument such as tweezers, and there is a high risk of damage during exfoliation, so the cell culture surface It becomes much more difficult to peel off from the cell. However, by using the peeling method of this embodiment, it is possible to peel quickly and easily, so that the risk of breakage even if the sheet is fragile can be minimized.

The cells that can be used in this method are not particularly limited as long as they are cells capable of forming a sheet-like cell culture (also referred to as "sheet-forming cells"), and any cells known in the art can be used. .. Specific examples thereof include, but are not limited to, skeletal myoblasts, skin cells, corneal epithelial cells, periodontal ligament cells, myocardial cells, hepatocytes, pancreatic cells, oral mucosal epithelial cells and the like. The peeling method of the present invention is particularly used for sheet-like cell cultures composed of skeletal myoblasts from the viewpoints of therapeutic usefulness and difficulty in handling by conventional techniques derived from fragility. preferable.

As described above, in a preferred embodiment of the present invention, the vibration of the liquid is caused by the reciprocating vibration of the container. In the present invention, the term "reciprocating vibration" means reciprocating motion in the uniaxial direction, and is preferably horizontal vibration (horizontal lateral vibration). Unless otherwise described below, the peeling method of the present invention shall be described using such a preferred embodiment.
In such an embodiment, when the frequency is constant, the exfoliation force acting on the sheet-like cell culture can be changed by changing the magnitude of the amplitude. On the contrary, when the amplitude is constant, the exfoliation force acting on the sheet-like cell culture can be changed by changing the frequency. Here, the "peeling force" as used herein means a force acting on the sheet-shaped cell culture to try to peel the sheet-shaped cell culture from the adhesive culture surface. When a peeling force is applied to the sheet-shaped cell culture, the adhesive force between the sheet-shaped cell culture and the adhesive culture surface becomes a resistance force, and as a result, a shearing force is generated in the sheet-shaped cell culture, that is, a breaking load is applied. It will be.

At the time of exfoliation, it is considered that the adhesive force between the sheet-shaped cell culture and the adhesive culture surface increases in proportion to the adhesive area between the sheet-shaped cell culture and the adhesive culture surface. Therefore, the adhesive force at the start stage of peeling is the largest. When the adhesive force is high, a large peeling force is applied, that is, vibration with a large amplitude increases the risk of damaging the sheet-like cell culture. Therefore, it is preferable to apply a small peeling force at the peeling start stage, that is, to vibrate with a small amplitude. However, it is considered that the adhesive strength decreases as the peeling progresses, so the risk of damage to the sheet-like cell culture decreases even when a large peeling force is applied, and conversely, the larger the peeling force, the lower the risk. The progress of peeling becomes faster. Therefore, it is preferable to increase the amplitude as the peeling progresses, that is, as the adhesive area decreases.

The amplitude may be controlled by any method. Examples of amplitude control are not limited to this, but for example, the progress of peeling is observed optically at any time by, for example, image analysis, an optical sensor, visual inspection, etc., and the amplitude is increased according to the progress of peeling. You may have. Further, since the progress of peeling is considered to correlate with the elapsed time from the start of peeling under the same conditions, the amplitude may be increased with the passage of time from the start of peeling.
The magnitude of the amplitude can vary depending on the shape and type of the vibrating container, the fragility of the sheet-like cell culture, the amount and type of liquid, and the like, and a person skilled in the art can appropriately determine an appropriate value. For example, when the sheet-like cell culture is skeletal myoblasts, an amplitude of about 0.1 cm to 10 cm is appropriate.

When the frequency is constant, it is considered that the peeling force is large when the amplitude is large, but conversely, when the amplitude is constant, it is considered that the peeling force is large when the frequency is large. Therefore, vibration at high frequencies increases the risk of damaging the sheet cell culture. Similar to the amplitude, the frequency can change depending on the shape and type of the vibrating container, the brittleness of the sheet-like cell culture, the amount and type of liquid, and the like, and a person skilled in the art can appropriately determine an appropriate value. For example, when the sheet-like cell culture is skeletal myoblasts, a frequency of about 5 rpm to 300 rpm is appropriate.

