JP3190145B2 - Animal tissue culture carrier and animal tissue culture method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a carrier for culturing animal tissues. More specifically, the present invention relates to a culture carrier suitable for producing a cultured animal tissue suitable for evaluating toxicity, side effects, pharmacological activity, and the like of a drug or the like.

[0002] The present invention also relates to a culture carrier suitable for producing a cultured animal tissue for use as a prosthetic material for a lesion or defect in a living body. The present invention also relates to a culture carrier suitable for producing a cultured animal tissue as a bioreactor for the purpose of producing a product of an animal tissue. Further, the present invention relates to a method for producing a cultured animal tissue having the above-mentioned object using the carrier.

[0003]

2. Description of the Related Art In recent years, animal tissue culture techniques have been developed in the fields of 1) artificial organs for replacing lesions and defects in living bodies, such as artificial skin, artificial liver, and artificial pancreas; 2) enzymes;
It is actively used in the field of bioreactors for producing useful cell products such as growth factors and vaccines, and 3) in the field of evaluating the biological activity of drugs.

The most important point when using cultured animal tissue for the above purpose is to culture and grow the cultured animal tissue while maintaining the same function as in a living body.

Methods for culturing animal tissues including cells include suspension culture, two-dimensional culture using a planar substrate, and three-dimensional culture embedded in a substrate such as a gel.

[0006] Tissues including cells constituting the living body,
It is well known that most organs do not express growth and normal function in the absence of a scaffold substrate. Therefore, suspension culture in a liquid medium is not suitable except for certain cells (eg, blood cells, cancer cells, etc.), and is completely unsuitable for culturing tissues and organs.

[0007] Therefore, at present, a substrate as a scaffold for tissues and organs including cells has been vigorously developed. The two-dimensional culture method of culturing on the surface of glass, plastic, etc. is a very efficient culture method in terms of replenishing nutrients and removing waste products, but it can maintain and express the functions of cells and tissues. It has a big problem that it is very difficult. This can be caused by your organization,
Organs do not exist in a planar state in a living body, but form a three-dimensional state, that is, a three-dimensional structure. The cells or tissue organs cultured on a flat surface adhere to the substrate, and the shape is significantly different from that in a living body and becomes flat. It has been suggested that such morphological differences affect the three-dimensional structure of the nucleus and change the expression form of the gene.

[0008] Furthermore, while cells in a living body are in close contact with adjacent cells to control gene expression by cell-cell interaction, on a flat surface, cell-cell contact is coarse and information between cells is lost. It is said that the exchange function is significantly reduced.

[0009] For these reasons, in recent years, three-dimensional culture methods have been actively developed, and many findings have been obtained that the expression and maintenance of functions of cultured cells and tissues are better than those of two-dimensional culture methods. Have been.

[0010] Among the three-dimensional culture methods, the most common one is gel-embedded culture (gel culture). Today, gel materials used for the gel culture are represented by agar (agarose), calcium alginate, and collagen.

However, the conventional gel culture method using these gel materials has the following major problems.

Alginate and collagen.
Agarose is a polysaccharide produced by seaweed, has a sol-gel transition temperature, shows a dissolved state or sol state at a temperature higher than the transition temperature, and changes to a gel state at a temperature lower than the transition temperature. Has properties. Utilizing this property, a method of dispersing animal tissue in a sol state, gelling by cooling the temperature below the sol-gel transition temperature, embedding the animal tissue in a gel, and culturing has been implemented. However, agarose has the following serious problems.

1) The melting temperature of agarose gel is very high, around 90 ° C., so that the tissue is thermally damaged when mixed with animal tissue. Also, when the cultured animal tissue is changed from the agarose gel to the sol state in order to be recovered or subcultured, the animal tissue is exposed to high temperatures and is extremely thermally damaged. Therefore, collection and passage of the culture from the agarose gel were substantially impossible. 2) Agarose is a natural polymer, and its physical properties, particularly its toxicity to cells, vary greatly from lot to lot.

On the other hand, in a gel culture method using an alginate gel, water-soluble sodium alginate is dissolved in a culture solution.
After mixing with animal tissue, the solution is brought into contact with a concentrated calcium chloride solution to ion exchange with calcium alginate having gel forming ability to form a gel. Alginate gels do not require high temperatures when embedding animal tissues as in agarose gels, and significantly reduce thermal damage, but have the following serious problems: .

