JP2013116045A - Method for creating three-dimensional cell aggregate, three-dimensional gel carrier for cell cultivation used in the same, and three-dimensional cell aggregate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for creating an arbitrary shaped three-dimensional cell aggregate without using a complex procedure.SOLUTION: There is provided the method for creating the three-dimensional cell aggregate. A three-dimensional article molded from a hydrophilic polymer gel capable of volume changes is used as a carrier. The carrier is inoculated with cells and the cells are cultured. The carrier volume changes, and the cultured cells thereby imparted with a three-dimensional structure is peeled off as a three-dimensional cell aggregate. A temperature-responsive polymer gel is used as the hydrophilic polymer gel. The hydrophilic polymer gel has a lower critical solution temperature (LCST), which contracts at critical temperature (Tc) or higher, cultivation of the cells is performed at lower critical consolute temperature or higher, and the cultured cells are peeled off from the hydrophilic polymer gel at the lower critical solution temperature or below.

Description

  The present invention relates to a method for producing a three-dimensional cell aggregate, a three-dimensional gel carrier for cell culture used therefor, and a three-dimensional cell aggregate.

  In conventional regenerative medicine, a method of inducing tissue regeneration in vivo by combining cells and scaffolds outside the body and implanting them in the body has been employed. However, in many cases, the scaffold material to be used is non-self, and there is a concern about harm to the living body. It may also cause a delay in tissue regeneration.

  On the other hand, a method using a cell sheet for the induction of tissue regeneration without using a scaffold has been developed (Patent Document 1). In the method of Patent Document 1, cells are cultured using a support material whose surface is coated with a polymer or copolymer whose upper critical solution temperature or lower critical solution temperature is in the range of 0 to 80 ° C., and the upper critical solution temperature. The cell sheet is recovered by peeling from the support material at a temperature equal to or higher than the lower critical solution temperature.

  However, since the cell sheet obtained by this method is a single layer, its strength is weak and its handling is difficult. Therefore, in recent years, a laminated cell sheet has been frequently used from the viewpoint of efficacy in clinical application.

Japanese Patent Publication No. 6-104061

  However, when stacking cell sheets, there is a problem that a complicated operation is required in which cell sheets are produced one by one and then the taken out sheets are stacked. In addition, since the cell sheet to be produced depends on the shape of the dish for producing the sheet, there is a problem that it is impossible to produce a sheet having an arbitrary shape. Further, what is obtained from the surface of the cell culture dish is a two-dimensional cell sheet, and there is a problem that it is not a technique capable of producing a three-dimensional cell aggregate capable of reproducing a living tissue.

  Therefore, the present invention solves the above problems and provides a method for producing a three-dimensional cell aggregate that can provide a three-dimensional cell aggregate that can be used as a tissue regeneration transplant material having any shape without complicated operations. Another object of the present invention is to provide a three-dimensional gel carrier for cell culture used therefor and a three-dimensional cell aggregate of an arbitrary shape obtained by the production method thereof.

In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, cells are seeded and cultured on a molded body of a hydrophilic polymer gel capable of changing volume, and the volume of the hydrophilic polymer gel is changed. The present invention has been completed by finding that cultured cells can be detached as a three-dimensional cell aggregate.
That is, the method for producing a three-dimensional cell assembly of the present invention uses a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume as a carrier, seeds and cultures the cells on the carrier, and changes the volume of the carrier. In this case, the cultured cells to which the three-dimensional structure is imparted are detached as a three-dimensional cell aggregate.

  In the production method of the present invention, it is preferable to use a temperature-responsive polymer gel as the hydrophilic polymer gel.

  In the production method of the present invention, the temperature-responsive polymer gel is a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher, and the lower critical solution temperature. It is preferable that the cells are cultured at a higher temperature, and the cultured cells are detached from the hydrophilic polymer gel at a temperature equal to or lower than the lower critical solution temperature. In addition, it is preferable to use a homopolymer or a crosslinked copolymer of N-alkyl-substituted (meth) acrylamide for the temperature-responsive polymer gel.

  In the production method of the present invention, the hydrophilic polymer gel is preferably molded using a resin matrix produced by using a three-dimensional printing method.

  In the production method of the present invention, it is preferable to apply an organic substance that promotes cell adhesion to the surface of the hydrophilic polymer gel prior to seeding the cells.

  Furthermore, another invention of the present invention provides a three-dimensional gel carrier for cell culture used in the above-mentioned method for producing a three-dimensional cell aggregate, and comprises a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume. It is characterized by being used as a template for imparting a three-dimensional structure to cultured cells.