The liquid in which the sheet-shaped cell culture is immersed is not particularly limited as long as it does not deteriorate the quality of the sheet-shaped cell culture (for example, it has cytotoxicity, contains manufacturing process impurities, etc.). In a preferred embodiment, the culture medium is used as the liquid because the step of replacing the liquid is not required. Examples of other liquids include, but are not limited to, Hanks Balanced Salt Solution (HBSS), Earl Balanced Salt Solution (EBSS), HEPES, Phosphate Buffered Saline (PBS), Electrolyte Infusion (Otsuka). Raw food Note), etc. In addition, for the purpose of reinforcing the sheet-shaped cell culture, it may be vibrated together with the support of the sheet-shaped cell culture. Examples of such a support include, but are not limited to, a thin film sheet made of a biomaterial such as collagen or fibrinogen, or a biodegradable polymer such as polylactic acid.

The amount of liquid to be put in the container is not particularly limited as long as the sheet-like cell culture can be immersed. The amount of the sheet-shaped cell culture that can be implanted can vary depending on the size of the container, the thickness of the sheet-shaped cell culture, the type of sheet-forming cells, and the like, and a person skilled in the art can immediately determine an appropriate amount. Generally, the range occupied by the liquid in the container is considered to be the range in which the sheet-shaped cell culture can move freely. From the viewpoint, it is preferable that the amount of liquid is as small as possible. Therefore, in a preferred embodiment, the amount of liquid is just the amount that the sheet-like cell culture is immersed. Specifically, for example, in the case of UpCell 3.5 cm (CellSeed Co., Ltd., model number: CS3007), the amount is about 1.55 ml, but the amount is not limited to this. Further, after the peeling is completed, the liquid amount may be adjusted in order to optimize for the subsequent steps.

The properties of the adhesive culture surface may change depending on the surrounding environment so that the sheet-like cell culture can be easily peeled off. In this embodiment, the "surrounding environment" of the adherent culture surface typically includes the temperature, pH, adhesion angle (hydrophilic or hydrophobic) of the liquid in the container, and the like. The "change in properties" of the adhesive culture surface means that the physicochemical properties of the adhesive culture surface change, preferably the properties involved in the adhesion between the adhesive culture surface and the sheet-like cell culture. .. Examples of property changes include, but are not limited to, changes such as hydrophilicity changing to hydrophobicity (or hydrophobicity changing to hydrophilicity). "Changes in properties depending on the surrounding environment" means that changes in physicochemical properties are caused by changes in the surrounding environment, typically the hydrophilicity changes in response to changes in temperature. The water absorption changes in response to a change in pH, and the like.

If the sheet-like cell culture is wrinkled or wrinkled after the peeling by the peeling method, a stretching step for eliminating the wrinkles or wrinkles may be continuously performed.

(2) Stretching method In the method for recovering the sheet-shaped cell culture of the present invention, when the sheet-shaped cell culture exfoliated from the adhesive culture surface has wrinkles or wrinkles, the sheet-shaped cell culture is stretched and wrinkled. Includes the process of eliminating kinks. Accordingly, the present invention relates to a method of spreading a sheet-like cell culture, comprising rotating the liquid in the container while the sheet-like cell culture is immersed in the liquid in the container in one aspect.

In the present invention, "stretching" a sheet-shaped cell culture means stretching and eliminating wrinkles and wrinkles generated in the sheet-shaped cell culture.
In the stretching method of the present invention, a liquid in which a sheet-shaped cell culture is immersed is rotated in a container to generate a centrifugal force in the liquid, and the centrifugal force and friction between the sheet-shaped cell culture and the liquid are generated. It is believed that the force pulls the sheet-like cell culture outwards, resulting in extension. Therefore, it is preferable that the liquid in the container rotates around the center of the container.

As a method for rotating the liquid, any method known in the art can be used as long as the sheet-like cell culture present in the liquid is not damaged. Specific examples thereof include, but are not limited to, methods such as rotating or rotating a container containing a liquid, rotating a stirrer or the like in the liquid, and the like. From the viewpoint of less risk of directly damaging the sheet-like cell culture, less risk of contamination, etc., the liquid inside is rotated by rotating or rotating the container without directly touching the liquid. The method is preferred. Considering the force transmission efficiency, it is most preferable to rotate and vibrate the container. In the present invention, "rotating" the container means rotating the container itself, and "rotating and vibrating" the container means rotating the container in a circular motion to vibrate.

The stretching method of the present invention is a force for stretching the centrifugal force generated when the liquid in which the sheet-shaped cell culture is immersed is rotated and the frictional force between the liquid and the sheet-shaped cell culture. Therefore, the force is not extremely strong and the sheet-like cell culture is not biased and loaded. Therefore, it can be suitably used particularly when the sheet-shaped cell culture is a fragile sheet-shaped cell culture.

