JP2007306856A - Method for freezing and preserving cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop new applications of vitrigel other than that as a cell culture base. <P>SOLUTION: A method for freezing and preserving cells is provided, comprising the step of culturing the cells on vitrigel to form a cell-cultured product in a state of being in adhesion to the vitrigel, or in an extended or proliferated state, and the step of preserving the cell-cultured product together with the vitrigel. The resulting frozen cells including vitrigel and cells adhered to the vitrigel are also provided. Such frozen cells can be produced by the method described above. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a frozen product of various cells using vitrigel and a frozen cell obtained thereby.

  After injecting the collagen sol into the culture dish and applying the optimal salt concentration, pH and temperature to gelation, it is thoroughly dried at low temperature and vitrified, and then rehydrated ( By rehydration), a technology has been established to convert the physical properties of collagen gel into a thin film with excellent strength and transparency with good reproducibility (Patent Document 1).

  Here, it has been reported that vitrification of heat-denatured protein proceeds in a process in which bound water gradually decreases after free water is removed in the drying step (Non-patent Document 1). If it is a hydrogel, gels of components other than collagen can be converted to stable new physical properties by rehydrating after vitrification, so new physical properties created through this vitrification process An academic term vitrigel was set as a term meaning a gel in a state (Non-patent Document 2).

  However, the use of such vitrigel is currently limited to use as a cell culture substrate, and development of new uses is awaited.

In general, cryopreservation of animal cells has been conventionally performed by freezing cells in a storage solution and storing them in liquid nitrogen. However, for example, in the case of primary cultured hepatocytes, conventional cryopreservation is extremely difficult, and it is often very poor when evaluated by an adhesion rate that is an index of cell physiology. Therefore, a method of cryopreserving with a carrier has been studied as a method for cryopreserving cells that are difficult to freeze by such conventional freezing methods. For example, primary hepatocytes (350 cells) immediately after separation from a living body are embedded in alginate gel beads with a diameter of 1 mm, and about 3,000 beads are placed in one vial and frozen in liquid nitrogen (about approx. 1 × 10 ⁶ cells / vial) (LIVERBEADS ^™ ; Biopredic Int. (Rennes France) / KC Corporation). However, when conventional cryopreservation methods are used, hepatocytes could not be separated from the beads after thawing and cultured in a monolayer state.
JP-A-8-228768 Takushi E, et al., Nature 345: 298-299, 1990 Takezawa T, et al., Cell Transplant. 13: 463-473, 2004

  This invention makes it a subject to develop the use of the new vitrigel other than the use as a cell culture substratum.

  The present invention succeeded in improving the viability of cells when thawed when various cells adhered, stretched or proliferated by culturing on Vitrigel were cryopreserved in liquid nitrogen.

  That is, the present invention includes a step of culturing cells on Vitrigel to form a cell culture that is adhered, stretched, or proliferated; and a step of storing the cell culture that is adhered, stretched or proliferated together with Vitrigel. A method for cryopreserving cells, comprising:

  The present invention also provides frozen cells, including vitrigel and cells attached on the vitrigel. As described above, such frozen cells are cultured on Vitrigel to form a cell culture that is adhered, stretched, or proliferated; and cells that are adhered, stretched, or proliferated together with Vitrigel. A method of cryopreserving cells, comprising the step of storing the culture.

  By utilizing the present invention, it becomes possible to freeze the cells in the cultured form of the cells under the culturing conditions, for example, cells in a state of adhesion, extension, or proliferation. Such a cryopreservation method for cells has not been put into practical use in the technical field so far, and a new cell cryopreservation method is provided.

  In addition, since the adherent, stretched or proliferated cells prepared by the method of the present invention use the vitrigel thin film as a culture support, the adherent, stretched or proliferated cells can be frozen and thawed. It has sufficient strength to preserve, transport, etc. with cells in the state of adhesion, extension, or proliferation.

