JP6332782B2 - Cell aggregate production method - Google Patents

Description

  The present invention relates to a method for producing a linear cell aggregate using a hollow fiber.

  In general, when cells are cultured using a culture container such as a dish, the cells adhere to and extend on the surface of the container and grow in a single layer. However, such monolayer cells may behave differently than in vivo cells. Therefore, a method called three-dimensional culture, in which cells are cultured in a state of an aggregate that is closer to the living body, is known. Conventionally, as such a three-dimensional culture method, a swivel culture method or a hanging drop method has been used. The swirl culture method is a method in which floating cells are brought into contact with each other by gradually swirling when cells are cultured, and a cell aggregate is gradually formed. The hanging drop method utilizes the fact that the cell suspension attached to the lower surface of the flat support is held in a state of hanging in the form of water droplets, and the cells descend by their own weight and spontaneously gather at the bottom of the suspended solution. This is a method for producing a cell aggregate. However, it has been difficult to control the size of the cell aggregate formed by these conventional methods. If the size of the cell aggregate becomes several hundred μm or more, nutrients and oxygen are not sufficiently supplied to the cells in the center, and a cell necrosis layer is generated in the center. It is important to control.

  In recent years, a method for producing a cell aggregate using a hydrogel fiber has been reported (Non-patent Document 1). In this method, a hydrogel fiber in which cells are entrapped and immobilized is first prepared, the fiber surface is cross-linked with a cross-linking agent, the inside of the fiber is liquefied, the cells are cultured, and finally the fiber is decomposed to control the size. Linear cell aggregates are obtained. However, this method has problems such as requiring a crosslinking agent having high cytotoxicity, the strength of the fiber liquefied inside is extremely low, and requiring many steps and being complicated.

  In addition, this inventor has reported preparation of an alginate gel hollow fiber by inject | pouring a dextran aqueous solution and a sodium alginate aqueous solution into a calcium chloride aqueous solution (nonpatent literature 2).

Nurazhani Abdul Raof et al., Biomaterials (2011) 32, p. 4498-4505 Takayuki Takei et al., Biochem. Eng. J. (2010) 49, p. 143-147

  An object of the present invention is to provide a simple method for producing a linear cell aggregate.

  As a result of intensive studies to solve the above problems, the present inventors have cultivated cells suspended in a biocompatible polymer-containing aqueous solution in a hollow portion of a hollow fiber made of an alginate gel, thereby obtaining a hollow fiber. The present inventors have found that a linear cell aggregate having the same diameter as the hollow diameter can be easily produced, and has completed the present invention.

That is, the present invention includes the following.
[1] Production of a linear cell aggregate including production of an alginate gel hollow fiber having a hollow diameter of 150 μm or less encapsulated in a hollow part with an aqueous solution containing a biocompatible polymer containing cells, and culturing the cell in the hollow part Method.
[2] The method according to [1], wherein the biocompatible polymer-containing aqueous solution is a polyvinyl alcohol-containing aqueous solution.
[3] The method according to [1] or [2], wherein the alginic acid gel is a calcium alginate gel.
[4] The alginate gel hollow fiber is produced by injecting a sodium-alginate-containing aqueous solution into a divalent cation-containing aqueous solution on the outside and a biocompatible polymer-containing aqueous solution containing cells into the inside of a double cylinder. The method according to any one of [1] to [3].
[5] The method according to [4], wherein the divalent cation-containing aqueous solution is a calcium chloride-containing aqueous solution.
[6] The method according to any one of [1] to [5], further comprising dissolving the alginate gel after cell culture.
[7] A linear cell aggregate filled in the hollow part of an alginate gel hollow fiber, produced by the method according to any one of [1] to [5].

  According to the present invention, a linear cell aggregate can be easily produced.