1) Reaction of the sodium alginate with a concentrated calcium chloride solution during gelation causes damage to animal tissues. In particular, calcium ions have strong physiological activity on tissues and cells. 2) In order to recover or subculture tissues and cells cultured in the gel, it is essential to dissolve the gel, but calcium chelates such as EDTA (ethylenediaminetetraacetic acid) used in this process The agent damages animal tissues. 3) Alginic acid gel has low mechanical strength, and is not suitable for the tank culture by the impeller method used for bead culture and the like. 4) In a medium having a high concentration of phosphoric acid, for example, an RPMI-1640 medium widely used in cell culture, calcium in the alginate gel reacts with phosphoric acid to form a precipitate of calcium phosphate, which makes culturing difficult and at the same time makes the gel brittle. become. 5) Alginic acid gel is formed by ion exchange reaction with calcium, so that gelation proceeds from the surface to the inside of the gel, and animal tissues and the like inside the gel hinder a uniform gelation reaction and tend to have a non-uniform structure. 6) Alginic acid is a natural polymer, and its properties, particularly its toxicity to cells, vary greatly between lots.

On the other hand, collagen, which is a main component of collagen gel, is a main component of animal tissues and plays a role in maintaining the strength, expressing functions, and maintaining the tissues. As a method of embedding tissue in collagen gel, acidic collagen separated from living tissue was dissolved in a medium at low temperature and low pH, then the pH was raised to neutral and the salt concentration was adjusted to physiological conditions. The most common method is to mix the tissue later, raise the temperature to 37 ° C., and gradually gel the collagen. In the case of the collagen gel, unlike in the case of the agarose gel and the alginate gel described above, the damage at the time of embedding the tissue is remarkably reduced, but there are the following problems.

1) In order to recover or passage cells or other tissues cultured in a collagen gel from the gel, it is essential to use a protease such as collagenase which dissolves the collagen gel. Fatal problems such as dysfunction of tissue or destruction of reconstructed structures such as colonies. 2) Since the gelation process of collagen proceeds slowly under the strictly controlled physiological conditions as described above, it is very difficult to impart a form such as granulation to the gel. 3) Collagen gel is also mechanically fragile, and at the same time, since collagen is a natural polymer, its properties vary greatly between lots.

As described above, the gel material used in the conventional gel culture has the unsolved fatal drawback of causing a great deal of damage to the tissue during tissue embedding and recovery or passage. Have been.

[0019]

SUMMARY OF THE INVENTION It is an object of the present invention to be able to easily embed animal tissues without damaging them, and to recover or passage them after culture without damaging the tissues. It is an object of the present invention to provide a carrier for animal tissue culture capable of carrying out the method and an animal tissue culture method using the same.

[0020]

Means for Solving the Problems As a result of intensive studies, the present inventor has a property completely different from that of the conventional gel material, that is, a sol-gel transition temperature. It has been found that the use of such a high molecular compound as a carrier for animal tissue culture is extremely effective in solving the above-mentioned problems.

The carrier for animal tissue culture of the present invention is based on the above findings, and more specifically, a polymer compound having a sol-gel transition temperature having a hydrophobic portion and a hydrophilic portion in the molecule. And a liquid state (sol state) reversibly at a temperature lower than the sol-gel transition temperature.

According to the present invention, there is further provided a polymer having a hydrophobic portion and a hydrophilic portion in the molecule, having a sol-gel transition temperature, and having a temperature lower than the sol-gel transition temperature. And a physiologically active substance for animal tissue culture, which comprises a carrier that reversibly exhibits a liquid state (sol state) and a composition for animal tissue culture.

According to the present invention, there is further provided a polymer having a hydrophobic portion and a hydrophilic portion in the molecule, having a sol-gel transition temperature, and having a temperature lower than the sol-gel transition temperature. A carrier which reversibly shows a liquid state (sol state) is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier, and animal tissue is mixed into the mixture. There is provided a method for culturing animal tissue, wherein the animal tissue is cultured in a gel state at a higher temperature.

According to the present invention, there is further provided a polymer compound having a hydrophobic portion and a hydrophilic portion in the molecule and having a sol-gel transition temperature, and a polymer having a sol-gel transition temperature. A carrier that reversibly shows a liquid state (sol state)
The carrier is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature, and an animal tissue and a physiologically active substance for animal tissue culture are mixed into the mixture, and the mixture is heated at a temperature higher than the sol-gel transition temperature. And a method for culturing animal tissue, wherein the animal tissue is cultured in a gel state.

