JPH03266980A - Base material for cell culture and production of cell aggregate using same - Google Patents

Abstract

PURPOSE: To provide a method dispensing with troublesome operations danger of pollution, capable of producing a cell aggregate manifesting excellent cell function by using a temperature sensitive polymeric compound having LCST lower than the culturing temperature.

CONSTITUTION: The substrate layer comprises the temperature sensitive polymeric compounds such as poly-N-substituted acrylamides having LCST lower than the culturing temperature by coating or graft polymerizing in a conventional way, on the previously prepared substrate of the desired shape such as membrane, sheet, etc. The culturing base is produced by sequentially coating the cell adhesion proliferator layers such as collagen, etc. The cell aggregate is obtained by culturing several cells on the culturing base at the temperature above LSCT to produce the cell aggregate, lowering the temperature lower than the LCST to eliminate the cell aggregates from the surface of the base. This process dissolves the troubles such as functional disorder caused by an eliminator, makes the cell aggregate capable of using as the alternatives of the in vivo tissue.

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a substrate suitable for cell culture. More specifically, the substrate is suitable for culturing cells that require subculturing in order to produce cell products.

Another aspect of the invention relates to a method of manufacturing a cell culture carrier.

The present invention relates to a method for producing cell aggregates such as cell sheets, cell clusters, and cell tubes.

Furthermore, the present invention relates to a cell aggregate suitable as a living tissue substitute for prosthesis of a defective part of living tissue, particularly cell tissue. It also relates to cell aggregates suitable for screening drug toxicity and pharmacological activity.

Today's cell culture technology is used for 1) production of cell products, 2) prosthetic materials for lesions and defects in living organisms, and 3) simulators for evaluating drug toxicity and pharmacological activity. has been done.

Below, we will provide an overview of the prior art, focusing on the above application fields.

Animal cells used in cell culture today are classified into two types. That is, adhesion-independent cells (anchorag)
e 1nd dependent cells) and adhesion dependent cells (anchorage dependent cells).
cells). The former adhesion-independent cells survive;
These are cells in which cell functions such as proliferation and substance production ability are normally expressed even in the absence of a matrix that serves as a cell scaffold. Typical examples include hybridomas formed from myeloma cells, lymphoma cells, etc.

On the other hand, the latter adhesion-dependent cells are cells in which cell functions such as survival, proliferation, and substance production ability cannot be expressed normally in the absence of a matrix, which is a cell scaffold.

Most normal diploid cells, including primary cultured cells, are adhesion dependent. Furthermore, many established cell lines that can proliferate indefinitely are known to exhibit adhesion dependence. For example, they produce useful cell products such as interferons, interleukins, various differentiation growth hormones such as erythroboedin, colony stabilizing factor, thromboboetin, and tissue plasminogen activator vaccines. Many established cell lines are also known to exhibit adhesion dependence. Therefore, the establishment of adhesion-dependent cell culture techniques is very important for the production of these useful cell products.

Generally, when cells are used for substance production, it is important to culture the cells in large quantities and at high density while maintaining high functionality. However, compared to microbial cells, animal cells are more susceptible to the accumulation of waste products and insufficient supply of nutrients such as oxygen, making it extremely difficult to maintain their functions during large-scale, high-density culture. .

In the case of adhesion-independent cells, suspension culture methods are considered to be the most appropriate. If the suspension culture method is performed under stirring,
This is because it is possible to quickly remove cellular waste products and efficiently supply nutrients, and it is easy to scale up the device for the purpose of mass production and high density production. However, in the case of adhesion-dependent cells, the suspension culture method cannot be applied because a substrate is required as a scaffold for the cells. Therefore, various culture vessels for adhesion-dependent cells have been developed. For example, when culturing small amounts of cells in the laboratory,
In general, cell culture vessels such as dish-type, flask-type, and plate-type vessels are widely used, but these vessels are unsuitable for culturing cells in large quantities and efficiently. For this reason, the following measures have been taken to make the area of the substrate to which cells adhere larger relative to the total volume suitable for mass culture.

■Roller bottle method, in which glass pins for cell culture are rotated to grow cells on the entire wall surface; ■Multi-tray method, in which plates for cell adhesion are arranged parallel to the culture solution and the culture solution is circulated between them; ■Plastic film Coil culture method, in which a spiralized material is inserted into a cylinder and rotated horizontally to allow cells to adhere, and then the culture solution is circulated between the films. Hollow fiber culture method that supplies nutrients and removes waste from the hollow fiber membrane by attaching cells to the outer surface and circulating the culture medium inside the hollow fiber: ■Cells are attached to the filled glass piece and the gaps between them are Filled glass beads method in which the culture solution is circulated: ■ This is a pseudo-suspension culture method (microbead culture method) in which microbeads are suspended in the culture solution, cells are attached to the surface of the microbeads, and cultured under stirring.

As mentioned above, in order to efficiently produce large quantities of useful cell products by culturing cells in large quantities and at high density,
Developments have traditionally focused on the shape of culture devices and culture substrates. However, in recent years, it has been found that the properties of the culture substrate are significantly related to the activity or function maintenance and expression of adhesion-dependent cells. It has been found that it is almost impossible to culture cells while maintaining their activity and specific functions for a long period of time only by efficiently supplying nutrients or efficiently removing cellular waste products. Therefore, in recent years, attempts have been made to improve cell activity and functional maintenance by variously changing the properties of culture substrates. Currently, polytythrene is widely used as a material for cell culture containers due to its optical transparency, non-toxicity, good mechanical properties and moldability, and low cost.

However, the surface of boritithrene has strong hydrophobicity and has a serious drawback in that cell adhesion is significantly inhibited in order to maintain cell activity. Therefore, a cell culture substrate has been developed and widely used in which cell adhesion and proliferation are improved by subjecting the polytythrene surface to a corona discharge treatment to introduce anionic groups to the surface and imparting hydrophilic properties. However, it has become clear that it is difficult to express and maintain the specific functions of cells with this level of improvement. Research has been conducted to improve functions such as cell adhesion, proliferation, differentiation, and substance production ability.

That is, it is a method of combining an extracellular matrix with a cellular substrate.

By the way, research on the function of extracellular matrices in vivo has progressed rapidly in recent years, and it is now possible to play not only the conventionally known simple passive role of supporting and immobilizing cells, but also active role of supporting and immobilizing cells. It has now been discovered that it also has a control function.

For example, it has been discovered that there are more than 10 types of collagen, which is the main component of the extracellular matrix, and each type of collagen is synthesized by specific cells, localized in specific tissues, and controls different cellular functions. It is becoming clear that this role plays a role. Furthermore, it has been found that even within the same type of collagen, cell functions are affected by modifications such as modification of the higher-order structure or introduction of various functional groups.

In addition, the second components of the extracellular matrix, such as fibronectin, laminin, vitronectin, proteoglycans, and glycosaminoglycans, have specific binding sites for collagen and cell membranes, and are used for adhesion of cells to the matrix. plays an important role. Note that each adhesive protein specifically binds to a certain type of cell or collagen.

