JPH05168470A - Cell agglomerated body and sheet consisting of multiple kinds of cells and manufacture thereof - Google Patents

Abstract

PURPOSE: To obtain cell granules or cell sheets constructed with several kinds of cell types by constituting the granules or sheets with several kinds of cell types of controlled sizes and controlled cell composition.

CONSTITUTION: The objective cell granules or sheets are cell aggregates having multilayer structures and formed from several kinds of cell types by inoculating and culturing them in turns. Cell aggregates or their sheets constituted with several kinds of cell types having controlled size and controlled cell composition. This cell aggregates and sheets are useful as a medical product as a reinforcing material for living body repairing or restoration.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell mass and a sheet composed of a plurality of cell types and a method for producing the same. More specifically, the present invention relates to a cell aggregate and a sheet having a desired size and a plurality of cell types having a desired cell composition. INDUSTRIAL APPLICABILITY The cell mass or sheet of the present invention is useful in that it can efficiently produce a cell product, and is useful for a living body such as a prosthesis or drug for repairing a defective portion or a lesion portion of a living tissue. It is also useful in that it can be used as a simulator for evaluating the effect of.

[0002]

2. Description of the Related Art Organs or tissues of a living body are formed by many types of cell aggregates, and their specific functions are considered to be expressed and controlled by the interaction between heterogeneous cell aggregates. ing. For example, blood vessels are intima,
It consists of the media and adventitia, with one layer of vascular endothelial cells in the intima, smooth muscle cells in the media and fibroblasts in the adventitia. It is known that various cells secrete various chemical mediators or hormones and control the functions of other cells. For example, hormones targeting smooth muscle cells are secreted from vascular endothelial cells and suppress the contractile action which is a function of smooth muscle cells. In addition, vascular endothelial cell growth factor is secreted from fibroblasts to promote the proliferation of endothelial cells.

On the other hand, the skin is composed of an epidermal layer and a dermis layer inside thereof, and each layer is composed of epidermal cells and fibroblasts. Then, fibroblasts secrete a factor that controls the proliferation and differentiation of epidermal cells, and epidermal cells secrete a factor that promotes the proliferation of fibroblasts. It is known to be effectively controlled.

The liver is also formed from sinusoidal endothelial cells, stellate cells, etc. in addition to the main hepatocytes, and the respective cell aggregates control the liver function by mutual close contact with each other. It is believed that

Therefore, for culturing cells while maintaining the functions possessed in the living body, a co-culture containing heterogeneous cells coexisting in the living body is more preferable than a culture system consisting of only one kind of cells. The system is believed to be more advantageous.
For example, it has been reported that a co-culture system of Langerhans islet cells and fibroblasts significantly increases insulin secretory capacity from Langerhans islet cells as compared with a culture system of Langerhans islet cells alone (Rabionovitch, A. et a.
l., DIABETES (1979) 28: 1108). It has also been reported that the co-culture system of hepatocytes and fibroblasts significantly prolongs the lifespan of hepatocytes compared with the culture system of hepatocytes alone (Katsuri Yoshito, 4th Primary Culture Hepatocyte Study). Meeting abstract, (1988)
11).

[0006] Most of the co-culture methods that have hitherto been carried out were a method of seeding a mixture of various cell types on a cell culture substrate and culturing, or a method of sequentially seeding and culturing each cell type. That is, the conventional co-culture has been based on a two-dimensional co-culture system, but it has been pointed out that such two-dimensional culture has various problems. First, although two-dimensional culture is useful for the process of cell proliferation, it is not suitable for the process of cell differentiation. Considering that the process of cell differentiation is an essential process for most cells to express their specific functions in vivo,
It is hard to say that a two-dimensional co-culture system that is not suitable for the process of differentiation is satisfactory. Further, most cells in the living body are present during the three-dimensional construction, but there is a principle problem in effectively culturing the cells having such a three-dimensional structure by the two-dimensional culture. That is, when cells are cultivated using a two-dimensional culture system, the two-dimensional cell substrate binds and binds the cells so strongly that the cells reproduce a specific structure similar to that in vivo. It becomes difficult to express the same function as in the living body. Further, the conventional two-dimensional co-culture system has a major drawback that it is impossible to collect the cultured cells in a useful form such as a sheet or a lump. This is because the cell releasing agents such as trypsin and EDTA used in the conventional recovery method destroy the intercellular bond necessary for maintaining the shape of the cell sheet or the aggregate. To use co-cultured cells as a replacement prosthesis for injured or diseased body parts, or as a tissue or organ model for measuring the biological activity of a test substance, a cell sheet It is necessary to maintain the morphology of lumps and lumps.