If the sheet-like cell culture to be stretched is a fragile sheet, it may be wrinkled or twisted because it is difficult to pinch it with an instrument such as tweezers and there is a high risk of damage during stretching. When it occurs, it becomes much more difficult to eliminate it. However, by using the stretching method of this embodiment, it is possible to stretch quickly, easily and with a low risk of breakage.

The cells that can be used in this method are not particularly limited as long as they are sheet-forming cells, and any cells known in the art can be used. Specific examples thereof include, but are not limited to, skeletal myoblasts, skin cells, corneal epithelial cells, periodontal ligament cells, myocardial cells, hepatocytes, pancreatic cells, oral mucosal epithelial cells and the like. The stretching method of the present invention is particularly used for sheet-like cell cultures composed of skeletal myoblasts from the viewpoints of therapeutic usefulness and difficulty in handling by conventional techniques derived from fragility. preferable.

The liquid for immersing the sheet-shaped cell culture is not particularly limited unless it deteriorates the quality of the sheet-shaped cell culture (for example, it has cytotoxicity, contains impurities in the manufacturing process, etc.), and the exfoliation of the above (1) is performed. A liquid similar to the liquid used in the method can be used. Since the stretching method of the present invention is usually carried out after exfoliating the sheet-shaped cell culture as needed, in a preferred embodiment, the liquid used for the exfoliation is used as it is.

The amount of liquid to be put in the container is not particularly limited as long as the sheet-like cell culture can be immersed. The amount of the sheet-shaped cell culture that can be implanted can vary depending on the size of the container, the thickness of the sheet-shaped cell culture, the type of sheet-forming cells, and the like, and a person skilled in the art can immediately determine an appropriate amount. Generally, the range occupied by the liquid in the container is considered to be the range in which the sheet-shaped cell culture can move freely, but the range of motion is wide because it is necessary to stretch and eliminate the wrinkles and wrinkles of the sheet. Therefore, it is preferable that the amount of liquid is as large as possible. Specifically, for example, in the case of UpCell 3.5 cm (CellSeed Co., Ltd., model number: CS3007), the amount is about 1.55 ml, but the amount is not limited to this. Further, in another preferred embodiment, it is used as it is without adjusting the liquid amount after the peeling step.

As described above, the step of performing the peeling method of the present invention and, if necessary, the step of continuously performing the stretching method of the present invention makes the sheet-shaped cell culture simple, quick and reduces the risk of breakage, and wrinkles, It can be collected in a high quality state without kinks.
Although the peeling method and the stretching method of the present invention have been described as a preferred embodiment according to the recovery method of the present invention, of course, these peeling methods and the stretching methods can also be used as independent methods. Therefore, the peeling method and the stretching method of the present invention may be used alone or in combination with another method.

(3) System The present invention relates to a system for executing the above recovery method in one aspect.
The system of the present invention peels and / or peels the sheet-shaped cell culture by vibrating at least (i) the storage unit for storing the culture container in which the sheet-shaped cell culture is cultured (ii) the culture container in the storage unit. Or it has a vibrating part to extend.

The shape of the storage portion of the culture container is not limited, and may be, for example, a warehouse-like one, or may simply fix the culture container to the shaking table. The warehouse shape means that one or more shelving members for arrangement can be provided in a closed space where temperature and humidity can be controlled as a storage unit, and one or more culture containers can be arranged on the shelf members.

In one embodiment, the storage unit has a control unit that can control the surrounding environment of the culture vessel. The surrounding environment in this embodiment is not limited to this, and examples thereof include temperature, pressure, humidity, and CO ₂ concentration. The control unit is an input unit for inputting settings to be controlled, a recording unit for recording set values, a measurement unit for measuring the current state of the surrounding environment, and an output unit for outputting information measured by the measurement unit. And so on. For example, in one embodiment of the present invention, a culture dish having an adhesive culture surface whose properties change depending on the surrounding environment (for example, a temperature-responsive culture dish) can be used as the culture container, and the surrounding environment of the culture container can be used. By controlling the range suitable for peeling, it is possible to improve the peeling efficiency.