For example, fibronectin primarily binds to fibroblasts and type I and type II collagen, and laminin has unique binding sites to epithelial cells, endothelial cells, and type II collagen, respectively. Furthermore, in addition to the extracellular matrix mentioned above, there are other substances that have a large effect on cell functions, such as gelatin, which is a heat-denatured product of collagen, lectin, which specifically binds to sugar chains on cell membranes, and adhesive properties, which are binding sites for fibronectin, etc. Oligopeptides, cell adhesion proteins obtained from mussels, etc. are known. An example of a culture substrate in which the factors that control cell adhesion and proliferation described above are combined with a culture substrate is a culture substrate coated with collagen (K, Yosl+1zato, et al., Anna
ls of P Iastic Surgery, 13
．． 9.1984), and also fibronectin-coated substrates (F, Grinnell, Expl.

Ce1l Res, Su2, 51, 1976), and a substrate coated with cell adhesion proteins obtained from mussels (P, TP Icciano, et al., Dev.
lop+'s ental B 1oloHy 22゜24
．． (1986) and others have been developed, and improvements in cell adhesion and proliferation effects have been recognized. Furthermore, instead of the extracellular matrix derived from living organisms, a substrate coated with polystyrene having galactose terminals in the side chains (Toshihiro Akaike et al., Artificial organs,
17, 227, 1988) was developed and has been shown to particularly improve adhesion and maintain viability of hepatocytes. Conventionally,
It has become possible to culture cells, which were extremely difficult to adhere to due to harsh culture conditions, by using culture substrates whose surfaces have been treated in the manner described above. As mentioned above,
Development of cell culture methods that enable efficient replenishment of nutrients and efficient removal of waste products, and development of culture substrates that incorporate cell adhesion and growth factors to maintain specific cell functions. Despite progress in development, current technology has significant problems as described below.

An important characteristic of adhesion-dependent cells is that once the cells adhere to the culture substrate, proliferate, and completely cover the substrate surface, a function called contact inhibition is activated, stopping further cell proliferation. It is a nature. Therefore, in order to further proliferate cells, it is necessary to transplant the cells to a new culture substrate, which is called passage.

Conventionally, proteolytic enzymes such as trypsin and collagenase, and EDTA, which is a complex compound of calcium ions, have been generally used for passaging operations to detach cells that have adhered to the substrate surface and grown. These cell detachment agents cause serious disturbances in cell function and cell culture processes as described below.

1) Conventional cell detachment agents not only destroy the bond between the culture substrate and cells, but also destroy all bonds between cells. Tight junctions are the connections between cells.
It is known that there are three types of bonding modes, called gap junctions and desmosomes, and when cells gather together to form tight junctions, they act as a barrier against the permeation of substances as a cell aggregate, and act as a barrier to the penetration of substances inside the living body. A positive environment is maintained, and when gap junctions are formed, information and substances are exchanged between adjacent cells through these connections.

On the other hand, desmosomes mechanically hold the cell collection itself so that tight junction and gap junction connections are maintained. Therefore, it is difficult for a cell to express its activities and functions by itself, and it is thought that functions are expressed only when a society is formed by the above-mentioned intercellular connections (BA 1berts, et al, “Mo
1ecular Biology of tbeCe
l l”, 3rd edn, Garland P
ubl 1sbiB, Inc.

New York & London, P. 673
．． 1983). Therefore, conventional cell detachment agents cause fatal damage during passage even to cells that have been cultured in a highly functional state.

2) Receptors for various signal substances such as hormones and neurotransmitters are present on cell membranes, and cells are remotely controlled by the specific reactions of these signal substances and receptors. Conventional cell detachment agents such as proteolytic enzymes have been shown to destroy these cell membrane receptors (C, Sung, et al., B
1ocl+emP barmocol, 38,6
96°1989). Therefore, it is almost impossible to control the functions of cells passaged by conventional techniques using signal substances having specific physiological activities.

3) Serum is generally added as a nutrient to cell culture media, and since serum contains a strong trypsin inhibitor, cells must be thoroughly washed with a buffer before passaging before trypsin treatment to inhibit trypsin. It is necessary to remove the substance.

This washing step not only complicates the operation but also becomes a serious source of contamination in several aspects for cell culture. As mentioned above, despite advances in culture substrates that use extracellular matrices and other factors that promote the expression of cell adhesion, proliferation, and differentiation, conventional methods of collecting and subculturing cells are difficult to achieve. If a cell detachment agent is used, the high functionality of the cells that has been maintained will be significantly impaired due to the reasons (1) and (2) above. This problem is a major obstacle to the technology for culturing cells that maintain high functionality.

On the other hand, conventional cell detachment procedures also have fatal drawbacks in large-scale culture techniques. That is, it is said that if the initial cell concentration of adhesion-dependent cells in the culture solution is too low, the cells will not be able to sufficiently exhibit their proliferation and substance production functions even if they adhere to the substrate. In cases where primary culture cells or normal diploid cells are particularly difficult to collect, culturing them in a large volume of culture medium from the beginning will result in too low a cell concentration, so it is necessary to use a small-capacity culture device to carry out the step-by-step process. Since cells must be passaged repeatedly, the capacity must be increased. That is, when culturing in large quantities, it is usually necessary to perform cell collection and passage several times. Therefore, for reason (3), conventional cell detachment methods lead to complicated operations and equipment, and a great risk of contamination, which are fatal flaws especially when culturing in large quantities.

On the other hand, apart from its use in the production of cell products, there are other uses of cell culture technology, such as 1) prosthetic materials for lesions or defects in living organisms, and 2) toxicity and pharmacological activity of drugs. Although this is a simulator for evaluation, the conventional method of collecting cells using cell detachment agents has fatal flaws in these methods as well.

The most important characteristics when using cultured cells as a prosthetic material are a size that fits the lesion and excellent self-retention properties. However, in the conventional cultured cell recovery method using a cell detachment agent, all bonds between cells are broken and the cells become scattered, so excellent self-retention properties could not be expected at all.

On the other hand, when using cultured cells as a simulator for evaluating drug activity, the most important characteristic is the drug sensitivity of the cells. As mentioned above, conventional cell detachment agents cause fatal damage to various glue receptors on the cell membrane surface, making it impossible to develop a highly sensitive simulator for drug evaluation using conventional methods. Ta.

As outlined above, conventional cell collection and passage methods using trypsin and the like have serious drawbacks that significantly impair the effective use of today's cell culture techniques.

As mentioned above, the purpose of the present invention is to solve the problem of deterioration of cell function, which is a problem during conventional cell recovery and passage in cell culture technology.
The object of the present invention is to provide a culture substrate that solves problems such as complexity of operation and risk of contamination, and a method for producing the same. Another object of the present invention is to provide a cell aggregate that uses the culture substrate to maintain good cell function and has excellent self-supporting properties, and a method for producing the same.

The purpose of the above is to develop a novel cell culture substrate, its manufacturing method,
This has been achieved by the present invention, which provides a method for producing a cell aggregate using the cell culture substrate described above, and a cell aggregate produced by the method.

The cell culture substrate of the present invention has L CS lower than the culture temperature.
It is characterized by being made of a temperature-sensitive polymer compound having T. Here, L CST (LowerCri
tical 5solution Temperat
ure) refers to the transition temperature between hydration and dehydration of a temperature-sensitive polymerized material.