Therefore, the following two types of methods have been investigated as a method for forming a cell sheet or a lump without using a cell releasing agent. One is a method in which cells are suspended in culture on a culture substrate coated with a non-adhesive substance such as agar or agarose, and the cells are naturally aggregated to form a cell aggregate (Ca.
rlsson, J., et al., Spheroids in Cancer Research
(1984), 1, editedby Acker, H., et al., Springer-Ve
rlag). This method was also examined using a substrate coated with polyhydroxyethyl methacrylate (Landry, J. et al., J. Cell Biol. (1985).
101: 914). The other method is to prepare cell aggregates by utilizing the fact that cells cultured on a weakly cell-adhesive substrate on which cells can adhere weakly spontaneously detach without using a cell detaching agent. is there. Proteoglycan coated substrate (Koide, N., et al., Biochem. Biophys. Res. Commum.
(1985) 161: 385) and positively charged polystyrene (Koide, N., et al., Experimental Cell Research.
(1990) 186: 227) is used as the weakly cell-adhesive substrate. These two methods were also applied to multiple co-culture systems. The first method using an agar-coated substrate produced a cell mass composed of HeLa cells and fibroblasts (Sasaki, T., et al., Cancer Researc.
h (1984) 44: 345). A second method using a proteoglycan-coated substrate produced a cell mass composed of hepatocytes and vascular endothelial cells (Matsushima, K., et.
al., Artificial organs (1990) 19: 848).

The major problems of the conventional methods for producing the above-mentioned cell aggregates or sheets are as follows.

(1) The conventional method is not always applicable to all types of cells. This is because the production of cell aggregates and sheets by these methods strongly depends on the aggregability and detachability of each cell, and the aggregability and detachability greatly differ depending on the cell type.

(2) It was almost impossible to control the total cell number related to the size of cell aggregates and sheets by the conventional method. In addition, it is impossible to control the size of cells, which form cell aggregates or sheets, due to agglutination or desorption of cells accidentally or unexpectedly. For this reason, the size distribution of the cell aggregates and sheets prepared by the conventional method was very wide.

(3) When a cell aggregate or sheet composed of a plurality of cell types is produced by the conventional method, it is very difficult to control the composition of each cell type constituting the cell aggregate or sheet. Met.

The above-mentioned problem is that the obtained cell mass or sheet is used for repairing a damaged or diseased living body part, or a substitute for a tissue or an organ for evaluating the biological activity of a test substance. It is especially serious when used as.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to provide a cell aggregate or sheet composed of a plurality of cell types, which solves the above-mentioned problems. Further, it is to provide a method for producing the cell aggregate or sheet.

[0014]

The above objects have been achieved by the present invention, which provides a cell mass or sheet composed of a plurality of cell types having a controlled size and a controlled cell composition. Furthermore, the present invention has been achieved by providing a method for forming the cell aggregate or sheet, which comprises the following steps. The method for producing cell aggregates or sheets of the present invention comprises (i) LCST lower than the cell culture temperature.
A plurality of cell types are seeded and cultured on a cell culture substrate consisting of a temperature-sensitive polymer compound having the following and a cell adhesion / growth factor, and (ii) a plurality of cell types grown on the cell culture substrate Detaching from the cell culture substrate by lowering the temperature below the LCST of the cell aggregate consisting of (iii)
The detached cell aggregates are subjected to suspension culture on a cell non-adhesive substrate to form cell aggregates or sheets.

The cell culture substrate used in the present invention comprises a temperature-sensitive polymer compound and a cell adhesion / growth factor.