  In the three-dimensional gel carrier of the present invention, the hydrophilic polymer gel is preferably a temperature-responsive polymer gel.

  In the three-dimensional gel carrier of the present invention, the temperature-responsive polymer gel is preferably a homopolymer or a crosslinked copolymer of N-alkyl-substituted (meth) acrylamide.

  Furthermore, another invention of the present invention provides a three-dimensional cell aggregate produced by using the above-described method for producing a three-dimensional cell aggregate, and a three-dimensional molded article of a hydrophilic polymer gel capable of changing volume. It is characterized by comprising an aggregate of cells that are cultured with a template and given a three-dimensional structure by the template.

  According to the production method of the present invention, the cultured cells can be detached as a three-dimensional cell aggregate by expanding the hydrophilic polymer gel, so that the three-dimensional cell aggregate can be produced without complicated operations. It becomes possible. Further, since the hydrophilic polymer gel can be formed into an arbitrary shape, a three-dimensional cell aggregate having an arbitrary shape can be produced by using the hydrophilic polymer gel as a carrier. In addition, a three-dimensional cell aggregate capable of reproducing a living tissue can be produced.

It is a photograph which shows the external appearance of the hydrophilic polymer gel used for this invention, A is the gel which used polyethyleneglycol dimethacrylate (PEG-DMA) for the crosslinking agent, B is a crosslinking agent with N, N'-bisacrylamide (MBAA). Shows the gel used. It is a graph which shows the relationship between the swelling degree of the hydrophilic polymer gel shown by A of FIG. 1, and temperature. It is a graph which shows the relationship of length standard expansion coefficient of hydrophilic polymer gel shown by A of FIG. 1, and time. It is a photograph which shows swelling and shrinkage | contraction of the hydrophilic polymer gel shown by A of FIG. The left shows a state at 25 ° C. and the right shows a state at 37 ° C. It is a graph which shows the contact angle of the bubble in 25 degreeC and 37 degreeC of the hydrophilic polymer gel shown by A of FIG. It is an optical microscope which shows the state of the cell of the hydrophilic polymer gel surface shown by A of FIG. 1, A is a control (the organic substance which promotes cell adhesion is not apply | coated), B is gelatin (1 weight%). What was applied, C was applied with FN at a concentration of 1 μg / ml, and D was applied with FN at a concentration of 2 μg / ml. It is a graph which shows the relationship between the cell number at the time of culture | cultivation, and time. It is a model figure of the mother block produced using graphic modeling software, and shows the mother die which has a plurality of parallel ridges, and the mother die which has a plurality of hemispherical convex parts. It is the photograph which shows the external appearance of the resin-made mother mold produced using the three-dimensional printing method, and the upper mold shows the mother mold which has a plurality of parallel ridges, and the lower mold which has a plurality of hemispherical convex parts. . It is a photograph showing the appearance of a gel molded body polymerized in a resin mold and taken out from the resin mold, with a mold having a plurality of parallel grooves on the upper side and a mold having a plurality of hemispherical recesses on the lower side Show. It is a photograph which shows the external appearance of the gel casting_mold | template containing a cultured cell. It is the photograph which shows the external appearance of the cell aggregate | assembly taken out from the gel casting_mold | template, the upper side has shown the rod shape and the lower side has shown the hemispherical cell aggregate. It is a graph which shows the change of the length of a rod-shaped cell aggregate when not applying with the case where FN is applied to a gel mold.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The method for producing a three-dimensional cell aggregate of the present invention uses a molded article of a hydrophilic polymer gel having a three-dimensional structure and a changeable volume as a carrier, seeded and cultured on the carrier, and the volume of the carrier. The cultured cells to which the three-dimensional structure is imparted are peeled off as a three-dimensional cell aggregate by changing the above.

  As used herein, “three-dimensional cell aggregate” refers to an aggregate of cells having a thickness of a single layer or more and having an arbitrary three-dimensional shape. Arbitrary three-dimensional shapes refer to all shapes including planar shapes such as films and sheets, rectangular shapes, spherical shapes, columnar shapes, curved shapes, and the like.

  On the other hand, the “cell sheet” used in the present specification refers to a single-layered cell aggregate having only a two-dimensional spread.

(Polymer gel)
The hydrophilic polymer gel used in the present invention is obtained by crosslinking a hydrophilic polymer with a crosslinking agent, and has a three-dimensional structure and can be changed in volume. The hydrophilic polymer gel used in the present invention serves not only as a carrier for culturing cells but also as a template for imparting a three-dimensional structure to cultured cells.
Here, “volume changeable” means that in an aqueous solution, the volume changes in response to an external stimulus and before and after receiving the stimulus. Examples of the external stimulus include temperature change, light irradiation, electric field application, pH change, solute concentration change, solvent composition change and the like. A temperature change is preferable as the external stimulus. This is because it has little effect on cells and is easy to operate by simply controlling the ambient temperature.