FIG. 1 is a schematic view showing a method for producing an alginate gel hollow fiber in which a PVA aqueous solution is encapsulated in a hollow part. The cross section of the fiber production as seen from the side is shown. The symbols indicate 1. outer cylinder, 2. inner cylinder, 3. calcium chloride aqueous solution, 4. PVA aqueous solution, and 5. sodium alginate aqueous solution, respectively. FIG. 2 is a photograph showing an alginate gel hollow fiber in which a PVA aqueous solution is encapsulated in a hollow portion. FIG. 3 is a graph showing the influence of the flow rate of the aqueous solution and the inner diameter of the outer cylinder on the outer diameter of the fiber at the tip of the double cylindrical tube. FIG. 4 is a graph showing the influence of the PVA flow rate ratio on the outer diameter and hollow diameter of the fiber. FIG. 5 is a photograph showing the formation of cell aggregates in the hollow gel fiber. A is a photograph on day 0 of culture, and B is a photograph on day 10 of culture. FIG. 6 is a photograph showing adhesion of linear cell aggregates to a petri dish for cell culture. A is a photograph on culture day 0 and B is a photograph on culture day 1.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a method for producing a linear cell aggregate. According to this method, a linear cell aggregate having a controlled diameter, particularly a thin linear cell aggregate having a diameter of 150 μm or less in which oxygen and nutrients can be supplied and no necrotic cell layer is generated can be easily prepared. . Specifically, the present method includes producing an alginate gel hollow fiber having a hollow diameter of 150 μm or less encapsulating a biocompatible polymer-containing aqueous solution containing cells in a hollow part, and culturing cells in the hollow part. .

  In the present invention, the linear cell aggregate refers to an aggregate of a plurality of cells having a linear structure as a whole. In the present invention, the linear refers to a structure having a sufficiently long length compared to the diameter, and the ratio of the length to the diameter is 2 times or more, for example, 5 times or more or 10 times or more, preferably 100 times or more. Say. In the present invention, the diameter of the linear cell aggregate is preferably 150 μm or less, particularly preferably 10 to 150 μm, and may be, for example, 20 to 120 μm or 30 to 100 μm. On the other hand, the length of the linear cell aggregate is not particularly limited, and may be 300 μm or more, for example, 500 μm to 1 m, 1 mm to 50 cm, or 3 mm to 10 cm.

  The cells in the present invention can be appropriately determined according to the intended use of the produced linear cell aggregate. The cell in the present invention is preferably a cell having an anchoring-independent growth ability (a cell that can proliferate without adhering to the scaffold). Examples of the cell having an anchorage-independent growth ability include cancer cells. Examples of cancer cells derived from liver cancer, stomach cancer, lymphoma, squamous cell carcinoma, neuroblastoma, kidney cancer, endometrial cancer, cervical cancer, pancreatic cancer, colon cancer (colon cancer, cecal cancer or rectal cancer), etc. However, the cell is not particularly limited. The cell used in the present invention is preferably an animal cell, and examples of the animal include mammals including primates and non-primates, birds, reptiles, amphibians, insects and the like. The animal is preferably a mammal including a human. Examples of mammals include, but are not limited to, humans, monkeys, cows, pigs, sheep, horses, mice and the like. Specific examples of cells in the present invention include liver cancer cell line Hep G2, gastric cancer cell HGC-27, colon cancer CACO-2 and the like.

  In the present invention, the alginic acid gel refers to a gel formed by forming a chelate structure between a carboxylic acid group or the like in a molecule of alginic acid and a polyvalent metal ion. In a preferred embodiment, the alginic acid gel is a calcium alginate gel in which a carboxylic acid group in a molecule of alginic acid and the like and a calcium ion form a chelate structure to form a gel.

  The alginic acid gel hollow fiber in the present invention is a microfiber having a hollow part mainly composed of alginic acid gel. In the present invention, the hollow diameter (that is, the diameter of the hollow portion) and length of the hollow fiber can be appropriately determined according to the diameter and length of the linear cell aggregate to be produced. The hollow diameter of the hollow fiber is preferably 150 μm or less, particularly preferably 10 to 150 μm, and may be, for example, 20 to 120 μm or 30 to 100 μm. The length of the hollow fiber is not particularly limited, and may be 300 μm or more, for example, 500 μm to 1 m, 1 mm to 50 cm, or 3 mm to 10 cm. The outer diameter of the hollow fiber is not particularly limited, but may typically be 100 to 600 μm, for example 200 to 500 μm. The outer diameter of the hollow fiber is preferably such that the ratio of the hollow diameter to the outer diameter (hollow diameter / outer diameter) is 0.8 or less in order to increase the strength of the fiber.