  For example, primary cultured hepatocytes are extremely important for pharmacokinetic studies in the development of new drugs, and it is possible to construct a universal system that can use cultured hepatocytes from the same individual (especially humans) at different research institutions. I'm in a hurry. When primary cultured hepatocytes are cryopreserved by the method of the present invention, this is an effective means for achieving this object.

BEST MODE FOR CARRYING OUT THE INVENTION

  The present invention includes culturing cells on Vitrigel to form a cell culture that is adhered, stretched, or proliferated; and preserving the cell culture that is adhered, stretched, or proliferated together with Vitrigel. It has been clarified that a method for cryopreserving cells can be provided.

  The vitrigel that can be used in the present invention can be produced according to a conventionally known method for producing a vitrigel (see, for example, JP-A-8-228768 or Japanese Patent Application No. 2006-25682). The shape of the vitrigel that can be used in the present invention may be any shape, for example, a thin film shape, a thread shape, a tubular shape, or a rod shape.

For example, when producing a thin-film vitrigel, specifically, a hydrogel solution in a sol state is injected into a culture dish, which is stored and gelled in the plate. After completion of gelation in the plate, the moisture (free water) in the gel was removed (dry) under constant temperature and humidity conditions with the plate, and further, the bound water was dried (vitrified) over time. The temperature and time for performing drying can be appropriately selected by those skilled in the art. For example, the drying can be performed at a temperature of 10 ° C. and a humidity of 40%. The dry gel thin film after completely removing free water is allowed to proceed through a vitrification process that slowly removes bound water while being stored at room temperature (see Non-Patent Document 2). Thereafter, UV irradiation of 8000 J / m ² is performed as necessary. The vitrigel thin film thus prepared is then used after being rehydrated with PBS.

  For example, when producing a vitrigel having a shape such as a thread, a tube, or a rod, the gel is formed by pouring hydrogel into a mold having a shape such as a thread, a tube, or a rod to form a gel such as a thread, a tube, or a rod. Make it. The long axis direction of the shape of such a hydrogel is set at an angle of 60 to 90 degrees with respect to the horizontal, dehydrated and dried under constant temperature and humidity conditions, and vitrified to form a filament, tube, or rod A vitrigel having a shape such as can be produced. The vitrigel thin film thus produced is then used after being rehydrated with PBS (see Japanese Patent Application No. 2006-25682).

  The composition of the hydrogel used as a raw material when producing the vitrigel of the present invention is not particularly limited as long as it is vitrified by dehydration and drying. The composition of hydrogel that can form vitrigel includes collagen such as type I collagen and type III collagen, or matrigel (base membrane component extracted from EHS tumor, mainly type IV collagen, laminin, and heparan sulfate proteoglycan) In addition, extracellular matrix components such as agar, agarose, acrylamide and the like are known. In the present invention, it is preferable to use collagen such as type I collagen, type III collagen, or a combination thereof.

  The target cells are cultured on the above-described vitrigel, and a cell culture in a state where the cells adhere, extend or proliferate to the vitrigel is prepared. As the conditions for performing such cell culture, known culture conditions for each cell can be used as they are. Any cells may be used as the cells that can be used in the present invention, and for example, primary hepatocytes, embryonic stem cells, or fibroblasts can be used.

  In the present invention, when a cell “adheres”, “extends”, or “proliferates” on Vitrigel, it indicates the relationship between Vitrigel and cells to be cultured. ”,“ Proliferation ”, or a combination thereof. For example, adhesion-dependent cells adhere to Vitrigel, but after adhering there are cells that expand (“adhesive” and “extend” cells) or cells that remain attached but do not extend (cells that only “adhere”) ) Is also present. In the case of stretched cells, there are cells that proliferate after spreading (cells that “adhere”, “stretch”, and “proliferate”) and cells that do not proliferate (cells that “adhere” and “stretch”). Exists. Depending on the cell type, the cell morphology on Vitrigel varies, but specifically, rat primary hepatocytes (eg, mature hepatocytes) and mitomycin C-treated fibroblasts are ”Will“ extend ”but not“ proliferate ”. In addition, mouse ES cells “grow” after “adhesion” and “grow” in an island shape while aggregating.