The temperature-sensitive polymer compound solidifies above the LCST and dissolves in the aqueous solution immediately below the LCST. Therefore, it is possible to detach the cultured cells by culturing a plurality of cells at a temperature of LCST or higher and then lowering the temperature to LCST or lower. However, most cells cannot grow by adhering to the temperature-sensitive polymer compound itself, so that a cell adhesion / growth factor is also present in the cell culture substrate. By using such a culture substrate, the cell aggregate formed on the substrate is not decomposed into individual cells, and the number of the cells is not reduced, and the aggregate is used as a culture substrate. Can be detached from.

In step (i) of the production method of the present invention, a plurality of cell types may be seeded and cultured at the same time, or a plurality of cell types may be seeded separately or sequentially and cultured. When a plurality of cell types are inoculated and cultured at the same time, a plurality of cell types directly adheres to the culture substrate, whereas when inoculated separately and sequentially, only one cell type is cultured. Only adheres to the culture substrate. Since the adhesiveness of cells strongly depends on the cell type, it is difficult to control the total cell number and cell composition of cell aggregates by the cell composition ratio at the time of seeding if a method of seeding and culturing multiple cell types at the same time is adopted. Sometimes. Further, since the cell desorption ability also depends on the cell type, it may be difficult to simultaneously desorb a plurality of cells in step (ii). Therefore, when seeding and culturing a plurality of cells having different adhesiveness and desorption ability, it is preferable to adopt a method of seeding and culturing these cells separately or sequentially.

The most preferred method is to seed the first cell type and culture until confluent, and then seed and culture the second cell type. By adopting such a method of seeding and culturing sequentially, it is also possible to produce a multilayer structure composed of each cell type. According to the present invention, it is possible to produce a cell aggregate consisting of virtually any number of layers. Preferably about 10 layers, more preferably 2
It is a cell aggregate consisting of ~ 5 layers. In the desorption step of step (ii), the confluent first cell type completely wraps other cell types like a scarf in the desorption step,
It is also possible to effectively prevent the cells from separating from the cell aggregate.

The total number of cells in the finally obtained cell aggregate or sheet and the number of cells of each cell type can be easily and accurately controlled by adopting a method of sequentially seeding and culturing. The number of confluent first cell types on the cell substrate can be easily and accurately calculated by a known means such as a phase contrast microscope before seeding the second cell types. The cell number of the second cell type that is subsequently seeded and cultured can also be measured by various methods. For example,
A method of directly calculating the number of cells of the second cell type attached to the first confluent cell layer by a phase contrast microscope, or the number of cells of the unadhered second cell type suspended in the culture solution Then, a method of calculating by subtracting from the number of cells at the time of seeding the second cell type can be mentioned.

In the step (iii), the cell aggregate detached in the step (ii) is subjected to suspension culture on a cell non-adhesive substrate to be converted into a cell aggregate or a sheet.
One of the preferred non-cell-adhesive substrates is coated with a temperature-sensitive polymer compound.

Cell types that can be seeded and cultured in the present invention include fibroblasts, hepatocytes, endothelial cells, pancreatic cells,
Examples thereof include epidermal cells, bone cells, nerve cells, muscle cells, kidney cells and brain cells, but the present invention is not limited to these cell types.

The method of the present invention is characterized in that cell aggregates and sheets having a desired size and cell composition can be produced. As is clear from the examples described below, the number of each cell can be easily controlled by adjusting the surface area of the cell culture substrate and the seeding density of each cell. Moreover, according to the method of the present invention, cell aggregates and sheets having various morphologies can be produced. For example, by setting appropriate conditions, it is possible to produce a cell aggregate or sheet having a structure in which a specific cell completely covers another cell, or a structure in which a specific cell partially covers another cell. . Among the cell aggregates produced using such a method of the present invention, cell aggregates consisting of fibroblasts that cover all or part of the central part consisting of pancreatic Langerhans islets or pancreatic Langerhans islet cells are particularly preferable. Has excellent effect. This cell mass has an excellent insulin secretory capacity and its activity is maintained for a long period of time, and therefore has extremely high utility value as an artificial organ. As described above, the cell aggregates and sheets produced by the method of the present invention are expected to be highly useful as medical products such as prostheses for bioremediation.