Further, in one aspect of the system of the present invention, the storage portion can keep the periphery of the culture vessel in an environment suitable for cell culture. Therefore, the housing in such an embodiment can function as a cell culture incubator. In this embodiment, it is also possible to culture the sheet-forming cells using the storage portion as an incubator, and after the sheet formation is completed, directly proceed to the step of exfoliating the sheet-like cell culture. Further, the storage portion of the present invention can, in one embodiment, store the sheet-like cell culture before and / or after exfoliation at a constant temperature.

The vibrating portion may be inside or outside the storage portion, and may be one that vibrates only the culture container or one that vibrates the entire storage portion. Further, when the storage portion has a warehouse shape, the shelf member may be vibrated. The type of vibration needs to be at least reciprocating vibration, and in a preferred embodiment, further rotational vibration is possible. Examples of the vibration machine that can be used in the present invention include a double shaker NR-3 manufactured by TAITEC.
From the viewpoint of reducing damage to the seat, it is preferable not to apply vibration more than necessary. Therefore, the vibrating unit may be provided with a mechanism for stopping the vibration after a certain period of time, or the vibration and / or the vibration time may be controlled by the control unit.

As described above, the control unit can be involved not only in controlling the surrounding environment but also in controlling vibration. Further, even when some control (for example, input value by the input unit or output control of the measured value by the measurement unit) is required for other members, the control unit can also be used. Therefore, the control unit may take charge of all the controls in the system of the present invention. As the control unit, input unit, recording unit, measurement unit, and output unit, members known in the art can be used. Typically, the control unit is a CPU, the input unit is a keyboard, touch panel, etc., the recording unit is a magneto-optical disk, flash memory, etc., the measurement unit is a sensor, timer, etc., and the output unit is a monitor, printer, etc. However, it is not limited to this.

Although the system in the present invention has been described above, various forms can be considered for each part. Therefore, to the extent that the object of the present invention is achieved, modified embodiments of the present invention are also included in the scope of the present invention, and such modifications are easily understood by those skilled in the art.

The present invention will be described in more detail below with reference to specific aspects of the present invention, but the present invention is not limited to these specific examples.

Example 1. Vibration exfoliation (1) Preparation of sheet-shaped cell culture Sheet-shaped skeletal myoblast culture was prepared as follows.
As skeletal myoblasts, human skeletal myoblasts isolated from human skeletal muscle samples are grown in MCDB131 medium (manufactured by GIBCO) containing FBS, collected, and cryopreserved in a cryopreservation solution containing DMSO. Was used.
The cryopreserved human skeletal myoblasts were thawed in a water bath set at about 37 ° C. and washed with a buffer solution containing human serum albumin. The cells were then placed in a temperature-responsive culture dish (UpCell ^(R) 3.5 cm dish, CellSeed) containing DMEM / F12 medium (GIBCO) containing human serum at a density of 6 × 10 ⁷ cells / cm ² . Was sown at 37 ° C. and cultured at 5% CO ₂ for 24 hours to form a monolayer sheet.

(2) Horizontal vibration peeling test The culture medium of the sheet-shaped cell culture prepared in (1) was discarded, and 1.55 mL of Hanks Equilibrium Salt Solution (HBSS) cooled to 4 ° C. was added. The constant temperature machine (EYELA LTI-600SD) was set to 25 ° C., and the double shaker NR-3 manufactured by TAITEC was used to vibrate horizontally at 100 rpm and an amplitude of 4 cm in the horizontal reciprocating direction. As a comparative example, the vibration was made into a rotational vibration of 74 rpm, the vibration was not made, the vibration was made into a lateral horizontal reciprocating vibration of 200 rpm and an amplitude of 4 cm, and the medium was made into a rotary vibration of 74 rpm without replacing with HBSS. The liquid volume was 2.5 mL, and the rotational vibration at 100 rpm and an amplitude of 4 cm was also observed. The results are shown in Table 1 and FIG.

When vibrated at 100 rpm and a horizontal lateral reciprocation with an amplitude of 4 cm, the peeling was completed 30 minutes after the start of the vibration (FIG. 1a). On the other hand, in the case of rotational vibration at 74 rpm (Comparative Example 1), peeling of the peripheral edge of the sheet was observed in 3 hours, but the central portion did not peel, and the peeling was completed in 4 hours (FIG. 1b). When no vibration was applied (Comparative Example 2), peeling was observed in 4 hours, and peeling was completed in 4 and a half hours (FIG. 1c). When vibrated at 200 rpm and a horizontal lateral reciprocation with an amplitude of 4 cm, the seat was damaged. When rotational vibration was performed at 74 rpm without replacing with cold HBSS, the peeling did not occur even after 7 hours had passed (FIG. 1d). When the liquid volume was 2.5 mL and rotational vibration was performed at 100 rpm, the peripheral portion of the seat was broken into pieces (Fig. 1e).
From the above, it was found that the peeling time of the sheet can be significantly shortened and no damage occurs due to the reciprocating vibration in the horizontal and lateral directions.