  The cell culture adhered, stretched or grown on the vitrigel in this way can be frozen together with the vitrigel. As a cryopreservation solution that can be used when freezing a cell culture, the same culture solution that is usually used when freezing a cell suspension can be used. For example, as a cryopreservation solution, a solution obtained by adding 10% dimethyl sulfoxide (DMSO) to a culture solution used for culturing cells can be exemplified. The cell culture placed in such a cryopreservation solution is frozen under the same temperature and time conditions as when freezing a normal cell suspension. For example, in addition to the method of freezing in a bicelle freezing container etc. in a freezer at -80 ° C. for 1 to 7 days, and then moving to liquid nitrogen and cryopreserving, the desired temperature and time using a program freezer A method of freezing under the above conditions and finally moving to liquid nitrogen and cryopreserving is known, but is not limited thereto.

Cell cultures cryopreserved in liquid nitrogen are used after thawing. When thawing the cell culture, the temperature of the cell culture is rapidly raised to 37 ° C, then the cryopreservation solution is converted to a normal culture solution and cultured in a 37 ° C, 5% CO ₂ incubator. continue.

  The present invention also revealed that frozen cells can be provided, including vitrigel and cells attached on vitrigel. As described above, such frozen cells are cultured on Vitrigel to form a cell culture that is adhered, stretched, or proliferated; and cells that are adhered, stretched, or proliferated together with Vitrigel. A method of cryopreserving cells, comprising the step of storing the culture.

Example 1: Freezing of rat primary hepatocytes In this example, hepatocytes were collected from rats, and after initial culture, cells that had been adhered and extended were prepared. The method of freezing was examined.

Rat hepatocytes were collected by applying the collagenase perfusion method. Hepatocytes collected in this way (viability 80.1%) contain 10% non-immobilized fetal bovine serum (FBS), 10 mM nicotinamide, 20 mM HEPES, 100 u / ml penicillin, 100 μg / ml streptomycin The cells were suspended in Dulbecco's modified Eagle medium (DMEM) so that the cell density was 2 × 10 ⁵ cells / ml. Cell suspensions were placed on a polystyrene culture dish (FALCON 3001; control group) or a type I collagen vitrigel thin film carrier (gel thin film group) with a circular nylon membrane support prepared in a polystyrene culture dish. Cultivation was started by seeding each ml, and culturing was performed in a 37 ° C., 5% CO ₂ incubator, and dead cells were removed by exchanging the culture solution at 3 hours of culturing. The gel thin film group was further cultured by peeling off the gel thin film carrier from the culture dish at 6 hours of culture.

  The vitrigel thin film used in the present invention was produced according to the method described in Non-Patent Document 2. That is, at 4 ° C, an equal volume of 0.5% type I collagen aqueous solution (CELLGEN I-AC, manufactured by Koken Co., Ltd.) and culture medium (10% non-immobilized fetal bovine serum (FBS), 20 mM HEPES, 100 u / A 0.25% type I collagen sol was prepared by mixing with ml Penicillin and Dulbecco's Modified Eagle Medium (DMEM) containing 100 μg / ml streptomycin. About 2.0 ml of the above-mentioned 0.25% type I collagen sol was injected into a polystyrene culture dish (FALCON 3001), which was kept at 37 ° C. for 2 hours to gel in the culture dish. After gelation is completed in the culture dish, the moisture (free water) in the gel is removed (dry) over 2 days in a constant temperature and humidity chamber with a temperature of 10 ° C and humidity of 40%. The combined water was dried (vitrified) over approximately two weeks. After the free gel is completely removed, the dried gel film is allowed to proceed through a vitrification process that slowly removes the bound water while being stored at room temperature over approximately two weeks. Thereafter, the vitrigel thin film was rehydrated with PBS and used in this example.