  In the present specification, a polymer gel whose shape and / or properties change in response to a temperature change is referred to as a temperature-responsive polymer gel. As a temperature-responsive polymer gel whose volume changes in response to a temperature change in an aqueous solution, a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher, and a Tc or higher temperature Hydrophilic polymer gels having an upper critical shared temperature (UCST) that swells at a known temperature are known. Here, it is known that a hydrophilic polymer gel having LCST becomes hydrophilic and swells at a temperature lower than the critical temperature, and conversely becomes hydrophobic and contracted at a temperature higher than the critical temperature. On the other hand, it is known that a hydrophilic polymer gel having UCST becomes hydrophilic and swells on the higher temperature side than the critical temperature, and conversely becomes hydrophobic and contracted on the lower temperature side than the critical temperature.

  In the present invention, both a hydrophilic polymer gel having LCST and a hydrophilic polymer gel having UCST can be used. When the cell aggregate is peeled from the polymer gel at a temperature higher than the cell culture temperature, a hydrophilic polymer gel having UCST can be used. Further, when the cell aggregate is peeled from the polymer gel at a temperature lower than the cell culture temperature, a hydrophilic polymer gel having LCST can be used. A hydrophilic polymer gel having LCST is preferable. This is because the general cell culture temperature is 37 ° C., and the cell aggregate can be detached from the polymer gel at room temperature (20 ° C. ± 5 ° C.).

  Here, LCST can be determined by visually observing two-phase separation. That is, the temperature at which the hydrophilic polymer gel is changed from transparent to white or from white to transparent is determined by immersing the hydrophilic polymer gel in an aqueous solution and gradually increasing or decreasing the temperature. Alternatively, a method can be used in which the size and weight of the gel are measured and the temperature at which the size or weight changes is obtained.

  As the hydrophilic polymer gel having LCST, the following water-soluble monomer homopolymers or cross-linked copolymers can be used. Examples of the water-soluble monomer include N-alkyl-substituted (meth) acrylamide and N, N-dialkyl-substituted (meth) acrylamide. Here, the alkyl group has 1 to 4 carbon atoms. Specific examples of these water-soluble monomers include N-isopropylacrylamide (32 ° C), N-isopropylmethacrylamide (43 ° C), Nn-propylacrylamide (22 ° C), Nn-propylmethacrylamide (27 ° C). ), N-cyclopropylacrylamide (43 ° C), N-cyclopropylmethacrylamide (60 ° C), N-ethoxyethylacrylamide (35 ° C), N-ethoxyethylmethacrylamide (45 ° C), N-ethylacrylamide (72 ° C), N, N-diethylacrylamide (32 ° C) and the like. In addition, the temperature in said parenthesis shows LCST of a homopolymer.

  LCST can be adjusted by copolymerizing these hydrophilic monomers and / or by adjusting the type and concentration of the crosslinking agent. LCST is preferably in the range of 37 ° C to 0 ° C. This is because if the temperature is lower than 0 ° C., the cell growth rate is extremely reduced or the cells are killed. Moreover, it is because it will become easy to peel at the time of culture | cultivation when it is higher than 37 degreeC.

  Examples of the copolymer include those containing two or more of the above water-soluble monomers, and examples thereof include a combination of N-isopropylacrylamide and N, N-diethylacrylamide.

  Moreover, in addition to said water-soluble monomer, a copolymer can also contain water-soluble monomers, such as acrylamide, acryloyl morpholine, N, N- dimethylaminopropyl acrylamide, as needed.

  The hydrophilic polymer gel used in the present invention is obtained by polymerizing water-soluble monomers such as N-alkyl-substituted (meth) acrylamide and N, N-dialkyl-substituted (meth) acrylamide in the presence of a crosslinking agent and a polymerization initiator. Can be obtained. As a crosslinking agent, a bifunctional crosslinking agent or a trifunctional crosslinking agent can be used. As the bifunctional crosslinking agent, bifunctional acrylamides such as N, N-methylenebis (meth) acrylamide, N, N-propylenebis (meth) acrylamide, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, Mention may be made of alkylene glycol di (meth) acrylates such as polyethylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylates. Examples of the trifunctional crosslinking agent include triallyl cyanurate and triallyl isocyanurate.