The substrate of the present invention is made of a temperature-sensitive polymerized material having an LCST lower than the culture temperature, and its shape, structure, and other constituent elements are particularly limited as long as it achieves the object of the present invention. Not done. Therefore, the substrate of the present invention may consist only of a polymerized material sensitive to a temperature lower than the culture temperature, or may consist of the polymer compound and a support. In addition, there are also facilities that contain other materials such as cell adhesion and growth factors, which will be described later. These materials and the polymer compound may be mixed or may form separate layers or parts. Furthermore, the attachment to the support may be by commonly used means such as coating or graft polymerization. Further, the substrate of the present invention can be in various shapes, such as a film, a plate, a particle, a fiber, a flake, a sponge, or a microbead.

The temperature-sensitive polymer compound used in the substrate of the present invention, which has an LCST lower than the culture temperature, is in a solid state at the cell culture temperature, and becomes soluble to the point where it dissolves in water by lowering the temperature below the LCST. It has characteristics.

Therefore, in the case of a base material made only of a temperature-sensitive polymer compound, the entire solid base material dissolves in water by lowering the temperature below the LCST. Furthermore, when combined with a support or the like, the portion containing the polymer compound changes from hydrophobic to hydrophilic. Furthermore, the base material made of a crosslinked polymer compound changes from a contracted state to an expanded state by lowering the temperature. All changes in the polymer compounds in these base materials are reversible.

Changes in the state of temperature-sensitive polymer compounds are said to be due to hydration and dehydration. Regarding this, see Haskin
S, M., et al., J. Macromol.
, Sci.

Chem, , A2(8), 1441.1968 gives an explanation using poly-N-isopropylacrylamide (PNIPAA+n), which is one of the polymer compounds, as an example. PNIPAA+n is a polymer compound with a negative solubility temperature coefficient in water. At low temperatures, a hydrate (oxonium hydroxide) that depends on hydrogen bonds between PNIPAAm molecules and water molecules is generated. However, this decomposes and dehydrates by increasing the temperature above the LCST, resulting in P
It is believed that NIP AAm molecules aggregate and precipitate.

Since the polymer compound of the base material of the present invention is in a solid state at the cell culture temperature, cells can proliferate without using the polymer compound as a scaffold. Then, when it is necessary to detach the proliferated cells, if the temperature is lowered to below the LCST, the polymer compound will be in a soluble state, so it can be easily detached without damaging the bonds between cells. can.

As described above, according to the present invention using a temperature-sensitive polymer compound, proliferating cells can be easily detached without going through a detachment step using a detachment agent or a preparation step for the detachment step.

Temperature-sensitive polymer compounds that can be used in the base material of the present invention include polyN-substituted acrylamide derivatives, polyN-substituted methacrylamide derivatives, and copolymers thereof, polyvinyl methyl ether, polyethylene oxide, etherified methyl cellulose, Examples include polyvinyl alcohol partial oxide. Particularly preferred is
They are polyN-substituted acrylamide derivatives, polyN-substituted methacrylamide derivatives, or copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partial acetate.

Preferred polymer compounds are listed below in descending order of LCST.

Poly-N-acryloylpiperidine/poly-N-n-propylmethacrylamide: Poly-N
-isopropylacrylamide; poly-N,N-diethylacrylamide/poly-N-isopropylmethacrylamide; poly-N-cyclopropylacrylamide;
Poly-N-acryloylpyrrolidine; Poly-N,N-ethylmethylacrylamide; Poly-N
-Cyclopropylmethacrylamide Poly-N-ethylacrylamide The above polymers may be used alone or may be copolymerized with other monomers. As the monomer to be copolymerized, either a hydrophilic monomer or a hydrophobic monomer can be used. Generally, when copolymerized with a hydrophilic monomer, the LCST increases, and when copolymerized with a hydrophobic monomer, the LCST decreases. Therefore, by selecting these, a polymer compound having a desired LCST can also be obtained.

Hydrophilic monomers include N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-
Methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate,
Acrylic acid, methacrylic acid and their salts, vinyl sulfonic acid, stylsulfonic acid, etc., which have an acidic group, and N,N-dimethylamine ethyl methacrylate, N,N-diethylaminoethyl methacrylate, N,N, which have a basic group. Examples include, but are not limited to, -dimethylamine propylacrylamide and salts thereof.

Since cell membranes are usually negatively charged, it is preferable to use a copolymer with a basic monomer in order to improve the adhesion of cells to a substrate through electrostatic interaction.

On the other hand, as hydrophobic monomers, acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate, glycidyl methacrylate, N-n
Examples include, but are not limited to, N-substituted alkylmethacrylamide derivatives such as -butylmethacrylamide, vinyl chloride, acrylonitrile, styrene, and vinyl acetate.

On the other hand, cell adhesion and
A growth factor is a substance that, when combined with a temperature-sensitive polymer 6, has the role of increasing cell adhesion and at the same time promoting cell proliferation. The most typical factor having such a function is an extracellular matrix, and various types of collagen, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, etc., which are the main components of the matrix, are preferable. In addition to the extracellular matrix, it also corresponds to gelatin, which is a heat-denatured collagen, lectins such as concanavalin A (ConA), which has an affinity for sugar chains on cell membranes, adhesive proteins derived from mussels, and the binding part between fibronectin and cells. Adhesive oligopeptides, thrombosbondin, etc. can be used.

The shape of the base material of the present invention is not particularly limited, and as described above, it can be in various shapes such as a film, a plate, a particle, a fiber, a flake, a sponge, or microbeads. The substrate for mass culture is particularly preferably membrane-like, plate-like, particulate or fibrous. In the case of microbeads, the particle size is preferably 50 to 300 μ'n.

For molding into these shapes, a conventional molding method for polymer compounds can be used. For example, a method in which a polymer compound is dissolved in water or an organic solvent and molded into a film or plate shape by a solvent casting method, or a liquid polymer compound cooled below the LCST is heated to a temperature above the LCST using a die. By extruding into water or an organic solvent that is immiscible with water to form a film, particles, or fibers, or directly forming a polymer compound into particles by suspension polymerization or precipitation polymerization, the desired product can be formed. Can be molded into any shape. When making microbeads,
The polymer compound may be dropped into an organic solvent that is immiscible with water, molded into microbeads, and then insolubilized.

When manufacturing a base material whose surface is coated with a polymer compound, it is possible to prepare the support in the desired shape in advance and coat the surface of the support with the polymer compound using a conventional method. .

When graft polymerizing a polymer compound onto the support surface, in addition to selecting a polymer compound that itself has a desired LCST, as described above, the desired polymer compound can be obtained by using a copolymerization grafting method with various monomers. The LCST can be adjusted to . When carrying out the copolymerization grafting method, both hydrophilic monomers and hydrophobic monomers can be used. Generally, the LCST of a graft copolymer increases when copolymerized with a hydrophilic monomer, and the LCS increases when copolymerized with a hydrophobic monomer.
T goes down. The hydrophilic monomers and hydrophobic monomers that can be used are as described above.

As the support when a polymer compound is graft-polymerized on the surface, materials that have been conventionally used as carriers for cell culture are preferred. For example, glass, polystyrene, polycarbonate, polymethacrylate, polystyrene, polypropylene, polyethylene, polyester, polyamide,
Examples include, but are not limited to, polyvinylidene fluoride, polyoxymethylene, polyvinyl chloride, polyacrylonitrile, polytetrafluoroethylene, polydimethylsiloxane, cellulose-based polymers, cross-linked dextran, cross-linked polyacrylamide, collagen, etc. .