In both the control group and the gel thin film group, the culture was continued for 24 hours in a 37 ° C., 5% CO ₂ incubator, and the culture solution was transferred to the culture 24 hours (phase contrast micrographs are shown in FIGS. 1A and 1B). Cryopreservation solution (Dulbecco's modified Eagle culture containing 10% dimethyl sulfoxide (DMSO), 10% non-immobilized fetal bovine serum (FBS), 10 mM nicotinamide, 20 mM HEPES, 100 u / ml penicillin, 100 μg / ml streptomycin The solution (DMEM) was replaced, and cryopreservation was started with a program freezer. In the control group, the primary hepatocytes attached to the polystyrene culture dish (FALCON 3001) are stored frozen with the polystyrene culture dish. In the gel thin film group, the primary hepatocytes attached to the vitrigel thin film carrier are stored frozen together with the vitrigel thin film carrier. As a result, the rat primary hepatocytes adhered and extended were cryopreserved.

The temperature and time program for cryopreservation is
Cool from 20.0 ℃ to 0.0 ℃ at a rate of -1.00 ℃ / min over 20 minutes,
After 2 minutes at 0.0 ℃,
Cool further from 0.0 ℃ to -8.0 ℃ at a rate of -2.00 ℃ / min over 4 minutes,
Further quenching from -8.0 ° C to -28.0 ° C at a rate of -50.00 ° C / min over 24 seconds,
Further cooling from -28.0 ° C to -33.0 ° C at a rate of -2.50 ° C / min over 2 minutes,
Then, increase the temperature from -33.0 ℃ to -28.0 ℃ at a rate of + 2.50 ℃ / min for 2 minutes,
Then, further cooling from -28.0 ° C to -60.0 ° C over 16 minutes at a rate of -2.00 ° C / min,
Next, further cooling from -60.0 ° C to -100.0 ° C over 4 minutes at a rate of -10.00 ° C / min,
After standing at −100 ° C. for 30 minutes, the cells were cryopreserved in liquid nitrogen, and finally the cells that had adhered and extended were cryopreserved in liquid nitrogen.

  In both the control group and the gel thin film group, the adherent / stretched cells were stored in liquid nitrogen, and on the 4th day, they were rapidly thawed and then the cryopreservation solution (10% non-immobilized fetal bovine serum (FBS)) , Replaced with Dulbecco's Modified Eagle Medium (DMEM) containing 10 mM nicotinamide, 20 mM HEPES, 100 u / ml penicillin, 100 μg / ml streptomycin, and resumed culture, and thawed at 1 hour of culture. In addition to observing the appearance of rat primary hepatocytes in an adherent / extended state with a phase-contrast microscope (Figs. 1C and 1D), commercially available LIVE / DEAD (green fluorescent light in live cells and bright red fluorescent light in dead cells) Cells were stained using a trademark viability assay kit (Invitrogen) (FIGS. 1E and 1F).

  As a result, the primary hepatocytes (control group) on the polystyrene culture dish (FALCON 3001) and the primary hepatocytes on the vitrigel thin film carrier (gel) were obtained under the phase-contrast microscope immediately after freezing. Both the thin film group) showed good adhesion / extension patterns (FIGS. 1A and 1B). However, when thawed after freezing for 4 days, at the first hour of thawing, as long as the phase-contrast micrographs are seen (FIGS. 1C and 1D), most of the cells were removed from the culture dish in the control group. In the group, it was morphologically revealed that about 60% of the cells remained adhered after thawing. In addition, fluorescence micrographs (Figures 1E and 1F) using a commercially available LIVE / DEAD ™ viability assay kit reveal that approximately 33% of the cells that adhere to the vitrigel film are viable. Became.