In the present invention described above, the hydrophilic portion of the polymer compound is necessary for the polymer compound to become water-soluble at a temperature lower than the above-mentioned sol-gel transition temperature, and the hydrophobic portion is required. It is necessary for the polymer to change to a gel state at a temperature higher than the sol-gel transition temperature.
In other words, bonding between the hydrophobic moieties is required for the formation of gel cross-linking points.

The carrier of the present invention utilizes the following properties of the bonds between the hydrophobic parts in the polymer compound, ie, the hydrophobic bonds.

[0027] Since the hydrophobic bond has a property that its binding strength becomes stronger with an increase in temperature, the strength of cross-linking and the cross-link density increase with an increase in temperature. (Temperature higher than the sol-gel transition temperature), it is possible to change to a gelled state. In addition, in the present invention, the sol-gel transition occurs reversibly due to the property that the temperature dependency of the hydrophobic binding force is reversible.

On the other hand, as described above, the hydrophilic portion in the polymer compound becomes water-soluble at a temperature lower than the temperature at which the animal compound is cultured (lower than the sol-gel transition temperature). At the same time as it is necessary to prevent the high molecular compound from agglomerating and precipitating due to excessive increase in the hydrophobic bonding force at a temperature higher than the above temperature,
It is also necessary to form a hydrogel state.

Hereinafter, the present invention will be described in more detail. (Sol-gel transition temperature) In the present invention, "sol state"
"Gel state" and "sol-gel transition temperature" are defined as follows. This definition is described in the literature (Polymer Jo
urnal, 18 (5), 411-416 (1986)).

1 mL of the polymer solution (polymer solution to be used as a carrier) is placed in a test tube having an inner diameter of 1 cm, and kept in a water bath at a predetermined temperature (constant temperature) for 12 hours. Thereafter, when the test tube is turned upside down, if the solution / air interface (meniscus) is deformed by the weight of the solution (including the case where the solution flows out), the polymer solution is not allowed to flow at the predetermined temperature. Defined as "sol state". On the other hand, if the solution / air interface (meniscus) does not deform due to the weight of the solution even when the test tube is turned upside down, the solution is defined as being in a “gel state” at the predetermined temperature. I do.

On the other hand, when the above-mentioned “predetermined temperature” is gradually increased (for example, at 1 ° C. increments) to determine the temperature at which the “sol state” changes to the “gel state”, the transition temperature obtained by this is calculated as follows. It is defined as a "sol-gel transition temperature" (at this time, the "predetermined temperature" may be lowered in steps of, for example, 1 ° C, and the temperature at which the "gel state" transitions to the "sol state" may be determined.

In the carrier of the present invention, the sol-gel transition temperature is higher than 0 ° C. and preferably 60 ° C. or lower, more preferably 4 ° C. to 50 ° C. (particularly 4 ° C. to 40 ° C.).
The following is preferred from the viewpoint of preventing thermal damage to animal tissues. Such a polymer compound having a suitable sol-gel transition temperature can be easily selected from the specific compounds described below according to the above-described screening method (sol-gel transition temperature measurement method).

In the animal tissue culturing method of the present invention, the above-mentioned sol-gel transition temperature (a ° C.) is determined by the temperature at the time of culturing (b ° C.) and the temperature at which the cultured animal tissue is collected or subcultured ( c.degree. C.).
That is, it is necessary that there is a relationship of b>a> c between the above three temperatures a ° C., b ° C., and c ° C. More specifically, (ba) is 1 to 40 ° C., more preferably 2 to 30 ° C.
° C, and (ac) is from 1 to 40.
C., more preferably 2 to 30C.

(Polymer Compound) The preferable sol-gel transition temperature as described above is, for example, the composition of the hydrophobic portion and the hydrophilic portion in the polymer compound used for the carrier of the present invention, the degree of hydrophobicity, and the hydrophilicity. It is possible to achieve this by adjusting the degree, molecular weight, etc., respectively.