The graft polymerization method of temperature-sensitive monomers to the support depends on the material,
The most suitable method can be selected depending on the shape and other factors.

The low-temperature plasma polymerization method can be suitably used when the support is in the form of a plate, film, or flat membrane. This method allows craft polymerization only on the surface of the support, does not impair the bulk properties of the support, and is suitable for polymers that are relatively hard to generate radicals, such as polypropylene, polyethylene, polytetrafluoroethylene, and polydimethyl. This is because it can be easily graft-polymerized to polymers such as siloxane, polyester, polycarbonate, and polymethyl methacrylate. When the support is in the form of a hollow fiber membrane or microbeads, the ozone oxidation method or the cerium ion method is suitable.

This method is most suitable as a graft polymerization method for microbeads such as cellulose polymer hollow fiber membranes, crosslinked dextrin, crosslinked polyacrylamide, and collagen.

The shape of the carrier with a polymer compound grafted onto its surface also varies.
It can take various shapes depending on the cell culture method, material characteristics, etc. For example, 1) plate-like or film-like as typified by culture dishes, 2) hollow fiber membrane or flat membrane type, 3)
) microbeads, etc.

Methods for forming a crosslinked structure include a method of introducing a crosslinked structure when monomers are polymerized, and a method of introducing a crosslinked structure after polymerization, but either method can be adopted in the present invention. .

Specifically, the former method is carried out by copolymerizing difunctional monomers. For example, N,N-methylenebisacrylamide, hydroxyethyl dimethacrylate, divinylbenzene, etc. can be used.

In the latter method, crosslinks are generally formed between molecules by irradiation with light, electron beams, or gamma rays.

A culture substrate consisting of a temperature-sensitive polymer compound, cell adhesion, and growth factors is mainly produced by the following method.

For molding the culture substrate, a conventional molding method for polymer compounds can be used. For example, a method in which a cell adhesion/growth factor and the polymer compound are dissolved in water or an organic solvent and formed into a membrane or plate shape by solvent casting; A mixture of adhesion and growth factors was added to the LCSTC using a cap.
It can be molded into a desired shape by extruding it into water at a temperature of 10°C or into an organic solvent that is immiscible with water, thereby molding it into a membrane, particles, or fibers. When forming microbeads, the mixture may be dropped into an organic solvent that is immiscible with water, molded into microbeads, and then insolubilized.

When producing the culture substrate of the present invention in which the surface of a support is coated with the mixture, a support of a desired shape can be prepared in advance, and the surface of the support can be coated by a conventional method.

Furthermore, when manufacturing a substrate having a polymer compound layer and a cell adhesion/growth factor layer on a support, it can be manufactured by a method such as sequentially coating each layer.

Note that the formation of the substrate layer on the support is not limited to coating, and can be performed by various methods such as graft polymerization.

On the other hand, the cell aggregate of the present invention can be obtained by culturing various cells on a culture substrate made of the temperature-sensitive polymer compound described above at a temperature higher than the LCST to form a cell composition such as a colony or a cell sheet. It can be obtained by lowering the temperature below the LCST and desorbing from the surface of the substrate by utilizing changes in the properties of the temperature-sensitive polymer. By using the base material of the present invention, cell aggregates of various shapes such as cell sheets, cell clusters, and cell tubes can be manufactured.

As already mentioned, conventionally, a detachment agent such as a proteolytic enzyme such as trypsin has been used to detach cell colonies or sheets formed on a culture substrate. For this reason,
The intercellular bonds formed on the substrate during culture were decomposed and the cells became scattered, making it impossible to obtain a cell sheet with good self-supporting properties. Furthermore, conventional detachment agents damage various glue receptors on the cell membrane and the cell membrane itself, thereby significantly reducing the activity and function of the recovered cells. When the culture substrate of the present invention is used, a cultured cell sheet can be recovered by a simple operation of simply lowering the temperature without using a conventional detachment agent. This not only eliminates the drawbacks of the conventional desorption method mentioned above, but also allows the desorption operation to be performed in a closed system without the need for washing steps, trypsin addition steps, etc. as in the conventional method. The risk of contamination, which can be fatal to cell cultures, can be significantly reduced.

Furthermore, the most appropriate cell adhesion/growth factor can be selected depending on the cell type. By combining the above factors into a temperature-sensitive polymer compound, it is possible to significantly promote adhesion and proliferation of cells to the surface of a culture substrate.

By the above method, for example, sheet-like cell membranes can be formed.1.
Ultimately, it may be collected as clusters. Of course, it is also possible to carry out a detachment operation without fixing the fixed part of the cell sheet cultured on the substrate, and to collect the cells while holding them in a sheet form. In addition, the cell density of clusters formed by natural aggregation from cell sheets is 10'a
cells/ml, which is the highest cell density that can be achieved in two-dimensional culture (10 cells/ml [
) is 100 times. That is, although it is a simple calculation, it means that it is possible to produce the same amount of cell products as in the past with an apparatus 1/100 the scale of the conventional one.

Furthermore, since conventional cell clusters are formed by chance during cell culture, it is difficult to adjust their size.
It has been difficult to obtain large quantities of clusters with a certain diameter. However, according to the method of the present invention, the size of the clusters can be adjusted by adjusting the surface area of the medium. That is, if the surface area of the medium is changed,
This is because the size of the cell sheet produced by culture changes depending on the size of the medium. According to the method of the present invention, it is possible to produce cell clusters of a desired size, preferably from several μm to several 11 μm. It should be noted that according to the present invention, it is possible to produce large cell clusters with diameters of several mm, which could not be obtained conventionally, and such large clusters can be transplanted into defective parts of living tissue. Useful as a prosthetic material.

Furthermore, the cell clusters produced by the method of the present invention survive for a long period of time and have the ability to proliferate. That is,
After culturing the cell clusters for a long period of time under normal cell culture conditions in a non-adhesive culture dish, the cell clusters are transferred to a cell-adhesive culture dish and cultured under the above culture conditions. They attach to the bottom and begin to proliferate again. Such long-term survival of the cell clusters of the present invention is achieved by maintaining intercellular connections.

Furthermore, the clusters can be stably cultured without aggregation using normal cell culture conditions under stirring in a spinner flask as in the suspension culture method. This means that the clusters can be used to produce large quantities of cellular products.

Hikari 1F and Bacteria 4 The culture substrate of the present invention addresses the serious problems of cell recovery and passage operations, such as cell dysfunction caused by detachment agents such as trypsin and other cell detachment enzymes, and cleaning operations. This solves the complexity of the process and the risk of contamination due to the complexity of the process, and allows cells to be recovered and subcultured through a simple process while maintaining their high functionality, resulting in the production of many useful cell products. This makes it possible to manufacture

The cell aggregate of the present invention is produced without using a detachment agent such as a cell detachment enzyme such as trypsin to remove the cultured cells from the substrate, so that the cell aggregates of the present invention can be produced without using detachment agents such as trypsin or other enzymes for cell detachment. There are no adverse effects such as damage, and the connections between cells are maintained. Therefore, it has excellent self-supporting ability and cellular function.