  In addition, cell adhesion rate and survival rate before freezing and after freezing and thawing were calculated from the area occupancy rate of adherent cells and viable adherent cells per unit area, respectively, and digitized (FIG. 2). As a result, in the control group, only 0.5% of viable cells remained attached to the culture dish after freezing and thawing compared to the adherent cells before freezing, but in the gel thin film group, approximately 20% of the cells were vitrigel. It became clear that it adhered as a living cell on the thin film carrier.

  From this result, it was revealed that primary hepatocytes adhered on a vitrigel thin film carrier can maintain the viability of the adherent cells even if they are frozen and thawed as they are.

Example 2: Freezing mouse embryonic stem cells (ES cells) In this example, mouse ES cell culture was performed to prepare adherent, extended, or proliferated cells, and then adhered, extended, or proliferated. The method of freezing cells was examined.

As mouse ES cells, 129SV mouse-derived ES cells (Dainippon Sumitomo Pharma Co., Ltd.), passage number 13 were used. For ES cells on a polystyrene culture dish (FALCON 3001; control group) or on a type I collagen vitrigel thin film carrier (gel film group) prepared by UV irradiation (8,000 J / m ² ) in a polystyrene culture dish 2.0 ml each of mitomycin C-treated primary mouse embryo fibroblasts as feeder cells suspended at a cell density of 1 × 10 ⁵ cells / ml in the culture medium was seeded for 8 hours at 37 ° C., 5% CO _2. Culture was performed in an incubator to prepare a feeder layer. Thereafter, 2.0 ml each of the above-mentioned ES cells suspended at a cell density of 5 × 10 ⁵ cells / ml in an ES cell culture medium was seeded on the feeder layer of each group at 37 ° C., 5% CO _2. Culture was performed in an incubator. ES cell culture solution is DMEM (GIBCO BRL # 11995-065) 400 ml, Antimyobic-Antimycotic (GIBCO BRL # 15240-062) 5.0 ml, 3.5 μl β-mercaptoethanol (SIGMA # M-6250) in PBS 5.0 ml , MEM non-essential amino acid (GIBCO BRL # 11140-050) 5.0 ml, nucleoside (adenosine 4 mg, guanosine 4.25 mg, cytidine 3.65 mg, uridine 3.65 mg, thymidine 1.2 mg in PBS) 5.0 ml, ESGRO (CHEMICON # ESG1107; 10e7 U / ml) 50 μl, and FBS (GIBCO BRL # 16141-079) 100 ml were added.

In both the control group and the gel thin film group, the culture was continued in a 37 ° C., 5% CO ₂ incubator for 48 hours, and the culture solution was added at 48 hours of culture (phase contrast micrographs are shown in FIGS. 3A and 3B). After changing to a cryopreservation solution (10% DMSO-containing ES cell culture solution), transfer to a bicell freezing container (Nippon Freezer Co., Ltd.) and freeze in an 80 ° C freezer for 5 hours before freezing in liquid nitrogen saved. In the control group, the cells attached to the polystyrene culture dish (FALCON 3001) were frozen and stored together with the polystyrene culture dish, and in the gel film group, the cells attached to the Vitrigel thin film carrier were stored together with the Vitrigel thin film carrier. -Mouse ES cells that were expanded and proliferated into islands were cryopreserved. Cryopreservation was continued for 6 days in liquid nitrogen.

  As a result, at 48 hours after seeding just before freezing, ES cells on a polystyrene culture dish (FALCON 3001) (control group) and ES cells on a vitrigel thin film carrier (gel thin film group) under a phase contrast microscope. ) Both showed good morphology that grew like islands (FIGS. 3A and 3B). However, when thawed after freezing for 6 days, at the first hour of thawing, as far as the phase contrast micrographs are seen (FIGS. 3C and 3D), all cells, including feeder cells, were detached from the plate in the control group. However, it was morphologically revealed that about 80% of the cells in the gel thin film group remained adhered after thawing. In addition, fluorescence micrographs (Figs. 3E and 3F) using a commercially available LIVE / DEAD ™ viability assay kit reveal that almost all adherent cells remaining on the vitrigel film are alive. became.