Specific examples of the polymer compound containing such a hydrophobic portion and a hydrophilic portion include, for example, a polyalkylene oxide block copolymer represented by a block copolymer of polypropylene oxide and polyethylene oxide; Etherified cellulose such as methylcellulose and hydroxypropylcellulose; chitosan derivatives (KRHolme. Et al, Macromolecules. 24 , 382)
8 (1991)); pullulan derivatives (Shigeru Deguchi, Polyme)
r Preprints. Japan. 19 , 936 (1990)); temperature-sensitive polymers represented by poly N-substituted (meth) acrylamide derivatives, partially acetylated polyvinyl alcohol, polyvinyl methyl ether, etc .; Conjugates with high molecular compounds (eg Takehisa Matsuda et al., Polyme
r Preprints. Japan, 39 (8), 2559 (199
0)) and the like, but are not limited thereto.

The temperature above Symbol high molecular compound in the present invention, the sol is set - varies depending gel transition temperature and the like, when used in a mixture with a predetermined medium as described below, generally 0.1~30wt %, Even 1-20
Preferably, the concentration is wt%.

(Animal tissue) "Animal tissue" in the present invention
The term “animal” refers to the tissues of animals including humans, and is used to include tissues and organs themselves, tissues and organ fragments, and cells or aggregates thereof separated from tissues and organs.

(Physiologically active substance for animal tissue culture)
Bioactive substances for animal tissue culture in
Has the effect of imparting some physiological activity to animal cells.
Substances, such as cell growth / differentiation factors, cell adhesion factors, etc.
Is used to mean Cell growth and differentiation factors
Tumor growth factor-β (TGF-β), platelet-derived growth factor
(PDGF), fibroblast growth factor (FGF), epithelialization
Long factor (EGF), interleukin group, etc.
You. These cell growth and differentiation factors are
(Carrier) 100 parts (parts by weight, hereinafter the same)
^-7-10^-3Part (and 10^-6-10^-FourPart) use about
Is preferred. Cell adhesion factors include collagen,
Ibronectin, vitronect Chin, la Minin, proteo
Glycan And extracellular matrices such as glycosaminoglycans
And corn and gelatin. These cell adhesion factors
100 parts of the above-mentioned polymer (carrier) is added to 0.1 part of the polymer.
About 0.1 to 50 parts (further 0.1 to 10 parts) can be used
preferable.

(Medium) In the present invention, a medium to be used in combination with the above-mentioned polymer compound includes:
A known animal tissue culture medium can be used without any particular limitation.

(Method of culturing animal tissue) A preferred example of a method of culturing animal tissue using the above-described polymer compound is described below.

First, the polymer compound is uniformly dissolved in a desired medium at a temperature lower than the sol-gel transition temperature of the polymer compound. At this time, the above-mentioned physiologically active substance for animal tissue culture can be simultaneously dissolved as appropriate. Next, animal tissues such as cells are mixed and dispersed in a medium solution of the polymer compound. Then, by raising the temperature of the mixture above the Ru-gel transition temperature, it is possible to gel the mixture quickly and easily. less than,
The above method will be described more specifically.

For example, as a method of molding a gel containing animal tissues into particles, a medium solution of the above-mentioned polymer compound in which animal tissues are mixed and dispersed at a temperature lower than the above-mentioned sol-gel transition temperature is prepared using a microdispenser or the like. A method in which the solution is dropped into a medium heated to a temperature higher than the sol-gel transition temperature to cause gelation; or a medium solution of the polymer compound in which animal tissue is mixed and dispersed is heated to a temperature higher than the sol-gel transition temperature. After addition in a large volume of liquid paraffin at low temperature and suspension under stirring, the suspended particles are gelled by raising the temperature of the suspension above the sol-gel transition temperature, and then the gel A method of adding a medium heated to a temperature higher than the sol-gel transition temperature to the particle suspension and performing centrifugation is used. In the latter method, the liquid paraffin layer is separated into an upper layer and the medium suspension layer of the gel particles is separated into a lower layer by centrifugation, so that the animal tissue-containing gel particles can be easily separated from the liquid paraffin.

On the other hand, when the gel is formed into a fiber, the medium solution of the high molecular compound is continuously extruded from the above-described microdispenser into a medium heated above its sol-gel transition temperature. Can be implemented.

When the gel is formed into a film or a sheet, the medium solution of the high molecular compound is used as the sol.
It can be carried out by spreading on a flat support heated to a temperature higher than the gel transition temperature and gelling.

As described above, examples of the method of imparting a form to a gel in which animal tissues are mixed and dispersed are not limited thereto.