The cell membrane itself of the present invention can be used as a living tissue substitute for prosthesis of a defective part of living tissue, particularly cell tissue. It has been chosen for its biocompatibility because it can be manufactured using cells from the transplant recipient. For example, when used for prosthesis of a defective part of skin tissue, epidermal cells proliferate quickly after transplantation, and the defective part does not form or shrink.

In addition, since the cell membrane receptor function is retained, when used as a simulator to screen the effects of drugs on cell tissues, it is possible to obtain results that closely reflect the effects when actually administered to living organisms. It is useful for drug development or pre-administration checks of drugs that require careful administration.

When cells are cultured by the method of the present invention, unlike conventional methods, no special desorption process or desorption agent is required for detachment from the medium. Detachment of cells from the medium can be performed simply by changing the temperature, making the operation extremely simple. In addition, detachment of cells with strong adhesion to the medium can be performed very well. Furthermore, it is easy to produce cell aggregates having desired areas ranging from small to large.

EXAMPLES The present invention will be described in more detail with reference to Examples below, but the scope of the present invention is determined by the claims and is not limited by the Examples below.

Dog 11yo”-: N-isopropylacrylamide monomer (NI PAAm, Easto+nan Ko
dak Co, ) 509 was dissolved in 500 mn of benzene and 2,2'-azobisisobutylnitrile (A I
B N) 0. 12 at 60°C using h as a polymerization initiator.
Polymerization was carried out under stirring in a nitrogen stream for an hour. The polymer was precipitated in benzene, so after decantation, the precipitate was dissolved in tetrahydrofuran (THF) and purified by precipitation using ethyl ether. The obtained poly-N isopropylacrylamide (P N I P AAm)
Various solutions (P N I P A Am concentration: I W
Table 1 shows the results of measuring /V$)NOL CST using the turbidity method.
Shown below.

It was found that PNIPA Am exhibited LCST in bovine serum and cell culture medium as well as in water.

5 6 Table 1. ．． LCST hot solution polymer concentration of various solutions of PNI PAAm: 5 W/V%) was prepared by turbidity method. ,, CST was measured and shown in Table 2. As the copolymerization ratio of n-BMA, a hydrophobic monomer, increases, L
It was observed that CST decreased.

Table 2. LCST (℃) of various polymers in various solutions 1
Takumi Uni: NIPAA m Monomer 501?
and n-butyl methacrylate (nBMA) monomer 3
．． 3g and A I B No. 2h as a polymerization initiator.
Dissolved in HF 500mff1, 50°C, 12 hours,
Polymerization was carried out under stirring in a nitrogen stream. The polymerization solution was concentrated twice using an evaporator, then subjected to precipitation purification using ethyl ether, and vacuum dried to obtain a flaky polymer (Cop, (NI PAAn+/BMA) -1>
I got it. Furthermore, n-
Butyl methacrylate 6.61? Cop using.

(N XPAA+n/BMA) Cop, (N I P A
A+n/BMA)-2 was obtained. Various m2 of each polymer: PNIP produced in Example-1 was mixed with Dulbecco's modified Eagle's medium (DMEM: GIBCO
A PNIPAAm test solution was prepared by dissolving it in 10% tallow serum (manufactured by Alumni Co., Ltd.) at a concentration of IW/V$. Also, Example-
The NIPAAm monomer used in 1 was added to the same medium as 1-/
A NIPAAm monomer test solution was prepared by dissolving it at a concentration of Vl. Moreover, additive-free DMEM itself was used as a control liquid. Next, human dermis-derived fibroblasts were dispersed in each of the test solutions described above at a concentration of about 1 x 105 cells (7 ml), and 2 mj! of each dispersion was dispersed. Plastic tissue culture tissue (Falcon, 35
mmφ).

Each of the dates was cultured in an air/15% carbon dioxide incubator at 25° C. for 1.3 days, and the degree of cell adhesion to the bottom of the dates and the degree of proliferation were observed using a phase microscope to determine the cytotoxicity of each substance. The results are shown in Table 3.

No cytotoxicity was observed.

Fire 1 salmon 2 points: Cop prepared in Example-2.

The cytotoxicity of (NI PAAm/BMA) 1 was evaluated using human dermis-derived fibroblast cells in exactly the same manner as in Example 3, and is shown in Table 4. However, the cell culture temperature is Cop,
(NIPAAm/BMA) 1 was set at 17° C. to dissolve in DMEH.

Table 4. Cop, (NIPAA+n/BMA) 1
Cytotoxicity ◎: Extremely good O: Good (NIP^^m/BM^)-
1 showed no cytotoxicity.

Support 1 Takumi Σ: PNIPAA++ produced in Example 1
Prepare a 0.5W/Vl aqueous solution of and sterilize it in an autoclave (
121°C for 20 minutes), the polymer was redissolved by cooling to 20°C. The polymer solution was added to a commercially available tissue culture plastic dish (Falcon,
After thoroughly wetting the bottom surface, the excess liquid was discarded and air-dried at room temperature in a clean bench to form a coating film of PNIP^^m on the bottom surface of the dish. All of the above coating operations were performed aseptically. Next, a cell dispersion was prepared using the human dermis-derived fibroblasts used in Example 3 and DMEM to a final concentration of approximately 2 x 10'' cells/mβ, and kept at 37°C. Approximately 2 cell suspensions were injected into the PNIP ring-coated dish kept at 37°C and cultured at 37°C in an air 15% carbon dioxide incubator for 7 days. After 7 days of culture, the cells were placed on the bottom of the dish Colony formation of fibroblasts was observed. By immersing the outside of the cell-proliferated dish in water cooled to 15°C, the cells adhered to the bottom of the dish, and the proliferated cells naturally detached from the bottom. It could be clearly observed under a phase contrast microscope.As a control experiment, P N
A cell proliferation experiment similar to the above was performed using a commercially available tissue culture plastic dish without IPA'Ar coating. No detachment was observed.

Shoji Fugon (: Cop prepared in Example-2, (NIP
^^m/BM^)-1 no 0.514/V$ aqueous solution was prepared, and Cop, (N I
A PAAn+/BMA1) coated dish was prepared. Next, using the human dermis-derived fibroblasts used in Example 5, cell culture was performed on the coated tissue using the same method, and the cell adhesion and proliferation rate was the same as on the PNIPAAm-coated tissue. Got a deitsch. When the outside of the cell-adhered dish was immersed in cold water at 10°C, a cell detachment phenomenon similar to that observed in Example-5 was observed. As a comparative example, a conventional cell detachment method was carried out as follows. Commercially available plastic tissue culture tissue (manufactured by Falcon, 35m+nφ)
Inside was a DMEM dispersion of human dermis-derived fibroblasts (HI
Cell concentration: approximately 2 x 105 cells/ml) 2 ml was added and cultured in the same manner as above. After 7 days of culture, the cells completely covered the bottom surface of the dates. In order to detach the cells attached to the bottom surface, the old medium in the dish was discarded, PBS2mZ was added, and the cell surface was washed. This operation was repeated twice. Next, discard the PBS and add trypsin/E.
DTA solution (0,051 Lipudin 0.53mM EDTA>
2J! After washing the cell surface with trypsin/E
D Discard the TA solution and add fresh trypsin/EDTA again.
Solution 2 [nl] was injected and the same procedure was carried out.