  By changing the type and concentration of the crosslinking agent, the LCST, transparency and expandability of the hydrophilic polymer gel can be adjusted.

  Generally, if the crosslinker concentration is increased, the LCST can be lowered. Therefore, the crosslinker concentration is adjusted so that the LCST is lower than the culture temperature. Of course, the LCST can be adjusted by selecting a monomer species for copolymerizing the hydrophilic monomer. For example, LCST can be lowered by copolymerizing with a more hydrophobic monomer.

  In the present invention, the transparency of the gel refers to the light transmittance at a wavelength of 500 nm at the cell culture temperature. Generally, when the crosslinker concentration is increased, the transparency is lowered. The transparency of the gel is not always essential, but the higher the light transmittance, the easier it is to observe the state of the cells during the cell culture period. The light transmittance is 70% or more, preferably 90% or more. As the crosslinking agent imparting such transparency, alkylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylate is preferable.

Moreover, in this invention, the expansibility of a gel means the degree of expansion | swelling of the volume of the gel in the temperature before and behind LCST of the hydrophilic polymer gel which has LCST, and can represent it with a swelling degree. In the present invention, when the temperature is lowered from a temperature A higher than LCST to a temperature B lower than LCST, the degree of swelling can be expressed by the following equation.
Swelling degree = (gel weight at temperature B / gel weight at temperature A)
Here, temperature A> temperature B.

  In the present invention, when the cell culture temperature is 37 ° C. and the temperature at which the cell aggregate is peeled from the gel is 25 ° C., the degree of swelling is preferably 1.1 or more. More preferably, it is 1.1-5.

Further, the expansion of the gel includes anisotropic expansion and isotropic expansion, but isotropic expansion is preferable. This is because, in anisotropic expansion, only the specific direction of the gel expands, and as a result, the cells are easily damaged at the time of peeling. Isotropic expansion can be represented by a change in gel length or a change in gel surface area. In the present invention, a length-based expansion coefficient or a surface area-based expansion coefficient represented by the following formula is used for a rectangular sheet-like gel. Here, the length means the length of the rectangular sheet in the x-axis direction or the y-axis direction, and the surface area means a two-dimensional plane defined by the length of the rectangular sheet in the x-axis direction and the length in the y-axis direction. The area.

Length standard expansion coefficient = (Gel length at temperature B / Gel length at temperature A) × 100 (%)

Surface area expansion coefficient = (Gel surface area at temperature B / Gel surface area at temperature A) × 100 (%)

The length standard expansion coefficient is preferably 110% to 200%. Moreover, the surface area reference | standard expansion coefficient is also the same, and 110%-200% are preferable. In any case, if it exceeds 200%, the cells are easily damaged. Further, since there is a possibility that the cell aggregate is damaged, it is not preferable to expand rapidly. It is preferable that the length-based expansion coefficient or the surface area-based expansion coefficient becomes 110% or more over at least 1 minute after changing the temperature. As such a crosslinking agent imparting isotropic expansibility, alkylene glycol di (meth) acrylate or polyalkylene glycol di (meth) acrylate is preferable.

  The hydrophilic polymer gel used in the present invention can be synthesized by a known aqueous solution radical polymerization. The polymerization is carried out in a polymerization vessel in the presence of a monomer such as the above hydrophilic monomer, the above crosslinking agent, and a polymerization initiator. In order to impart a three-dimensional structure as a template to the hydrophilic polymer gel, the polymerization container is preferably polymerized in a matrix described later having a predetermined three-dimensional structure. As the polymerization initiator, those generally known as water-soluble radical initiators can be used. Examples include hydrogen peroxide, persulfates such as ammonium persulfate and potassium persulfate, and hydroperoxides such as t-butyl hydroperoxide. The concentration of the polymerization initiator is 0.01 to 10 mmol% with respect to the water-soluble monomer.

  A particularly preferred hydrophilic polymer gel in the present invention is a homopolymer of N-isopropylacrylamide and polyethylene glycol dimethacrylate as a crosslinking agent, and the concentration of the crosslinking agent is based on that of N-isopropylacrylamide monomer. And 0.5 mmol% to 5 mmol%. This is because if the amount is less than 0.5 mmol%, the gel becomes cloudy, and if it exceeds 5 mmol%, the temperature responsiveness is lost. When the crosslinking agent concentration is 3.5 mmol%, this hydrophilic polymer gel has a light transmittance of 94% at a cell culture temperature of 37 ° C., and has high transparency. Further, when the temperature is lowered from 37 ° C. to 25 ° C., the cell aggregates are easily exfoliated due to isotropic expansion.