The temperature-sensitive polymer compound used in the cell culture substrate used in the present invention means LCST (Lower Critical).
It is a polymer compound characterized by having a Solution Temperature) and being in a hydrated state below LCST and changing to a dehydrated state above LCST. Examples of temperature-sensitive polymer compounds include poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polyethylene oxide, etherified methyl cellulose, polyvinyl alcohol partial acetic acid and the like. You can Particularly preferred are poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives or their copolymers, polyvinyl methyl ethers, and polyvinyl alcohol partial acetic acid.

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

Poly-N-acryloylpiperidine; Poly-N-n-propylmethacrylamide; Poly-N-isopropylacrylamide; Poly-N, N-diethylacrylamide; Poly-N-isopropylmethacrylamide; Poly-N-cyclopropylacrylamide; Poly-N-acryloyl Piperidine; poly-N, N-ethylmethylacrylamide; poly-N-cyclopropylmethacrylamide; poly-N-ethylacrylamide; the above polymer compounds may be used alone or may be copolymerized with other monomers. As the monomer to be copolymerized, a hydrophilic monomer,
Any of the hydrophobic monomers can be used. Generally, copolymerization with a hydrophilic monomer increases LCST, and copolymerization with a hydrophobic monomer decreases LCST. Therefore, by selecting these appropriately, the desired LCS
A polymer compound having T can be obtained.

The hydrophilic monomer has N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethacrylate, hydroxymethyl acrylate, and an acidic group. Acrylic acid, methacrylic acid and salts thereof, vinyl sulfonic acid, still sulfonic acid, etc., and N, N-dimethylaminoethyl methacrylate, N, N-diethylethylaminoethyl methacrylate, N, N-dimethyl having a basic group Examples thereof include, but are not limited to, aminopropyl acrylamide and salts thereof.

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

On the other hand, as the hydrophobic monomer, acrylate derivatives and methacrylate derivatives such as ethyl acrylate, methyl methacrylate and glycidyl methacrylate, N-substituted alkyl methacrylamide derivatives such as Nn-butyl methacrylamide, vinyl chloride, acrylonitrile, Examples thereof include, but are not limited to, styrene and vinyl acetate.

The temperature-sensitive polymer compound used in the present invention preferably has a molecular weight of 1.0 × 10 ⁵ or more.
More preferably, it is 1.0 × 10 ⁶ or more. The molecular weight referred to in the present invention refers to the average molecular weight obtained from the viscosity. For example, regarding poly-N-isopropylacrylamide, the relational expression between the average molecular weight (Mn) and the intrinsic viscosity ([η]) is [η] = 9,59 × 10 ⁻⁵ Mn ^0.65 (27 ° C. tetrahydrofuran solution) (Ito, RT Gero
nimo, Research Institute for Fiber and Polymer Materials No. 159 (19
88)).

On the other hand, examples of the cell adhesion / growth factor used in the present invention include extracellular matrix, adhesive oligopeptide which is a binding site for adhesive proteins such as gelatin, lectin, fibronectin, and mussel-derived adhesive protein. , Basic polymers and the like. The extracellular matrix is a substance existing between cells in the living body, and examples thereof include collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, thrombospondin and the like.

Examples of the basic polymer compound include polylysine, polyhistidine, protamine sulfate, polydimethylaminoethyl (meth) acrylate, polydimethylaminopropyl (meth) acrylate, polyethyleneimine and polyvinylpyridine.

The cell culture substrate used in the present invention is typically prepared by coating a temperature-sensitive polymer compound having an LCST lower than the cell culture temperature and a cell adhesion / growth factor on a support. can do.

As such a culture substrate, an aqueous solution of a mixture of a temperature-sensitive polymer compound and a cell adhesion / growth factor is prepared by LCST.
It is obtained by coating all or part of the support at a lower temperature and drying the coating layer. The culture substrate of the present invention can also be obtained by the step of immersing the support in an aqueous solution of a mixture of a temperature-sensitive polymer compound and a cell adhesion / growth factor at a temperature of LCST or lower and drying.