  The hollow fiber in this invention encloses the biocompatible polymer containing aqueous solution containing a cell in a hollow part. Biocompatible polymers that can be used in the present invention include those currently used as cell culture materials, as long as they do not have adverse effects such as irritation and cytotoxicity on cells. Any known natural or synthetic polymer may be used. Non-limiting examples of biocompatible polymers include polyvinyl alcohol, polyethylene glycol, polysaccharides, cellulose derivatives such as carboxycellulose, and dextran. The biocompatible polymer-containing aqueous solution used in the present invention may be a polyvinyl alcohol-containing aqueous solution.

  In the method of the present invention, a linear cell aggregate having the same diameter as the hollow diameter of the hollow fiber can be easily produced by sufficiently culturing cells in an aqueous solution containing a biocompatible polymer in the hollow portion of the alginate gel hollow fiber. can do. For this reason, this method is suitable for production of a linear cell aggregate with a controlled diameter.

  The hollow diameter of the hollow fiber may not be constant in one fiber, and may change in the middle. By using such a fiber, it is possible to produce a linear cell aggregate having a diameter that partially changes.

  In the present invention, the alginate gel hollow fiber encapsulating a biocompatible polymer-containing aqueous solution containing cells in the hollow part may be produced by any method, utilizing a hydrogel hollow fiber production method known to those skilled in the art. Can be produced. For example, an alginate gel hollow fiber may be prepared, and then a biocompatible polymer-containing aqueous solution containing cells may be injected into the hollow part, or a biocompatible polymer-containing aqueous solution containing cells at the same time as the preparation of the alginate gel hollow fiber. May be taken into the hollow portion.

  In a preferred embodiment, the alginate gel hollow fiber in which a biocompatible polymer-containing aqueous solution containing cells is encapsulated in a hollow part is a biocompatible solution containing cells in a divalent cation-containing aqueous solution and a sodium alginate-containing aqueous solution outside. It can be produced by injecting a polymer-containing aqueous solution into a double cylinder inside. When the alginate in the aqueous solution containing sodium alginate comes into contact with the divalent cation in the aqueous solution containing a divalent cation, the divalent cation is taken in and the aqueous solution gels. On the other hand, a biocompatible polymer-containing aqueous solution does not gel when it comes into contact with a divalent cation. Thereby, alginate gel fibers (hollow fibers) are formed so as to surround the liquid flow of the biocompatible polymer-containing aqueous solution. In this method, since the alginate gel hollow fiber is produced and the biocompatible polymer-containing aqueous solution containing cells can be simultaneously disposed in the hollow portion, the operation is simple and efficient.

  When a low-viscosity liquid such as water is used instead of the biocompatible polymer-containing aqueous solution in the same manner as in this embodiment, the liquid flow tends to be turbulent and it is difficult to form a hollow fiber well. . However, biocompatible polymer-containing aqueous solutions have a higher viscosity than water, so the turbulence of the liquid flow is suppressed and the laminar flow state is maintained, and as a result, a hollow fiber with a controlled size can be successfully produced. Can do.

  In the present embodiment, “injecting a sodium alginate-containing aqueous solution into a divalent cation-containing aqueous solution outside and a biocompatible polymer-containing aqueous solution containing cells into the inside of a double cylinder” means divalent cation-containing solution. Contains sodium alginate so that a double cylindrical flow with an aqueous solution containing sodium alginate on the outside and an aqueous solution containing a biocompatible polymer containing cells is formed in the aqueous solution. It means that an aqueous solution and a biocompatible polymer-containing aqueous solution containing cells are injected into a divalent cation-containing aqueous solution. In the present invention, “double cylindrical shape” means that a liquid flow of another aqueous solution is formed so as to surround a liquid flow of a predetermined aqueous solution in a tube shape, and a strict double cylindrical shape is formed. Does not mean only.