  In addition, the cell adhesion rate and survival rate before freezing and after freezing and thawing were calculated from the area occupancy rate of adherent cells and viable adherent cells per unit area, respectively, and digitized (FIG. 4). As a result, in the control group, only 0.2% of viable cells remained attached to the culture dish after freezing and thawing compared to the adherent cells before freezing. In the gel thin film group, as many as 82% of the cells were vitrigel thin film. It became clear that it adhere | attached as a living cell on a support | carrier.

  From these results, it was found that ES cells that adhere to and spread on a vitrigel thin film carrier and proliferate in an island shape can sufficiently maintain the viability of the proliferating cells even if they are frozen and thawed as they are.

Example 3: Freezing of mouse fibroblasts In this example, after culturing mouse fibroblasts to prepare adherent / extended cells, the method for freezing the cells in the adhered / extended state is described. Study was carried out.

As mouse fibroblasts, mitomycin C-treated primary mouse fibroblasts (Dainippon Sumitomo Pharma Co., Ltd.) were used. For ES cells on a polystyrene culture dish (FALCON 3001; control group) or on a type I collagen vitrigel thin film carrier (gel film group) prepared by UV irradiation (8,000 J / m ² ) in a polystyrene culture dish Seed 2.0 ml of mitomycin C-treated primary mouse fibroblasts suspended at a cell density of 1 × 10 ⁵ cells / ml in the culture medium, and culture in a 5% CO ₂ incubator for 24 hours at 37 ° C. This was done to produce cells that would become feeder layers.

In both the control group and the gel thin film group, the culture was continued for 24 hours in a 37 ° C., 5% CO ₂ incubator, and the culture solution was transferred to the culture 24 hours (phase contrast micrographs are shown in FIGS. 5A and 5B). After changing to a cryopreservation solution (10% DMSO-containing ES cell culture solution), transfer to a bicell freezing container (Nippon Freezer Co., Ltd.) and freeze in an 80 ° C freezer for 5 hours before freezing in liquid nitrogen saved. In the control group, the cells attached to the polystyrene culture dish (FALCON 3001) were frozen and stored together with the polystyrene culture dish, and in the gel film group, the cells attached to the Vitrigel thin film carrier were stored together with the Vitrigel thin film carrier. -The mouse fibroblasts treated with mitomycin C in an expanded state were cryopreserved. Cryopreservation was continued for 10 days in liquid nitrogen.

  As a result, fibroblasts (control group) on a polystyrene culture dish (FALCON 3001) and fibroblasts (gel) on a vitrigel thin film carrier were obtained under a phase contrast microscope at 24 hours after seeding just before freezing. Both the thin film group) showed a good form of adhesion and extension (FIGS. 5A and 5B). However, when thawed after freezing for 10 days, at the first hour of thawing, all the cells were detached from the culture dish in the control group as long as the phase contrast micrographs were seen (FIGS. 5C and 5D). In the group, it was morphologically revealed that about 50% of the cells adhered after thawing. In addition, fluorescence micrographs (Figs. 5E and 5F) using a commercially available LIVE / DEAD ™ viability assay kit reveal that almost all adherent cells remaining on the vitrigel film are viable. became.

  In addition, the cell adhesion rate and survival rate before freezing and after freezing and thawing were calculated from the area occupancy rate of adherent cells and viable adherent cells per unit area, respectively, and digitized (FIG. 6). As a result, in the control group, only 0.04% of viable cells remained attached to the culture dish after freezing and thawing compared to the adherent cells before freezing. In the gel thin film group, 53% of the cells were vitrigel thin film. It became clear that it adhere | attached as a living cell on a support | carrier.

  From this result, it was clarified that the feeder layer of mouse fibroblasts treated with mitomycin C formed by adhesion on a vitrigel thin film carrier can maintain the viability of feeder layer cells even if it is frozen and thawed as it is. .