After discarding all but 0.51 of the trypsin/EDTA solution,
After incubation at 7°C for 10 minutes, the cells slowly detached from the bottom of the dates. When observed under a microscope, it was observed that the cells were detached one by one.

The above-described conventional cell recovery process using trypsin was significantly more complicated to operate than the temperature difference recovery process using a temperature-sensitive dish, and at the same time, the risk of bacterial contamination was significantly higher.

Fugon Gaijigo: A 0.511I/vz aqueous solution was prepared using PNIPAAm prepared in Example-1, and a 0.45μ
After filtration sterilization using an Innofilter, the polymer aqueous solution was mixed with a 0.5 W/Vl aqueous solution of maggot dermal pepsin-solubilized type I collagen (sterilized, manufactured by Kouken Co., Ltd.) until the final concentration was 0. .25111/V$P NIP
A mixed solution of Am and 0.25 W/V% collagen was prepared. The L CST of the mixed solution was about 32°C as measured by a turbidity change measurement method. The mixed solution was added to a commercially available tissue culture plastic dish x (Fal
(manufactured by Con, Inc., 35+11111φ) and air-dried aseptically at room temperature in a clean bench. By the above method, collagen and P N I P are added to the bottom of dates.
Dishes coated with a 1=1 mixture of AAm at various thicknesses were obtained.

The thickness of the coating layer was varied depending on the volume of the mixed solution injected into the dish. Next, the human dermis-derived fibroblasts used in Example-3 were suspended and dispersed in DMEM (cell concentration: approximately 2 x 105 cells/mf).
+nj! After incubating at 37°C, it was injected into a dish coated with a PNIPA A+n/collagen mixture pre-incubated at 37°C.
The cells were cultured for 5 days in an air/15% carbon dioxide incubator at °C. Table 5 shows the relationship between the proliferation of cells on the bottom surface of the datesch and the thickness of the coating layer. On the other hand, the detachment of the grown cells was determined by taking out the dish from the 37° C. incubator, leaving it at room temperature, and observing it under a microscope. Table 5 shows the relationship between the thickness of the coating layer and detachability. As can be seen from Table 5, cell proliferation was almost independent of the thickness of the coating layer and was good. On the other hand, the detachability of cells improved with the thickness of the coating layer and was saturated at about 0.7 μIl1 or more.

Table 5. Relationship between coating thickness and cell proliferation and cell detachment of collagen and PNIPAAm in the same condition ◎ 0.9 ◎ ◎ ◎: Very good ○: Good
Mixtures were prepared using different compositions of collagen and collagen. Table 6 shows the composition ratio of collagen and PNIPAA+n. Examples using mixed liquids with various compositions
A dish having a coating layer thickness of approximately 0.9μ+n was prepared in exactly the same manner as in Example 7.

Next, human dermis-derived fibroblasts were cultured in the same manner as in Example 7, and the relationships between cell proliferation, cell detachment, and the composition ratio of collagen and PNIPAAm are shown in Table 6. As shown in Table 6, the cell proliferation rate improved with the collagen composition ratio, and the cell detachment rate improved with PNIP.
The properties improved as the composition ratio of AA+n increased. Preferred composition ratio that satisfies both (collagen/P N
I P A A + n> is approximately 0, 1.71. , O
~about 2.071. , O.

Table 6. Cell proliferation and cell detachment when changing the composition ratio of collagen and PHIPAAm with a constant coating thickness (0.9μ+n) ◎: Very good ○: Good △; Slightly poor ×: Poor Marugame Takumi y2: Collagen produced in Example-8/
F' N I P A A +n coating dateish (collagen/P N I P A Il + composition ratio: 1.O/1.0. Coating thickness approximately 0.9 μm
) using bovine pulmonary artery-derived endothelial cells (CPAE, A
merican Type CultureCol
fection) was cultured. CPAE cells in DMEM
The mixture was suspended and dispersed in a medium to a concentration of approximately 2.0 x 105/+nff1 and kept at 37°C. Of that, 2m (l of the above collagen/PNIPAA+n kept warm at 37°C in advance)
15% air injected into coating plate at 37℃
The cells were cultured in a carbon dioxide incubator for 4 days. Within 4 days, the cells completely covered the bottom of the dateish. When the dish in which the cells had grown was left at room temperature from a 37°C incubator, the process of spontaneous detachment of the cells from the bottom of the dish could be observed under a microscope.

and 1 Takumi Nirisara: Collagen produced in Example-8/
P N I P A M coating date (
Collagen/PN I PAAm composition ratio: 2.0/1.
0. Human epidermal cells (including keratinocytes) were cultured using a coating thickness of approximately 0.9 μIn. Human epidermal cells isolated by trypsinization from skin sections thinly excised with a dermatome were dispersed in F-12 medium (hydrococcicin and adenine supplemented) at a concentration of approximately 3 x 10 cells/Ol. 2 ml of the cell dispersion was kept at 37°C, poured into the coated dish previously kept at 37°C, and cultured at 37°C in an air 15% carbon dioxide incubator for 7 days.

Before replacing the medium, it was stored at 37°C. Culture medium was used every 2 days. After culturing for 7 days, the cells proliferated and completely covered the bottom of the dish. By immersing the outside of the dish in 10°C cooling water, the cells naturally detached and a good sheet of epidermal cells was obtained. did it.

m2LL= A 0.5W/V$ aqueous solution of amorphous atactic polymethyl vinyl ether (Tokyo Kasei) was prepared, sterilized in an autoclave at 121°C for 20 minutes, and then redissolved by cooling to room temperature. did. The LCST of the solution was determined by turbidimetry to be approximately 35° C. in PBS solution. The final concentration of the polymer aqueous solution and the 0.51+1/V% collagen aqueous solution used in Example-7 was 0.2! J/Vl polymer and 0.25W
A mixed solution of /Vπ collagen was prepared. A dish having a coating layer with a thickness of about 1 μm was prepared using the mixed solution in exactly the same manner as used in Example-7. Next, the human dermis-derived fibroblasts used in Example 3 were suspended and dispersed in DMEM (cell concentration: approximately 2×10
'Cells/J) 2 +nf was kept at about 40°C,
The cells were injected into the coated dishes kept at about 40°C, and cultured for 5 days at 40°C in an air 15% carbon dioxide incubator. After 5 days, the dish whose bottom surface was covered with cells was removed from the 40° C. incubator and left to stand at room temperature, whereupon the cell sheet naturally detached from the bottom surface of the dish and floated in the culture medium.

Fruit 11-knee L3- PNIPA produced in Example-1
A 0.5H/V$ aqueous solution was prepared using Am and 0.45μ
After filtration sterilization using a No. 0 filter, the polymer aqueous solution and gelatin (Iwaki Glass Co., Ltd.) 0.05 dollar aqueous solution were mixed together, and 400 μl of the mixed solution was added to a commercially available tissue culture plastic dish (35 m + nφ). ) and air-dried aseptically at room temperature in a clean bench.By the above method, gelatin and PNIPΔ^ were injected onto the bottom of the dateshu.
A sheet of fibroblasts coated with the mixture was prepared, human dermis-derived fibroblasts were cultured in exactly the same manner as in Example 7, and the fibroblast sheet was coated on the bottom of the dates by lowering the temperature to 10°C. was able to be detached and recovered.