Further, the culture substrate used in the present invention can also be prepared by forming a layer of a temperature-sensitive polymer on the surface of a support and then forming a layer of cell adhesion / growth factor on the layer. can get. The culture substrate used in the present invention can also be obtained by forming a layer of cell adhesion / growth factor on the surface of a support and then forming a layer of a temperature-sensitive polymer compound on the layer.

The mixing ratio of the temperature-sensitive polymer compound used in the present invention and the cell adhesion / growth factor is typically 1 :.
It is 0.1 to 1: 3, and this value changes depending on the type of cell adhesion / growth factor and cell type used.

The thickness of the coating layer after drying is 0.2 μm
Or more, preferably 0.5 μm or more, more preferably 1.0
It is at least μm. When the thickness of the coating layer is 0.2 μm or less, the desorption property of cells is remarkably deteriorated, so that it takes a very long time to desorb the cells, and as a result, the function of the cells becomes unstable.

The support used in the present invention is preferably transparent or translucent, and is preferably glass or plastic. Typical plastics include polystyrene, polycarbonate, polymethylmethacrylate, polypropylene, polyethylene, polyester, polyamide, polyvinylidene fluoride, polyoxymethylene, polyvinyl chloride, polyacrylonitrile,
Examples thereof include polytetrafluoroethylene and polydimethylsiloxane. The form of the support is not particularly limited, and it may be used in the form of a dish, plate, film, sheet or the like.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is determined by the description of the claims, and is not limited by the following examples. Absent.

[0039]

EXAMPLES Example 1 N-isopropylacrylamide monomer (NIPAA
m, Eastman Kodak Co.) 50 g was dissolved in 500 ml of benzene, and 2,2'-azobisisobutyronitrile (AIB
N) 0.2 g was used as a polymerization initiator, and 1 at 60 ° C.
Polymerization was carried out for 2 hours under stirring in a nitrogen stream. After decanting the polymer precipitates in benzene,
The precipitate was dissolved in tetrahydrofuran (THF) and purified by precipitation using ethyl ether to obtain poly-N-isopropylacrylamide (PNIPAAm). The PNIPAAm thus obtained had a number average molecular weight of 2.0 × 10 ⁶ . 0.5% (W / V) PN
LCST of IPAAm aqueous solution and 1.0% (W / V)
PNIPAAm phosphate buffer (PBS) LCST
Was 32 ° C. and 29 ° C., respectively. Bovine dermal pepsin solubilized type I collagen (sterilized, manufactured by Koken Co., Ltd.) 0.5% (W / V) aqueous solution was used, and PNIPAAm concentration was 0.25% (W / V),
An equal volume mixed solution having a collagen concentration of 0.25% (W / V) was prepared. About 9mm diameter holed mold and about 15mm diameter
Using the perforated mold of
By coating on the bottom surface of a commercially available hydrophobic dish (Falcon), the coating area is about 0.
A culture substrate of 6 cm ^{2 and} a culture substrate of about 1.8 cm ² (type I substrate) were prepared. The weight mixing ratio of collagen and PNIPAAm was 1: 1 and the thickness of the coating layer was about 2 μm. Separately, 0.5% (W / V) P on a commercially available tissue culture dish (Falcon) having a diameter of 35 mm.
A type II substrate was prepared by coating an aqueous solution containing only NIPAAm. The thickness of the coating layer is about 2
It was μm.