  By utilizing the present invention, it becomes possible to freeze the cells in the cultured form of the cells under the culturing conditions, for example, cells in a state of adhesion, extension, or proliferation. Such a cryopreservation method for cells has not been put into practical use in the technical field so far, and a new cell cryopreservation method is provided.

  In addition, since the adherent, stretched or proliferated cells prepared by the method of the present invention use the vitrigel thin film as a culture support, the adherent, stretched or proliferated cells can be frozen and thawed. It has sufficient strength to preserve, transport, etc. with cells in the state of adhesion, extension, or proliferation.

  For example, primary cultured hepatocytes are extremely important for pharmacokinetic studies in the development of new drugs, and it is possible to construct a universal system that can use cultured hepatocytes from the same individual (especially humans) at different research institutions. I'm in a hurry. When primary cultured hepatocytes are cryopreserved by the method of the present invention, this is an effective means for achieving this object.

FIG. 1 shows the appearance of primary hepatocytes (control group) on a polystyrene culture dish (FALCON 3001) and primary hepatocytes (gel thin film group) on a vitrigel thin film carrier before freezing and after freezing and thawing. . Here, FIGS. 1A and 1B are phase contrast micrographs of the state 24 hours after the start of culture before freezing, FIGS. 1C and 1D are phase contrast micrographs of the first hour after freezing and thawing, and FIGS. Fluorescence micrographs at 1 hour after freezing and thawing stained with a commercially available LIVE / DEAD ™ viability assay kit are shown respectively. FIG. 2 is a graph showing cell adhesion rates and survival rates of rat primary hepatocytes before freezing and after freezing and thawing. FIG. 3 is a view showing the appearance of mouse ES cells (control group) on a polystyrene culture dish (FALCON 3001) and mouse ES cells (gel thin film group) on a vitrigel thin film carrier before freezing and after freezing and thawing. . Here, FIGS. 3A and 3B are phase contrast micrographs of the state 48 hours after the start of culture before freezing, FIGS. 3C and 3D are phase contrast micrographs of the first hour after freezing and thawing, and FIGS. Fluorescence micrographs at 1 hour after freezing and thawing stained with a commercially available LIVE / DEAD ™ viability assay kit are shown respectively. FIG. 4 is a graph showing cell adhesion rate and survival rate of mouse ES cells before freezing and after freezing and thawing. Figure 5 shows freezing of mitomycin C-treated mouse fibroblasts (control group) on a polystyrene culture dish (FALCON 3001) and mitomycin C-treated mouse fibroblasts (gel film group) on a vitrigel thin film carrier. It is a figure which shows the external appearance before and freeze-thaw. Here, FIGS. 5A and 5B are phase contrast micrographs of the state 24 hours after the start of culture before freezing, FIGS. 5C and 5D are phase contrast micrographs of the first hour after freezing and thawing, and FIGS. 5E and 5F are Fluorescence micrographs at 1 hour after freezing and thawing stained with a commercially available LIVE / DEAD ™ viability assay kit are shown respectively. FIG. 6 is a graph showing the cell adhesion rate and survival rate of mouse fibroblasts treated with mitomycin C before freezing and after freezing and thawing.

Claims (6)

Culturing the cells on Vitrigel to form a cell culture that is attached, stretched or proliferated;
Storing the cell culture in a state adhered, stretched or expanded together with vitrigel;
A method for cryopreserving cells, comprising:   2. The method according to claim 1, wherein the vitrigel is a thin-film vitrigel.   The method according to claim 1 or 2, wherein the cells are primary hepatocytes, embryonic stem cells or fibroblasts.   Frozen cells, including vitrigel and cells attached on vitrigel.   5. The frozen cell according to claim 4, wherein the vitrigel is a thin-film vitrigel.   The frozen cell according to claim 4 or 5, wherein the cell is a primary hepatocyte, embryonic stem cell or fibroblast.