Tue 1 27 y: PNIP produced in Example-1 ^^
A 5% HIV aqueous solution was prepared using lfi and 0.45μ
It was sterilized by filtration using a filter. A solution was prepared by mixing the polymer aqueous solution and a fibronectin solution (derived from bovine plasma, Nitta Gelatin Co., Ltd., 0.5B/+1) at various ratios, and the solution at each mixing ratio was mixed with a commercially available datesch (35 mmφ). Inject it into the tank and clean it at room temperature.
,,,'),'Ii'h The mixture of nectin and PNIPAAm is about 0.9μ thick.
Example 8
The mixing ratio, cell proliferation and cell detachment were measured in exactly the same manner as in Table 7. As can be seen from Table 7, cell proliferation improved as the mixing ratio of fibronectin increased, but cell detachment was generally poorer than in the case of collagen.

Table 7. Cell proliferation and cell detachment when changing the composition ratio of fibronectin and PNIPAAm with a constant coating thickness (0.9 μm) ◎: Very good ○: Good △: Slightly poor X: Poor fruit 41 Example 221A Uni PNIP used in Example-13
^^ Infusion solution and Ce1l, an adhesive protein derived from mussels
-Tak” (Collaborative Inc., concentration 1nglvn1
．． 5% acetic acid solution) in various ratios, each mixed solution was poured into a 35+ om diameter plate and air-dried, and a mixture of Ce1l-Tak@ and PNIPAAm was coated with a thickness of about 0.9 μm in the composition ratio of seeds. The mixing ratio, cell proliferation and detachment were measured in exactly the same manner as in Example 13, and as shown in Table 8, the results were as follows for fibronectin (Table 7). ), but the cell proliferation was generally poor.

Table 8. Cell proliferation and cell removal when the composition ratio of Cell I-Tak" and PNIP^^ transport was varied with a constant coating thickness (0.9 μl 11) ◎: Very good ○: Good △: Slightly Defective X: Bad Fire” 1 Example 22 Y5-: PNI used in Example-13
PAAm and cancanavalin A, a type of lectin (
Aqueous solutions of ConA and Honen Oil Co., Ltd. were mixed at various ratios, each mixed solution was poured into a 35m+*φ datesh, air-dried, and ConA and PNIPAA were mixed at various mixing ratios.
Table 9 shows the mixing ratio, cell proliferation and detachment of a dish coated with a mixture of m to a thickness of about 0.9 μm.

As shown in Table 9, although cell adhesion was observed, almost no proliferation was observed.

* Adhesion was observed. ◎: Very good, almost no proliferation. O: Good not observed. Δ: Slightly poor ×: Bad husband 1 katsu 2 Ribo: 0, 5 W/V$P used in Example-7
Using NIPAA+n aqueous solution, commercially available dates (3
51φ) with a thickness of approximately 0.9 μn+ on the bottom surface.
Form a coating layer. Next, the fibronectin aqueous solution (concentration 0.1 B/ml) used in Example-13
Incubate 0μl at 37℃ in advance.
The solution was poured into a PNIPAA+n coated dish, coated uniformly on the bottom surface, and air-dried aseptically in a constant temperature bath at 37°C. By the above method, a PNIPAAn+ layer (thickness: about 0.9 μm) is first formed on the bottom of the datesh, and then a laminate coating layer is coated with a fibronectin coating layer (the coating density of fibronectin is about 5 μg/am2). Created. Using the coated dish, cell proliferation and detachment of proliferating cells were evaluated in exactly the same manner as in Example 7, and as a result, extremely good proliferation and detachment were observed.

Example 2217: Commercially available polyester film (
Toshishiku Co., Ltd.), approximately 100 μm thick, 5 cm x 5 cm) was inserted into the chamber of an internal electrode type plasma irradiation device (Samco International Co., Ltd.), and after exhausting the chamber to approximately 0.8 Torr, argon gas was introduced at a flow rate of 30
Introduced at mR/min, output 50 watts, frequency 13.
Immediately after plasma irradiation at 56 MHz for 15 seconds, a 5% aqueous solution of NIP A Am was introduced into the chamber, and the polyester film was immersed in the solution for 1 hour at room temperature.
Graft polymerization was carried out for 6 hours. Before introducing the monomer aqueous solution into the chamber, air dissolved in the aqueous solution was completely removed by sufficiently bubbling with argon. Films subjected to plasma polymerization, films subjected to plasma irradiation under exactly the same conditions without the introduction of the monomer, and films simply coated with P N I P A M by a normal solvent casting method were tested at 10°C and 40°C, respectively. The contact angle with respect to water at ℃ was measured and shown in Table 10. As shown in Table 10, no change in contact angle was observed at 10°C and 40°C in the case of untreated PET film and film exposed only to plasma irradiation, but in the case of PNIPAAm-coated film and plasma-grafted film. 10℃ and 40℃
Differences in contact angle were observed at 10°C and hydrophobicity at 40°C.

Example 1: Two swords: The plugged polyester film produced in Example 17 had a diameter of 34.5n+mφ.
A circular film was cut out, sterilized in an autoclave, and then inserted into a commercially available 35 m+oφ culture dish. Fibroblasts derived from human dermis were cultured using the dates in the same manner as in Example 7, and the proliferation of the cells was observed. The cell proliferation rate was almost the same as that of the coated date.

-19: NIP AA+*50+7. n-B
100 g of water in which 3.3 g of MA and 1.5 g of N,N'-methylenebisacrylamide were dissolved was mixed with 500 g of hexane.
0.13 g of ammonium persulfate and 0.13 g of tetramethylethylenediamine as polymerization initiators suspended in
2 at room temperature under a nitrogen stream with 300 μl of stirring using
Polymerization was performed for 4 hours to obtain crosslinked microbeads. Microbeads were washed with chilled water at 15°C and dried under vacuum. The average particle size of the microbeads in cooling water at 15°C was 210 μm, and when the temperature was raised to 37°C, they shrank and became opaque. After thoroughly washing 1 g of the microbeads with sterilized water, they were placed in Dulbecco's modified Eagle's medium (DMEM>/10% F etal C, which had been preheated to 37°C).
alf Seru+n (Fe2)), and further added mouse dermis-derived fibroblasts to approximately 1X10'
The mixture was added to a concentration of /ml, then transferred to a glass beaker and incubated in a carbon dioxide gas incubator at 37°C for 4 hours with stirring using a magnetic cooler.

After that, the microbeads in the glass beaker were taken out and the surface of the microbeads was washed with PBS heated to 37°C.
After washing twice, the microbeads were immobilized with 1% geltal dialdehyde-PBS heated to 37°C. The immobilized microbeads were washed with distilled water, dehydrated with an alcohol series, dried using a critical point drying method, gold-palladium was deposited, and cell attachment was observed using a scanning electron microscope. On the other hand, the microbeads taken out after incubation for 4 hours were left in a medium cooled to 15°C for 20 minutes, the medium was discarded, and the surface of the microbeads was washed twice with PBS cooled to 15°C. The microbeads were immobilized with 1% geltal dialdehyde in PBS cooled to 100 mL. Regarding the immobilized microbeads, cell adhesion was observed using a scanning electron microscope in the same manner as above. As a result, the number of cells immobilized at 15°C was significantly lower than that on the surface of microbeads immobilized at 37°C, and almost no adherent cells were observed. This fact indicates that cells are completely detached from the bead surface by cooling the microbeads.