  Such a double cylindrical injection of a sodium alginate-containing aqueous solution and a biocompatible polymer-containing aqueous solution containing cells into a divalent cation-containing aqueous solution uses, for example, a container having a double cylindrical tube inside. You may do it. For example, a double cylindrical tube is a tube having both ends opened as an inner cylinder (inner cylinder) constituting a double cylindrical tube, for example, an injection needle, and an outer cylinder (outer cylinder) constituting a double cylindrical tube. It may be constituted by a pipe having both ends opened, for example, a pipette tip. The inner cylinder inner diameter may be 200 to 600 μm, for example 300 to 500 μm or 450 to 500 μm, and the outer cylinder inner diameter may be 200 to 1000 μm, for example 240 to 910 μm. The inner cylinder inner diameter refers to the inner diameter of the tip from which the aqueous solution comes out of both ends of the inner cylinder. The outer cylinder inner diameter refers to the inner diameter at the tip of the outer cylinder from which the aqueous solution exits. The container provided with the double cylindrical tube may be, for example, one in which the inner tube and the outer tube are attached to one end of a tube having an inner diameter of 5 to 10 mm. In such a container, the outer cylinder and the inner cylinder may or may not be completely coaxial. The outer cylinder and the inner cylinder may be columnar, but are preferably frustoconical. For example, it is also preferable that the outer cylinder has a truncated cone shape and the inner cylinder has a columnar shape. A sodium alginate-containing aqueous solution from the outer cylinder, and a biocompatible polymer-containing aqueous solution containing cells from the inner cylinder, from a double cylindrical tube (preferably a double cylindrical tube fixed to the bottom of the container), for example (preferably upwards) ) By extruding, these aqueous solutions can be injected into the double cylinder into the divalent cation-containing aqueous solution accommodated in the outer section of the double cylindrical tube.

  In the embodiment using the double cylindrical tube as described above, the flow rate of the aqueous solution to be injected, the inner diameter of the cylinder (outer tube) injecting the sodium alginate-containing aqueous solution, and the biocompatible polymer flow rate ratio (biocompatible polymer-containing aqueous solution) Volume / (sodium alginate-containing aqueous solution amount + biocompatible polymer-containing aqueous solution amount)), for example, polyvinyl alcohol (PVA) flow rate ratio (PVA-containing aqueous solution flow rate / (sodium alginate-containing aqueous solution flow rate + PVA-containing aqueous solution flow rate)) Thus, the outer diameter and hollow diameter of the produced hollow fiber can be controlled.

  Specifically, the outer diameter of the hollow fiber can be controlled by the flow rate of the injected aqueous solution (that is, the aqueous solution containing sodium alginate and the aqueous solution containing the biocompatible polymer) and the inner diameter of the outer cylinder. Increasing the flow rate of the aqueous solution to be injected increases the outer diameter of the hollow fiber, and decreasing the flow rate decreases the outer diameter of the hollow fiber. Increasing the inner diameter of the outer cylinder increases the outer diameter of the hollow fiber, and decreasing the inner diameter of the outer cylinder decreases the outer diameter of the hollow fiber. On the other hand, the hollow diameter of the hollow fiber can be controlled by the biocompatible polymer flow rate ratio. Increasing the biocompatible polymer flow rate ratio increases the hollow diameter of the hollow fiber, and decreasing the biocompatible polymer flow ratio decreases the hollow diameter of the hollow fiber. In addition, when the biocompatible polymer flow ratio is constant, the ratio of the hollow diameter to the outer diameter is constant.

  The sodium alginate-containing aqueous solution, biocompatible polymer-containing aqueous solution, and divalent cation-containing aqueous solution used in the present embodiment are not limited to aqueous solutions containing only sodium alginate, biocompatible polymer, and divalent cation, respectively. It may be an aqueous solution further containing other components in addition to sodium, a biocompatible polymer and a divalent cation. For example, an aqueous solution in which any of sodium alginate, a biocompatible polymer, and a divalent cation (typically a divalent cation salt) is dissolved in a buffer solution is also included. Examples of the buffer include phosphate buffered saline containing no calcium or magnesium (PBS (−), pH 7.4), HBSS, and the like, but are not particularly limited. The sodium alginate-containing aqueous solution and the biocompatible polymer-containing aqueous solution in the present invention preferably do not contain polyvalent cations such as calcium and magnesium. This is because alginic acid in the aqueous solution containing sodium alginate is prevented from gelling before coming into contact with the aqueous solution containing divalent cations.

  The sodium alginate concentration of the aqueous solution containing sodium alginate used in the present embodiment may be 0.1 to 10% (w / v), preferably 0.5 to 3% (w / v), for example 1% (w / v). The biocompatible polymer concentration of the aqueous solution containing the biocompatible polymer may be 1-20% (w / v), preferably 5-15% (w / v), for example 10% (w / v). The divalent cation concentration of the divalent cation-containing aqueous solution may be 10 to 500 mM, preferably 50 to 200 mM, for example 100 mM.