On the other hand, the simplest and easiest method of separating the animal tissue cultured in the gel from the gel in order to recover or subculture it is to lower the temperature below the sol-gel transition temperature of the gel. In this manner, the gel is brought into a sol state to prepare an animal tissue suspension. According to this method, the cultured animal tissue can be separated from the gel without any damage by centrifugation or the like.

[0047]

The present invention will be described more specifically with reference to the following examples. The scope of the present invention is limited by the claims, and is not limited by the following examples.

Example 1 Hereinafter, a method for synthesizing and sterilizing the F-127 polymer used in this example will be described. (F-127 polymer synthesis method) 10 g of Pluronic F-127 (manufactured by Asahi Denka Kogyo KK) which is a block copolymer of polypropylene oxide and polyethylene oxide
Was dissolved in 30 mL of dry chloroform, 0.13 g of hexamethylene diisocyanate was added in the presence of phosphorus pentoxide, and the mixture was reacted at the boiling point of reflux for 6 hours. After evaporating the solvent under reduced pressure, the residue was dissolved in distilled water, and subjected to ultrafiltration dialysis using an ultrafiltration membrane having a molecular weight cut off of 100,000 to recover an aqueous solution of a high molecular weight polymer alone. The obtained aqueous solution is freeze-dried,
An F-127 polymer was obtained.

The above F-127 polymer was cooled under ice-cooling for 10 watts.
It was dissolved in distilled water at a concentration of t%. When this aqueous solution was gradually heated, the viscosity gradually increased from 21 ° C., and solidified at about 27 ° C. to form a hydrogel. When the hydrogel was cooled, it returned to an aqueous solution at 21 ° C. This change was repeatedly observed reversibly.

(F-127 Polymer Sterilization Method) In a 100 mL medium bottle, 5 g of the F-127 polymer obtained above was placed, and dissolved in about 40 mL of acetone (special grade).
An injection needle was placed on a silicone stopper, and a cannula fitted with a 0.2 μm filter (Myshoridisk H-13-5, manufactured by Tosoh Corporation) was used as the stopper. The stopper was put on the above-mentioned medium bottle and put into a bell jar, and the inside of the bell jar was depressurized with an aspirator to remove acetone. In order to completely remove acetone, the inside of the bell jar was further reduced in pressure by a vacuum pump. It was separately confirmed that the F-127 polymer thus treated was completely sterilized.

(Cell culture method) 5 g of sterilized F-127
The polymer was dissolved in 50 mL of RPMI 1640 medium (manufactured by Nissui Pharmaceutical, containing 10% fetal bovine serum) cooled to 4 ° C.
10% (w / v) F-127 polymer / RPMI 164
0 solution was prepared.

On the other hand, cholangiocarcinoma cells (MEC) used in the experiment
Was previously treated with a trypsin / EDTA solution, collected from the culture flask, centrifuged (220 × G, 5 minutes), and the supernatant was removed.

When the cell concentration is 2 × 10^Five cells / mL
Thus, the cholangiocarcinoma cells were converted to the 10% F-127 polymer /
Suspended in RPMI 1640 solution, 1 mL of this was
Diameter φ 35mm dish (Falcon 3001, Nippon Beck
Tons), and immediately incubate in a 37 ° C incubator (5
% Carbon dioxide / 95% air) for 10 minutes.
I did it. Preheat this dish to 37 ° C
Plate (micro warm plate, Kitasato Supply
And the solution dispensed is completely gelled.
After confirming, RPMI 1 which has been pre-warmed to 37 ° C
Add 1 mL of 640 medium to the top of the gel,
Cubbed.

In order to confirm the characteristics of the animal cell culture gel of the present invention, the following two experiments were performed.

(Cell recovery experiment) 1 hour after starting incubation
After a while, take out the dish from the incubator,
The gel in the dish is formed without dissolving in the added medium.
Shape. Prepared to recover cells from gel
Transfer the dish to the ice cubes
Was. Upon cooling for about 5 minutes, the gel turned into a sol. This sol
Collect the solution in a tube, centrifuge as above, and discard the supernatant.
Was removed, and the cells were suspended in 2 mL of fresh medium. This suspension
Sample 100 μL of the suspension and trypump
-Add 100μL (manufactured by GIBCO) and use this as a sample
Then, count the number of living cells with a TATAI hemocytometer,
The cell recovery was determined by the following equation.Cell recovery using this gel The rate was 100%.