Addition: PNIP^Δ produced in Example-7
Human dermis-derived fibroblasts were cultured in the same manner as in Example 7 using a datesch (35zn+nφ) coated with an isostatic compound of 0.0 and collagen to a thickness of about 0.9 m, and after 5 days, When the cells completely covered the bottom of the dish, the dish was taken out of the 37°C incubator and left at room temperature.When the cell sheet was not completely detached from the bottom of the dish, the old medium was discarded and washed with the same medium. After removing the dissolved polymer and collagen, add 2 ml of fresh medium to a hydrophobic dill, :L (Falcon, 35+n+).
nφ) and cultured for about 2 days to obtain floating spherical fibroblast clusters.

After maintaining the suspended cell clusters in a carbon dioxide gas incubator for an additional 7 days, the clusters were transferred to a hydrophilic cell culture dish (manufactured by Falcon) and cultured under the same conditions as above. The clusters adhered to the bottom surface of the cultured dates and repopulated in a single layer, and completely covered the bottom surface of the dates after about 10 days.

Figure 1 shows that fibroblasts derived from human dermis are PNTPAA+n
/ This is a photograph showing the state in which the collagen has grown on the dates and completely covered the bottom surface of the dates.

FIG. 2 is a photograph showing a state in which the cell sheet was partially detached from the bottom surface of the dish by lowering the temperature to below the LCST. The right end of this figure shows the cells covering the bottom of the dates, the center shows the cells detached from the bottom of the dates and turning over, and the upper left shows the bottom of the dates that appeared after the cells detached.

FIG. 3 is a photograph showing a cell cluster prepared by transferring the detached cell sheet to a hydrophobic dish.

FIG. 4 is a photograph showing that cell clusters stored in a hydrophobic dish were transferred to a hydrophilic dish, and the clusters re-adhered to the bottom of the dish on the second day.

FIG. 5 is a photograph showing a state in which the cells were cultured in a hydrophilic dish for 10 days, repopulated, and completely covered the bottom surface of the dish.

As a control experiment, a collagen solution (0.25χ concentration) 4 that was not used in Example 20 was placed on the bottom of the cell culture dish used in Example 20.
00μβ was injected and air-dried at room temperature in a clean bench.
We created a date dish whose bottom surface was coated with collagen. Human dermis-derived fibroblasts were cultured on this datesch in the same manner as in Example 20. After 4 days, the cultured cells completely covered the bottom surface of the tissue. From this point on, the cells were not detached from the bottom of the dish at all even though the dish was kept at a culture temperature of 37°C to 15°C for 20 minutes.

Therefore, in order to detach the cells attached to the bottom surface, the old medium in the dish was discarded, PBS2+np was added, and the cell surface was washed. Next, throw away the PBS and try
TA solution (0,051 subunits, 0.53+nM ED
T8) After washing the cell surface by adding 2 ol, discard the trypsin/EDTA solution and add fresh trypsin/EDTA again.
TA solution 2 "nl was injected and the same procedure was carried out. After discarding all but 0.5 mN of l-lipudin/EDTA solution,
After 0 minutes of incubation, the cells detached from the bottom of the dateschew. However, observation under a microscope showed that the cells were detached one by one, and no cell sheets were observed to form. Furthermore, when the suspension of detached cells was transferred to a new hydrophobic culture plate (manufactured by Falcon) and cultured, the formation of the spherical fibroblast clusters observed in Example 20 was not observed at all. .

After culturing, the detached human dermis-derived fibroblast cell sheet was transferred to a hydrophobic tissue sheet at the same time as detachment, and the cells aggregated in about 2 days, resulting in a diameter of approximately do not form cell clusters of l+nm. The clusters were stored in a hydrophobic dish at 37°C in an air/15% carbon dioxide incubator for about 3 months, and then transferred to a hydrophilic dish for tissue culture and cultured. The cells again adhered to the bottom of the dish and started repopulating. This fact suggests that the cell clusters are extremely well preserved.

[Brief explanation of drawings]

Figure 1 shows that fibroblasts derived from human dermis are PNTPAA+n
/ This is a photograph showing the state in which the collagen has grown on the dates and completely covered the bottom surface of the dates. FIG. 2 is a photograph showing a state in which the cell sheet was partially detached from the bottom surface of the dish by lowering the temperature to below the LCST. The right end of this figure shows the cells covering the bottom of the dates, the center shows the cells detached from the bottom of the dates and turning over, and the upper left shows the bottom of the dates that appeared after the cells detached. FIG. 3 is a photograph showing a cell cluster prepared by transferring the detached cell sheet to a hydrophobic dish. FIG. 4 is a photograph showing that cell clusters stored in a hydrophobic dish were transferred to a hydrophilic dish, and the clusters re-adhered to the bottom of the dish on the second day. FIG. 5 is a photograph showing a state in which the cells were cultured in a hydrophilic dish for 10 days, repopulated, and completely covered the bottom surface of the dish. (4 others) JP-A-3 266980 (18)

Claims (1)

[Claims] 1. A cell culture substrate comprising a temperature-sensitive polymer compound having an LCST lower than the culture temperature. 2. Claim 1, wherein the polymer compound is selected from the group consisting of polyN-substituted acrylamide derivatives, polyN-substituted methacrylamide derivatives, or copolymers thereof, polyvinyl methyl ether, or polyvinyl alcohol partial acetate. Substrate for cell culture. 3. The cell culture substrate according to claim 1, further comprising a cell adhesion/growth factor. 4. The cell culture according to claim 3, wherein the cell adhesion/proliferation factor is selected from the group consisting of extracellular matrix, gelatin, lectin, mussel-derived adhesive protein, adhesive oligopeptide, and thrombosbondin. Base material for use. 5. The cell culture substrate according to claim 3, wherein the extracellular matrix is selected from the group consisting of collagen, fibronectin, vitronectin, laminin, proteoglycan, and glycosaminoglycan. 6. The cell culture substrate according to claim 1, wherein the polymer compound is coated on the surface of the support. 7. The cell culture substrate according to claim 1, wherein the polymer compound is graft-polymerized on the surface of the support. 8. The cell culture substrate according to claim 1, wherein the polymer compound has a crosslinked structure. 9. The cell culture substrate according to claim 1, which has a membrane, plate, particle, fiber, flake, sponge, or microbead shape at the cell culture temperature. 10. The cell culture substrate according to claim 3, comprising a layer comprising a mixture of the polymer compound and the cell adhesion/growth factor, and a support. 11. The cell culture substrate according to claim 3, comprising a support, a layer comprising the polymer compound, and a layer comprising the cell adhesion/growth factor. 12. The method for producing a cell culture substrate according to claim 11, which comprises coating the surface of a support with the polymer compound, and further coating the surface of the coated polymer compound with the cell culture/growth factor. 13. A cell assembly characterized in that cells are cultured using the cell culture substrate according to any one of claims 1 to 11, and after cell proliferation, the temperature is lower than LCST to detach the proliferating cells from the substrate. How the body is manufactured. 14. A cell aggregate produced by the method of claim 13.