  In this embodiment, the divalent cation in the divalent cation-containing aqueous solution may be, for example, a calcium ion, a magnesium ion, a barium ion, or a strontium ion. The divalent cation-containing aqueous solution is preferably a calcium ion-containing aqueous solution. Examples of the calcium ion-containing aqueous solution include a calcium salt-containing aqueous solution such as a calcium chloride-containing aqueous solution, a calcium phosphate-containing aqueous solution, a calcium carbonate-containing aqueous solution, and a calcium sulfate-containing aqueous solution, and a calcium chloride-containing aqueous solution is preferable.

In the method of the present invention, the biocompatible polymer-containing aqueous solution containing cells is preferably a biocompatible polymer-containing aqueous solution in which cells are suspended. The density of the cells in the aqueous solution containing the biocompatible polymer can be appropriately adjusted depending on the cells, but is typically 1.0 × 10 ^{6 to} 1.0 × 10 ⁸ cells / ml, for example 1.0 × 10 ⁷ cells / ml. It's okay.

  The produced hollow fiber may be wound and collected using a roller or the like. In particular, the fiber can be continuously prepared by simultaneously winding and collecting while producing the hollow fiber, and a long fiber can be produced.

  After the prepared hollow fiber is placed in the medium, the cells can be cultured in the hollow part of the fiber. As a medium, a medium suitable for culturing cells to be used can be appropriately selected. For example, Dulbecco's modified Eagle medium supplemented with 10% (v / v) fetal calf serum may be used.

  The period during which the cells are cultured in the hollow portion of the hollow fiber can be appropriately changed depending on the cell proliferation ability, the diameter and length of the linear cell aggregate to be produced, and the like. For example, the culture period can be 5-15 days. The culture period can be determined by observing the cell growth state with a microscope. If it is observed that the cells are growing without any gaps in the hollow part of the hollow fiber, the culture is preferably terminated.

  The method for producing a linear cell aggregate according to the present invention may further include dissolving the alginate gel after cell culture. The dissolution is performed using a metal ion chelating agent such as citric acid (eg trisodium citrate), ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic acid (EGTA), or alginic acid hydrolase such as alginate lyase. It can be performed by immersing the fiber in an aqueous solution. The cell aggregate after the alginate gel is dissolved may be collected by centrifugation.

  The method for producing a linear cell aggregate according to the present invention does not require a highly cytotoxic cross-linking agent, and has an advantage that the diameter of the cell aggregate can be controlled more accurately. The hollow fiber in the present method is strong and strong, and the fiber preparation process has the advantage that it does not adversely affect cell viability.

  The present invention also provides an alginate gel hollow fiber produced by producing an alginate gel hollow fiber having a hollow diameter of 150 μm or less enclosing a biocompatible polymer-containing aqueous solution containing cells in a hollow part, and culturing the cells in the hollow part. It also includes a linear cell aggregate filled in the hollow portion of the fiber. Thus, since the form with which the hollow part of the hollow fiber is filled with the linear cell aggregate is high in strength, it is suitable for distribution. The linear cell aggregate in this form can be used by dissolving the alginate gel as needed by the user.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
(Outer diameter and hollow diameter control of hollow gel fiber)
Sodium alginate (Kimika Co., Ltd.) was dissolved in purified water to a concentration of 1% (w / v) to prepare a sodium alginate aqueous solution. Polyvinyl alcohol (PVA, complete saponification type, polymerization degree: 400 to 600, Wako Pure Chemical Industries, Ltd.) was dissolved in purified water to a concentration of 10% (w / v) to prepare a PVA aqueous solution. Using a double cylindrical tube (consisting of a stainless steel injection needle as an inner tube and a plastic pipette tip as an outer tube) fixed to the bottom of a glass tube containing 100 mM calcium chloride aqueous solution (Fig. 1) The syringe containing the sodium alginate aqueous solution was connected to the outer cylinder of the double cylindrical tube, and the syringe containing the PVA aqueous solution was connected to the inner cylinder of the double cylindrical tube using a silicon tube and a stainless pipe, respectively. Thereafter, the sodium alginate aqueous solution and the PVA aqueous solution were pushed upward into a 100 mM calcium chloride aqueous solution from the syringe through a double cylindrical tube using a syringe pump (FIG. 1). The extruded sodium alginate aqueous solution is composed of an alginate gel in which the alginic acid dissolved therein is gelled by taking in calcium ions in the surrounding calcium chloride aqueous solution, and as a result, the PVA aqueous solution is encapsulated in the hollow portion. A hollow gel fiber was produced (FIG. 2). The produced hollow gel fiber was wound up and collected with a roller.