(Experiment for Evaluating Environment in Gel) Cells were cultured for 10 days according to the culture method described above. The culture medium was exchanged by exchanging 1 mL of the medium added onto the gel three times a week. At this time, fresh medium was added after warming to 37 ° C.

During this time, the morphology of the cells in the gel was observed under a phase-contrast microscope (Nikon). Micrographs at this time (magnification: 117 times) are shown in FIGS. 1 to 4 (FIG. 1: culture start 2).
(Time, FIG. 2: 2 days after the start of culture, FIG. 3: 7 days after the start of culture, FIG. 4: 10 days after the start of culture). As is clear from the photograph, it was found that the cells grew in the gel, formed colonies, and the colonies grew larger.

In this experiment, by cooling the gel, the formed colonies could be collected as they were, and the colonies did not fall apart.

To confirm the cell growth in more detail,
The cells were collected by the collection method described above, and the number of viable cells was counted. However, in the method described above, since cells are collected as colonies, trypsin / EDT is used in accordance with a conventional method.
The colonies were separated by treating with the solution A, and then counted. The cell growth rate was determined by the following formula. The results are shown in the graph (dotted line) in FIG. The cells proliferated satisfactorily and were found to be about 400% by day 10.

From the above results, 1) Once the F-127 polymer medium solution is gelled, it does not dissolve in the newly added medium at 37 ° C. in the culture at 37 ° C. 2) Cells can grow in the gel. 3) The gel is not toxic to cells. 4) By cooling the gel, the gel is changed to a sol, and 100% of the cultured cells can be recovered. 5)
It was found that colonies formed in the gel could be recovered intact as colonies. Therefore, the gel of the present invention was found to be suitable as an animal cell culture gel.

Example 2 The same gel as that used in Example 1 was used, and a bovine dermal pepsin solubilized type I collagen solution (KOKEN CELL) was used.
GEN I-PC. (Manufactured by Koken Co., Ltd.) was used for the experiment. Final concentration 10% (w / v) F-127 polymer / 0.5%
(W / v) collagen / RPMI 1640 solution. Since the solution could be mixed at 4 ° C., there was no fear of denaturation of the collagen. The cells were cultured in the same manner as in Example 1 except that the above-mentioned F-127 polymer / collagen medium solution was used instead of the F-127 polymer medium solution of Example 1. .

(Cell Recovery Experiment) When carried out in the same manner as in Example 1, the same results as in Example 1 were obtained.

(Experiment for Evaluating Environment in Gel) Using the same cells as in Example 1, an experiment was conducted in the same manner as in Example 1. FIGS. 6 to 9 show micrographs (magnification: 117 times) of the morphology of the cells in the gel (FIG. 6: 2 hours after the start of culture, FIG. 7: 2 days after the start of culture, FIG. 8: 7 days after the start of culture, FIG. 9: 10 days after the start of culture). As is clear from the photograph, it was found that the cells grew in the gel, formed colonies, and the colonies grew larger.

FIG. 5 is a graph (solid line) showing the cell growth rate. The cells were found to be about 500% on day 10 of culture. Therefore, it was found that collagen could be added to this gel for culturing cells. In addition, since mixing can be performed at a low temperature, there is no fear of denaturation of the protein.

Comparative Example 1 Agarose conventionally used for cell culture was used as a comparative example. It is known that an agarose aqueous solution gels at 35 ° C. or lower, and must be heated to 80 ° C. or higher to melt the gel.

An aqueous solution of 6% (w / v) agarose (type IV, manufactured by Sigma) was sterilized in an autoclave and immediately cooled in a 40 ° C. warm bath. On the other hand, a double concentration medium (RP
MI1640) was warmed to 40 ° C. and mixed with an agarose solution to prepare a 3% agarose / RPMI 1640 medium. Cells were immediately suspended and cultured in the same manner as in Example 1 except that the agarose medium solution obtained as described above was used instead of the F-127 polymer medium solution of Example 1.
The same cells as in Example 1 were used, and the culturing operation was performed in the same manner as in Example 1.

(Cell Recovery Experiment) The gel was heated to about 85 ° C. to melt the gel, and an attempt was made to recover the cells. However, all the recovered cells were dead. Further, the gel was broken using a homogenizer to recover the cells, but the gel and the cells could not be separated cleanly. Therefore, it was impossible to recover cells from this gel.