(Polymer gel molding)
For the formation of the hydrophilic polymer gel, a method of polymerizing in a polymerization vessel having a three-dimensional structure or a method of cutting the polymerized gel can be used, but a method of polymerizing in a polymerization vessel having a three-dimensional structure is used. It is preferable to use it. For the polymerization vessel having a three-dimensional structure, a resin matrix molded by a known resin molding method such as a casting method, a compression method, an injection molding method, an extrusion method, a three-dimensional printing method can be used. It is preferable to use a resin matrix produced by the original printing method. The three-dimensional printing method is a method for producing a three-dimensional structure by continuously depositing or forming a thin layer material. For example, a liquid or powder monomer is sprayed onto a substrate, a print head is moved based on a stepped pattern, and a curing agent is sprayed by ink jet to produce a single layer, and the same procedure is repeated to laminate a plurality of layers in a predetermined three-dimensional manner. A resin matrix having a structure is produced. According to this three-dimensional printing method, even a matrix having a complicated shape can be manufactured with higher accuracy and lower cost than in the past. Therefore, it becomes possible to reproduce the fine form of the transplanted site such as the defect site and accurately give the arbitrary shape to the three-dimensional cell aggregate.

  As the resin matrix, a known thermosetting resin or photocurable resin used in the three-dimensional printing method can be used.

(Production of three-dimensional cell aggregates)
The cells are cultured by seeding the cells on a carrier made of a molded body of a hydrophilic polymer gel and culturing the cells in a predetermined medium for a predetermined period. In order to produce a three-dimensional cell aggregate, it is necessary to culture cells at a high density, and the cell concentration is 0.5 × 10 ⁶ to 2 × 10 ⁶ cells / ml, preferably 1 × 10 ⁶ cells / ml. A culture solution of ˜2 × 10 ⁶ cells / ml is seeded on a three-dimensional gel carrier of polymer gel. The medium and culture conditions can be selected according to the type of cells to be cultured.

  The cultured cells are detached as a three-dimensional cell aggregate by changing the volume of the hydrophilic polymer gel as a carrier. For example, when a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher is used, a hydrophilic polymer gel having a Tc lower than the culture temperature (for example, 37 ° C.) is used. When the temperature of the hydrophilic polymer gel is lowered to a temperature lower than the LCST after completion of the culture, the hydrophilic polymer gel swells and the cultured cells are detached accordingly. As a specific example, when a poly (N-isopropylacrylamide) gel (LCST: 32 ° C.) is used as the hydrophilic polymer gel, the gel is cultured at 37 ° C., and the gel temperature is lower than the LCST, that is, 32 ° C. or lower. By lowering, the cultured cells are detached from the gel.

  Cells that can be cultured using the present invention include ES cells (Embryonic stem cells), iPS cells (induced pluripotent stem cells), stem cells such as mesenchymal stem cells, various epithelial systems, mesenchymal somatic cells, All animal cells, such as tumor cells. In addition to animal cells, all cells can be cultured regardless of eukaryotic or prokaryotic, such as insects, plants or bacteria.

  Further, prior to seeding cells, it is preferable to apply an organic substance that promotes cell adhesion to the surface of the formed hydrophilic polymer gel. By attaching an organic substance that promotes cell adhesion to the surface of the hydrophilic polymer gel, cell adhesion to the hydrophilic polymer gel can be promoted to promote growth. Moreover, the molding accuracy of the cell aggregate to be produced can be increased. The organic substance that promotes cell adhesion is a substance involved in adhesion between cells or between cells and extracellular matrix, and includes laminin, collagen, fibronectin, gelatin, and various peptides such as RGD, IKVAV, and YIGSR. In the present invention, fibronectin or gelatin is preferred. The concentration of fibronectin is 0.5 μg / ml or more, preferably 1.0 μg / ml or more.

(Three-dimensional cell assembly)
A three-dimensional cell aggregate obtained by using the production method of the present invention is cultured using a molded article of a hydrophilic polymer gel having a three-dimensional structure and having a variable volume as a template, and the three-dimensional structure is imparted by the template. It consists of an aggregate of cells.

  The size and thickness of the three-dimensional cell aggregate of the present invention are not particularly limited, and can be appropriately selected according to the application.