  In the production of this hollow fiber, the flow rate of each aqueous solution extruded from the double cylindrical tube and the inner diameter of the outer tube are changed while keeping the PVA flow rate ratio (PVA aqueous solution flow rate / (sodium alginate aqueous solution flow rate + PVA aqueous solution flow rate)) constant. Thus, the influence of the flow rate of the aqueous solution at the tip of the double cylindrical tube and the inner diameter of the outer cylinder on the hollow diameter and outer diameter of the hollow gel fiber was evaluated. In addition, by fixing only the flow rate of the aqueous solution at the tip of the double cylindrical tube (17.9 cm / s) and the inner diameter of the tip of the double cylindrical tube (outer cylinder inner diameter) (910 μm) and changing only the PVA flow rate ratio, the PVA flow rate ratio is The influence on the hollow diameter and outer diameter of the fiber was evaluated. Detailed experimental conditions are shown in Table 1. The flow rate of the aqueous solution at the tip of the double cylindrical tube is equal to the flow rates of both the sodium alginate aqueous solution and the PVA aqueous solution.

  The results are shown in FIGS. When the flow rate of the aqueous solution at the tip of the double cylindrical tube and the inner diameter of the tip of the double cylindrical tube (outer cylinder inner diameter) are changed, the outer diameter of the fiber tends to increase as the aqueous solution flow rate increases. When the flow rate of the aqueous solution was the same, the outer diameter of the fiber tended to increase as the inner diameter of the outer cylinder increased (FIG. 3). In addition, if the flow rate of the aqueous solution at the tip of the double cylindrical tube and the tip inner diameter (outer cylinder inner diameter) of the double cylindrical tube are changed while keeping the PVA flow rate constant, the ratio of the hollow diameter to the outer diameter is maintained. It remained. From the above, it was possible to control the outer diameter of the fiber by changing the flow rate of the aqueous solution at the distal end of the double cylindrical tube and the inner diameter (outer cylinder inner diameter) of the distal end of the double cylindrical tube.

  Next, when the flow rate of the aqueous solution at the tip of the double cylindrical tube and the inner diameter (outer tube inner diameter) of the double cylindrical tube were fixed and only the PVA flow ratio was changed, the outer diameter of the fiber did not change, but the hollow The diameter tended to increase as the PVA flow ratio increased (Fig. 4). From the above, it was possible to control the hollow diameter of the fiber by changing the PVA flow rate ratio. In addition, for example, a hollow gel fiber with a hollow diameter of 104 ± 13 μm is produced when the flow rate of the aqueous solution at the tip of the double cylindrical tube is 17.9 cm / s, the inner diameter of the double cylindrical tube tip (outer cylinder inner diameter) is 910 μm, and the PVA flow ratio is 0.01. As can be seen from FIG. 4 (FIG. 4), a hollow gel fiber having a hollow diameter of 150 μm or less could be produced by changing the fiber production conditions as described above.

[Example 2]
(Evaluation of cytotoxicity by fiber production operation)
Dissolve sodium alginate (Kimika Co., Ltd.) in phosphate buffered saline (PBS (-), pH 7.4) that does not contain calcium and magnesium to a concentration of 1% (w / v). Prepared. PVA (completely saponified type, degree of polymerization: 400 to 600, Wako Pure Chemical Industries, Ltd.) was dissolved in PBS (−) to a concentration of 10% (w / v) to prepare a PVA-containing aqueous solution. The reason why PBS (-) containing no calcium and magnesium was used is to prevent alginic acid from gelling at this stage. In a PVA-containing aqueous solution, Hep G2 cells, which are human liver cancer-derived cell lines, were suspended to a density of 1.0 × 10 ⁷ cells / ml. Using a double cylindrical tube (consisting of a stainless steel injection needle as the inner tube and a plastic pipette tip as the outer tube) fixed to the bottom of a glass tube containing 100 mM calcium chloride-containing aqueous solution (pH 7.4) (Fig. 1), a syringe containing a sodium lginate-containing aqueous solution is placed in the outer cylinder of the double cylindrical tube, and a syringe containing a PVA-containing aqueous solution is placed in the inner cylinder of the double cylindrical tube. Connected using. Thereafter, the aqueous solution containing sodium alginate and the aqueous solution containing PVA are pushed upward into a 100 mM calcium chloride-containing aqueous solution (pH 7.4) through a double cylindrical tube from the syringe using a syringe pump, thereby removing the aqueous solution containing PVA containing cells. A hollow gel fiber (animal cell-containing hollow gel fiber) made of alginate gel encapsulated in the hollow part was prepared. The produced hollow gel fiber was wound up and collected with a roller.