(3) Peeling test by other vibration
The culture medium of the sheet-shaped cell culture prepared in (1) was discarded, and 1.55 mL of Hanks Equilibrium Salt Solution (HBSS) cooled to 4 ° C. was added. A constant temperature machine (EYELA LTI-600SD) was set at 25 ° C., and a horizontal reciprocating vibration was performed in the horizontal direction at 30 rpm and an amplitude of 4 cm using a TAITEC double shaker NR-3. As another example, the one that was shaken at a speed of 2.8 seconds / reciprocating using AS ONE's Hot Rocking Mixer, and the one that was shaken horizontally at 250 rpm using Biosan's PST-60HL Plus. Also observed. The results are shown in Table 2 and FIG.

In the case of horizontal lateral shaking at 30 rpm and an amplitude of 4 cm (Example 2), the peeling was completed in about 2 hours from the start of shaking (FIG. 2a). When the liquid was reciprocally vibrated by rocking instead of horizontal lateral vibration (Example 3), the peeling was completed in about 4 hours from the start of rocking (FIG. 2b). Further, when the frequency was increased instead of decreasing the amplitude due to the horizontal eccentric vibration (Example 4), the peeling was completed in about 1 hour from the start of the vibration (FIG. 2c). No damage to the sheet-like cell culture was observed in either case.

Example 2. A sheet-shaped cell culture was prepared and exfoliated in the same manner as in Extension Experiment Example 1. A sheet-like cell culture that was wrinkled due to exfoliation was used in the stretching experiment.
A constant temperature machine (EYELA LTI-600SD) was set at 25 ° C., and a container was installed in the center of the TAITEC double shaker NR-3, which was rotated and vibrated at 70 rpm. As a comparative example, we also observed a container installed at the end of a rotary shaker under the same conditions and a container subjected to horizontal and lateral reciprocating vibration at 100 rpm and an amplitude of 4 cm. The results are shown in FIG.
When installed in the center of the shaker, the sheet was completely stretched by vibrating for 2 minutes (Fig. 3a). When installed at the end of the shaker, the seat leaned to one side (Fig. 3b). In the case of horizontal and lateral reciprocating vibration, it did not extend.

According to the method of the present invention, the formed sheet-shaped cell culture can be peeled off easily and without damage in a short time. In addition, even if some wrinkles or wrinkles occur during exfoliation, it can be easily stretched, and a high-quality sheet-shaped cell culture can be provided.

Claims (7)

This is a method of peeling a sheet-shaped cell culture from the adhesive culture surface, in which the sheet-shaped cell culture adhered to the adhesive culture surface is immersed in a liquid in a container, and the liquid is mixed with the liquid and the sheet-shaped cell culture. The frictional force generated between the object and the cell culture material vibrates so as to act perpendicularly to the outer edge of the surface to which the sheet-like cell culture and the cohesive culture surface are adhered, and the vibration causes the container. The method described above, which is caused by horizontal eccentric vibration at an amplitude of 0.1 cm to 4 cm and a frequency of 100 rpm to 250 rpm. The method according to claim 1, wherein the vibration is generated by vibrating the container with a horizontal eccentric vibration at an amplitude of 0.1 cm to 4 cm and a frequency of 250 rpm. The method of claim 1 or 2 , wherein the sheet-like cell culture is a sheet-like cell culture that exhibits a breaking load of less than 10 N in a tensile test. The method according to any one of claims 1 to 3 , wherein the liquid is an amount of liquid that causes the entire sheet-shaped cell culture to be immersed in the liquid when left to stand. The method according to any one of claims 1 to 4 , wherein the liquid is a Hanks balanced salt solution. The method according to any one of claims 1 to 5 , wherein the properties of the adhesive culture surface change depending on the surrounding environment. A method for producing a sheet-shaped cell culture, which comprises peeling the sheet-shaped cell culture from the adhesive culture surface by the method according to any one of claims 1 to 6 .

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