For example, the following uses are considered for the three-dimensional cell aggregate of the present invention.
(1) Transplant material for tissue regeneration Based on one or more types of cells, it can be used as a transplant material reflecting the size and form of the defect site. It can also be used as a transplant material that induces tissue regeneration.
(2) Artificial embryo production material based on iPS cells and ES cells There is a possibility that an artificial early embryo of a specific size can be produced based on a certain number of cells.
(3) Tool for in vitro examination of tissue morphogenesis process Tissue morphogenesis by using not only early embryos using ES cells, but also cells that have undergone differentiation to some extent and using them to produce cell aggregates It can be used as a study tool.
(4) Materials for constructing artificial tumor tissue It is also possible to construct an artificial tumor tissue based on tumor cells and use it as a tool for reproducibly examining the state of neovascularization at the transplant site and the likelihood of metastasis. .
(5) Drug screening tool It is possible to produce various cell-derived structures and use them as a tool for examining the effect on cells in the type and amount of drug and administration period in a three-dimensional environment. In addition, it is easy to control the size of the cell aggregate and the number of cells, and it can be applied to many specimens.
(6) Artificial biofilm construction material An artificial biofilm (bacteria flora) is prepared based on bacteria that exhibit infectivity to living bodies. It can be applied as a tool to know the mechanism of bacteria and infection in special environments.

EXAMPLES Although this invention is demonstrated further in detail using an Example, this invention is not limited to a following example.
Synthesis Example 1
(Preparation of poly (N-isopropylacrylamide) gel)
N-isopropylacrylamide (NIPAAm) (manufactured by Kojin Co., Ltd.) was recrystallized from hexane and used. As a crosslinking agent, polyethylene glycol dimethacrylate (PEG-DMA) (manufactured by Sigma) was used. 0.79 g of NIPAAm was dissolved in 10 ml of ultrapure water, and then 16 mg of ammonium persulfate (APS) (manufactured by Nacalai Tesque) as an initiator (1 mol% based on the total monomers) and N, N, N, N-tetramethylethylenediamine 10 μl of (TEMED) (manufactured by Nacalai) was added. The mixed solution was poured into a glass mold (size: 100 × 100 × 1 mm) and left in an incubator at 37 ° C. for 2 hours. The polymerized gel was removed from the mold and washed with distilled water for 72 hours and then with alcohol (70% solution) for 48 hours. The resulting gel had an LCST of 32 ° C.

Synthesis Example 2
A gel was prepared in the same manner as in Synthesis Example 1 except that N, N′-bisacrylamide (MBAA) (manufactured by Sigma) was used as the crosslinking agent instead of PEG-DMA. The resulting gel had an LCST of 32 ° C.

(Characteristic evaluation of gel)
In order to evaluate the light transmittance of the gel, the light transmittance at 500 nm was measured using an ultraviolet-visible spectrophotometer (V-550 manufactured by JASCO Corporation).

In order to evaluate the ease of peeling of the cell aggregate from the gel, the swelling property of the gel was measured. The gel temperature was changed in the range of 25 ° C. to 37 ° C., and the degree of swelling was calculated using the following formula.
Swelling degree = (gel weight at each temperature except 37 ° C./gel weight at 37 ° C.)

  In order to evaluate the hydrophilicity / hydrophobicity of the gel, the contact angle of bubbles was measured by an underwater gas method using a contact angle meter (Drop Master 500 manufactured by Kyowa Interface Science Co., Ltd.). In the underwater gas method, 10 μl of air was contacted from below while the gel was immersed in water, and the contact angle after 30 seconds was measured at room temperature.

Experimental Example 1 (Examination of optimal conditions for cell culture on gel)
In order to examine the optimum conditions for cell culture on the gel, osteoblast MC3T3-E1 (RIKEN, Japan) was used as the cell, and the cell was cultured on a gel coated with an organic substance that promotes cell adhesion. All operations other than the incubator were performed on a hot plate controlled at 37 ° C.

  The gel synthesized in Synthesis Example 1 was used as the gel. As the organic substance that promotes cell adhesion, human fibronectin (FN) (0 to 2 μg / ml, manufactured by BD Biosciences) or gelatin (1% by weight / ml, manufactured by Wako Pure Chemical Industries, Ltd.) was used. The gel was immersed in a phosphate buffered saline (PBS) solution in which FN or gelatin was dissolved, and incubated for 6 hours.

Next, 2 × 10 ⁴ cells / ml of cells were seeded at 37 ° C. on the surface of an organic pre-coated gel that promotes cell adhesion. Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum was used as the medium, and the cells were cultured for 10 days in a saturated humidity and 5% CO ₂ atmosphere.

  In order to evaluate the state of cell attachment and proliferation during culture, the number of cells was measured using a hemocytometer.

  The adhered cells and the cell sheet detached from the gel were observed using an optical microscope (Nikon TE2000, CoolSNAP) equipped with a CCD camera.