Comparative Example 2 Collagen was used as a comparative example. Collagen solution is p
It is known that gelation occurs at 37 ° C with H7,
It is known that enzymes must be used to break down the gel.

The collagen solution used in Example 2 was used. The collagen solution was diluted with a medium having a concentration twice as high as 4 ° C. and a medium having a normal concentration, and 0.2% collagen / RPMI 16
Forty solutions were prepared. The cells were suspended at a low temperature in the same manner as in Example 1, except that the collagen medium solution obtained above was used instead of the F-127 polymer medium solution of Example 1,
Cultured. The cells and operation were the same as in Example 1.

(Cell recovery experiment) The gel was treated with 0.2% collagenase (manufactured by Wako Pure Chemical Industries) / Hanks solution (manufactured by GIBCO, containing 1% serum) for about 10 minutes. Cell recovery is about 9
It was 0%.

After culturing in the gel for 10 days, a cell recovery experiment was attempted in the same manner as in Example 1. After 10 days of culture, the cells had formed colonies. When an attempt was made to collect the colonies by the operation described above, more than half of the colonies were broken up by collagenase. Therefore, the formed colonies could not be efficiently collected.

[0072]

As described above, according to the present invention, embedding of animal tissue in a gel and separation of animal tissue from the gel can be easily carried out by a temperature change within a physiological temperature range. As a result, it is possible to embed and / or separate from the gel without damaging the animal tissue at all.

[Brief description of the drawings]

FIG. 1 is a micrograph of cells in a gel 2 hours after the start of culture (Example 1).

FIG. 2 is a micrograph of cells in a gel two days after the start of culture (Example 1).

FIG. 3 is a micrograph of cells in a gel 7 days after the start of culture (Example 1).

FIG. 4 is a micrograph of cells in a gel 10 days after the start of culture (Example 1).

FIG. 5 is a graph showing the growth rate of cells in Examples 1 and 2. In this graph, the dotted line is 10% F
The case using the -127 polymer medium solution (Example 1) is shown, and the solid line shows the case using the 10% F-127 polymer + 0.05% collagen medium solution (Example 2).

FIG. 6 is a micrograph of cells in a gel 2 hours after the start of culture (Example 2).

FIG. 7 is a micrograph of cells in a gel two days after the start of culture (Example 2).

FIG. 8 is a micrograph of cells in a gel 7 days after the start of culture (Example 2).

FIG. 9 is a micrograph of cells in a gel 10 days after the start of culture (Example 2).

Claims (8)

(57) [Claims] 1. A high molecular compound having a hydrophobic part and a hydrophilic part in a molecule and having a sol-gel transition temperature,
A carrier for animal tissue culture, which reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature. 2. The carrier for animal tissue culture according to claim 1, wherein the sol-gel transition temperature is higher than 0 ° C. and 60 ° C. or lower. 3. At a temperature higher than the sol-gel transition temperature.
Once the carrier has gelled, it will react with the newly added water at that temperature.
3. The animal according to claim 1 or 2, which is substantially insoluble.
Tissue culture carrier. 4. The method according to claim 1, wherein the polymer compound has a plurality of
4. The method according to claim 1, which has a hydrophobic portion.
Carrier for animal tissue culture. 5. A high molecular compound having a hydrophobic part and a hydrophilic part in a molecule and having a sol-gel transition temperature,
An animal tissue culture composition comprising a carrier that reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature and a physiologically active substance for animal tissue culture. 6. A polymer compound having a hydrophobic part and a hydrophilic part in a molecule and having a sol-gel transition temperature,
A carrier which reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier; A method for culturing an animal tissue, comprising mixing the tissue, setting the mixture in a gel state at a temperature higher than the sol-gel transition temperature, and culturing the animal tissue. 7. A high molecular compound having a hydrophobic part and a hydrophilic part in a molecule and having a sol-gel transition temperature,
A carrier that reversibly exhibits a liquid state (sol state) at a temperature lower than the sol-gel transition temperature is mixed with a predetermined medium at a temperature lower than the sol-gel transition temperature of the carrier; And a physiologically active substance for animal tissue culture, wherein the mixture is in a gel state at a temperature higher than the sol-gel transition temperature to culture the animal tissue. 8. The gel containing the cultured animal tissue,
The method for culturing animal tissue according to claim 6 or 7 , wherein the animal tissue is collected and / or subcultured in a liquid state (sol state) at a temperature lower than the sol-gel transition temperature.