  Next, it was evaluated whether the cells were damaged in the process of encapsulating animal cells in hollow gel fibers. First, the prepared animal cell-containing hollow gel fiber was immersed in an aqueous solution of trisodium citrate (pH 7.4) to dissolve the gel part, and then centrifuged to collect cells. The viability of the collected cells was measured by trypan blue dye exclusion method. Using the survival rate, the “ratio of cells not damaged by the fiber manufacturing operation” (= survival rate of cells after inclusion × 100 / survival rate of cells before inclusion) was calculated. As a result, the “ratio of cells not damaged by the fiber production operation” was 97%. From this, it was thought that there was almost no influence on the cell of the animal cell inclusion hollow gel fiber preparation process.

[Example 3]
(Culture of animal cells encapsulated in hollow gel fiber)
In order to examine whether Hep G2 cells proliferate in the hollow part of the hollow gel fiber and can form a cell aggregate of the same diameter as the hollow diameter, 10% ( v / v) Cells were cultured in Dulbecco's modified Eagle medium supplemented with fetal calf serum. FIG. 5 shows photographs of observation of animal cell-encapsulated hollow gel fibers on day 0 and day 10 of culture. As the culture time elapses, it was confirmed that the cells were proliferating in the hollow portion, and after 10 days of culture, a cell aggregate having the same diameter as the hollow diameter could be formed. The length of the cell aggregate was about 5 cm.

  Next, after culturing, the gel gel portion was dissolved using an aqueous solution of trisodium citrate (pH 7.4), and cell aggregates were collected and cultured again. FIG. 6 shows the results of observation of the cell aggregate culture. It was confirmed that the formed cell aggregate adhered to the petri dish for cell culture on the first day after culturing. From this, it was shown that the linear cell aggregate obtained by this method can be cultured after that.

  The cell aggregate produced by the method of the present invention can be used for drug discovery screening, elucidation of cell-cell interactions, and the like. It can also be used as a substitute for laboratory animals.

1 Outer cylinder 2 Inner cylinder 3 Calcium chloride aqueous solution 4 PVA aqueous solution 5 Sodium alginate aqueous solution

Claims (7)

An alginate gel hollow fiber having a hollow diameter of 150 μm or less encapsulating a biocompatible polymer-containing aqueous solution containing cells in a hollow portion, and culturing cells in the hollow portion to form a linear cell aggregate having the same diameter as the hollow diameter A method for producing a linear cell aggregate including the production of alginate gel hollow fiber, wherein the alginate gel hollow fiber comprises a bivalent cation-containing aqueous solution, a sodium alginate-containing aqueous solution outside, and a biocompatible cell containing cells. A method comprising injecting a water-soluble polymer-containing aqueous solution into the inside of a double cylinder, wherein the biocompatible polymer-containing aqueous solution is a polyvinyl alcohol-containing aqueous solution . The method of claim 1 , wherein the alginate gel is a calcium alginate gel. The method according to claim 1 or 2 , wherein the divalent cation-containing aqueous solution is a calcium chloride-containing aqueous solution. The method according to any one of claims 1 to 3 , wherein the cell is a cell having an anchorage-independent growth ability. The method of claim 4 , wherein the cell is a cancer cell. Further comprises dissolving alginate gel after cell culture method according to any one of claims 1-5. Claim 1 produced by the method according to any one of 5, alginic acid gel hollow linear cell clumps filled in the hollow portion of the fiber.

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