  In order to confirm the adhesion between the cells and the matrix in the obtained cell sheet, both the bottom surface of the obtained cell sheet and the gel surface from which the cell sheet was peeled off were used using anti-FN and Sigma fluorescent immunostaining of FN. Went about. Digital images of gel templates and cell aggregates were taken and analyzed using image analysis software (Image J, NIH).

(Statistical processing)
Data were statistically processed with the ANOVA test. For comparison, the Student t test was used with a 99% confidence interval. Data points and error bars in the graph indicate the mean and standard deviation in each experiment.

  The adhesion surface of the cell sheet with the gel was observed with a scanning electron microscope (JEOL 6350, manufactured by JEOL Ltd.).

(result)
(1) Gel characteristics The transparency of the gel at 37 ° C., which is a general cell culture temperature, was examined. A and B in FIG. 1 are external photographs of a gel using PEG-DMA as a crosslinking agent and a gel using MBAA as a crosslinking agent, respectively. Table 1 shows the transmittance values of the A and B gels at a wavelength of 500 nm.

  The gel using PEG-DMA as a crosslinking agent was transparent at 37 ° C. and the light transmittance was 94%. On the other hand, the gel using MBAA changed from transparent to white when the temperature was raised from 15 ° C to 37 ° C.

  From this, it was found that when PEG-DMA is used as a cross-linking agent, transparency can be secured at a normal culture temperature of 37 ° C.

  FIG. 2A is a graph showing the relationship between the temperature and the degree of swelling of a gel using PEG-DMA as a crosslinking agent, and the degree of swelling is calculated based on the gel weight at 37 ° C. From this graph, the degree of swelling at 25 ° C. was about 4.

  FIG. 2B is a graph showing the relationship between the length-based expansion coefficient of gel using PEG-DMA as a crosslinking agent and time at a temperature of 25 ° C. The gel-length expansion coefficient was about 110% in 1 minute, 120% in 10 minutes, about 130% in 20 minutes, and became almost constant after about 60 minutes, and was about 137%.

  FIG. 2C is an appearance photograph showing a reversible expansion / contraction state of a gel using PEG-DMA as a cross-linking agent, with the left side at 25 ° C. and the right side at 37 ° C. The surface area standard expansion coefficient accompanying the temperature change from 37 ° C. to 25 ° C. calculated from this photograph was about 140%.

  FIG. 2D is a graph showing the contact angle of bubbles at 25 ° C. and 37 ° C. of a gel using PEG-DMA as a cross-linking agent. The contact angle did not change between 25 ° C. and 37 ° C. and had a large angle of about 150 degrees. The contact angle indicates the degree of hydrophilicity / hydrophobicity of the material surface, and this result indicates that there is no significant change in the hydrophilicity / hydrophobicity of the gel surface between 25 ° C and 37 ° C.

  From the above results, it can be seen that the gel using PEG-DMA as a crosslinking agent shows a large expansion coefficient when the temperature is lowered from 37 ° C. to 25 ° C., but there is no change in the hydrophilicity / hydrophobicity of the gel surface. It was.

(2) Effect of organic substance that promotes cell adhesion FIG. 3 is an optical microscope image showing the state of cells on the gel surface, A is a control (without applying organic substance that promotes cell adhesion), and B is gelatin. (1 wt%) applied, C applied FN at a concentration of 1 μg / ml, and D applied FN 2 μg / ml. FIG. 4 is a graph showing the relationship between time and the number of cells, where ◇ indicates A, ○ indicates B, Δ indicates C, and □ indicates the result for D. FIG. After 10 days, the number of cells was almost the same, but it was found that the number of cells adhered to the gel can be increased in a shorter number of days by applying FN or gelatin to the gel surface. Furthermore, it has been found that FN has a greater effect of increasing the number of cells than gelatin. In the case of FN, a sufficient effect can be obtained at a concentration of 1 μg / ml or more. Therefore, a gel coated with FN at a concentration of 1 μg / ml was used in the subsequent experiments.

(3) Cell sheet A cell sheet was prepared using a gel using PEG-DMA as a crosslinking agent as a carrier.
When the seeded cells were confluent on the gel and the gel temperature was lowered from 37 ° C. to 25 ° C., the cell sheet could be detached from the gel without losing its shape.

  Further, when the adhesion surface between the gel surface and the gel of the cell sheet was observed by immunofluorescent staining, FN was detected only on the bottom surface of the cell sheet and not on the gel surface. This shows that according to the production method of the present invention, the cultured cell aggregate can be detached and recovered without damaging, and the recovered cell aggregate completely contains the extracellular matrix.

Experimental Example 2 (Production of three-dimensional cell aggregate)
A gel serving as a template for preparing a three-dimensional cell aggregate was prepared using a resin mold. The resin mold was designed using graphic modeling software (FreeForm, Sensable, MA), and produced using a three-dimensional printing system (Eden, Objet, Israel).

  FIG. 5A is a model diagram of a mother die produced using the graphic modeling software described above, and shows a mother die having a plurality of parallel ridges and a mother die having a plurality of hemispherical protrusions. FIG. 5B is a photograph showing the appearance of a matrix made of a photocurable resin produced using the above-described three-dimensional printing system, with the upper side having a plurality of parallel protrusions and the lower side having a plurality of hemispheres. A matrix having a convex portion is shown. FIG. 5C is a photograph showing the appearance of a gel molded body (hereinafter also referred to as a gel mold) which is polymerized in the resin mold and taken out from the resin mold. It has been transcribed. The upper side shows a mold having a plurality of parallel grooves, and the lower side shows a mold having a plurality of hemispherical recesses.

Osteoblast MC3T3-E1 is seeded at a cell concentration of 1 × 10 ⁶ cells / ml in a gel template having a plurality of parallel grooves shown in FIG. 5C, cultured at 37 ° C. for 48 hours, and the temperature is lowered to 25 ° C. The aggregate was peeled from the gel mold to obtain a rod-shaped cell aggregate. The gel mold used was one with and without FN coating.

(result)
FIG. 5D is a photograph showing the appearance of a gel template containing cultured cells. FIG. 5E is a photograph showing the appearance of the cell aggregate taken out from the gel mold, in which the upper side shows a rod-like shape and the lower side shows hemispherical cell aggregates of various sizes.

  FIG. 5G is a graph showing changes in the length of rod-shaped cell aggregates when FN is applied to a gel template and when FN is not applied (“length (%)” in the figure indicates the gel template). Represents the ratio of the length of rod-shaped cell aggregate to the length). When FN was applied to the gel template, the length of the rod-shaped cell aggregate was 91% of the length of the groove of the gel template, whereas it was 65% when FN was not applied. From this, it was found that in order to produce a cell aggregate in accordance with the shape of the gel template, it is preferable to apply FN to the gel template.

  As is apparent from the above description, according to the present invention, it is possible to easily produce a three-dimensional cell aggregate having an arbitrary shape, which has been difficult to produce. The three-dimensional cell aggregate obtained by the present invention not only provides a new tissue regeneration tool, but can also be used as a tool for pseudo-tumor tissue and bacterial infection model construction and drug screening. .

Claims (11)

  A three-dimensional molded body of a hydrophilic polymer gel capable of changing the volume is used as a carrier, cells are seeded and cultured on the carrier, and a cultured cell to which a three-dimensional structure is imparted by changing the volume of the carrier is three-dimensional. A method for producing a three-dimensional cell aggregate that peels as a cell aggregate.   The method according to claim 1, wherein a temperature-responsive polymer gel is used as the hydrophilic polymer gel.   The temperature-responsive polymer gel is a hydrophilic polymer gel having a lower critical solution temperature (LCST) that shrinks at a critical temperature (Tc) or higher, culturing cells at a temperature higher than the lower critical solution temperature, The production method according to claim 2, wherein the cultured cells are peeled from the hydrophilic polymer gel at a temperature equal to or lower than the lower critical solution temperature.   The production method according to claim 3, wherein the temperature-responsive polymer gel is a homopolymer or copolymer cross-linked product of N-alkyl-substituted (meth) acrylamide.   The production method according to claim 1, wherein the hydrophilic polymer gel is molded using a resin matrix produced using a three-dimensional printing method.   The preparation method according to claim 1, wherein an organic substance that promotes cell adhesion is applied to the surface of the polymer gel prior to seeding the cells.   A three-dimensional gel carrier for cell culture, comprising a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume and used as a template for imparting a three-dimensional structure to cultured cells.   The three-dimensional gel carrier for cell culture according to claim 7, wherein the hydrophilic polymer gel is a temperature-responsive polymer gel.   The three-dimensional gel carrier for cell culture according to claim 8, wherein the temperature-responsive polymer gel is a homopolymer or a crosslinked product of N-alkyl-substituted (meth) acrylamide.   The three-dimensional gel carrier for cell culture according to claim 7, wherein an organic substance that promotes cell adhesion is attached to the surface.   A three-dimensional cell assembly comprising a collection of cells cultured with a three-dimensional molded body of a hydrophilic polymer gel capable of changing volume as a template and provided with a three-dimensional structure by the template.