Fibroblasts derived from human dermis are Yoshizato
Collected and maintained by the method of Yoshizato, K., et al.
Al., Biochem. Biophys. Acta (1980) 627: 23). The primary cultured hepatocytes were 0.05% (W / W) from a 5-week-old male rat (Charles River Japan, Inc.) weighing about 150 g.
Harvested by perfusing the liver with V) collagenase. Fibroblasts derived from human dermis are 10% (W /
V) The cells were suspended in a medium (DMEM) supplemented with FBS, 20 mM HEPES, 100 U / ml penicillin and 100 mg / ml streptomycin, and heated to 37 ° C. The human dermis-derived fibroblast suspension was seeded at a concentration of about 4.0 × 10 ^4.
/ Surface area cm ² were seeded at about 0.6 to about 1.8 cm ² Type I substrates on each. By culturing at 37 ° C. for 3 days in a humidified incubator containing 5% carbon dioxide and 95% air, the fibroblasts were grown to a confluent state.
Confirmation of the confluent state and measurement of the density of the fibroblasts in the confluent state were performed by an inverted phase contrast microscope. Next, a suspension medium of rat primary culture hepatocytes preheated to 37 ° C. was seeded on the confluent fibroblast monolayer at seeding concentrations of about 1.0 × 10 ⁴ and about 5.0 × 10 ⁴ , respectively. . It was confirmed by inverted microscope observation that 90% or more of the hepatocytes were adhered to the confluent fibroblast layer after co-culturing for 60 minutes. Then, the culture dish was taken out from the 37 ° C. incubator to room temperature of 25 ° C. and left as it was for about 5 minutes. By this operation, the fibroblast monolayer with hepatocytes attached was completely detached from the type I substrate as its cell aggregate. The detached cell aggregate is washed twice with cold PBS to prevent contamination with PNIPAAm and collagen dissolved in the culture medium and once with a cold medium, and then preheated to 37 ° C. II Transferred to medium in substrate. Formation and maintenance of cell aggregates was performed at 37 ° C. on a Type II substrate in a humidified incubator with 5% carbon dioxide, 95% air. The medium was replaced every 3 days.

The number of fibroblasts and hepatocytes in the cell mass was measured by the following method. Cell aggregates cultured for 4 days were fixed by immersing them in 10% formalin neutral buffer for 60 minutes at 4 ° C. It was then dehydrated and embedded in paraffin wax. A thin layer with a thickness of about 2 μm was cut out from the vicinity of the center of the cell aggregate, and after removing the wax, hematoxylin-eosin (H
E) Staining was performed. Wax-removed thin layer sections for immunostaining were immersed in methanol containing 0.3% by weight of H ₂ O ₂ for 30 minutes to remove endogenous peroxidase activity. The sections were then immersed in goat serum for 60 minutes and then rabbit IgG against rat albumin (Cappel, Organon Teknika Corporation) (P
Diluted 500 times with BS) for 30 minutes. Then 3 in a biotinylated goat antibody against rabbit IgG
Soak for 0 minutes, and avidin-biotin-peroxidase complex (ABC) (Vector L in a humidifier at 37 ° C).
aboratories, Inc.) for 30 minutes. The bound peroxidase contained 0.01% by weight H ₂ O _{2 in an} amount of 0.01%.
Detection was by immersion in 5 mg / ml DAB (Dojindo Laboratories) for 2 minutes. The obtained sample section was neutralized and stained with hematoxylin and dried.

The numbers of fibroblasts and hepatocytes were calculated from the micrographs of the HE-stained section and the above-mentioned indirect immunoperoxidase-stained section, respectively. The diameters of all cell aggregates were measured from phase contrast micrographs.

The numbers of hepatocytes and fibroblasts in all cell aggregates and the size of the aggregates are shown in Table 1. As is clear from Table 1, the number of fibroblasts and hepatocytes can be easily and accurately controlled by adjusting the surface area of the type I substrate and the seeding density of hepatocytes, respectively. It is also possible to accurately control the diameter of cell aggregates by the total cell number in the method of the present invention.

[0044]

Example 2 Method for Lacy of Pancreatic Langerhans Islets (Lacy, PE, et.
al., DIABETES (1967) 16:35) from WKA rats weighing approximately 300 g. The diameter of the obtained Langerhans island was 200 to 500 μm.

The Type I substrate used in Example 1 was 0.25.
% (W / V) PNIPAAm and 0.25% (W / V) collagen mixture aqueous solution was prepared by coating on a Falcon dish with a diameter of 35 mm. The mixing weight ratio of collagen and PNIPAAm in the coating layer was 1: 1 and the thickness of the coating layer was about 2 μm. The type II substrate was prepared by the method used in Example 1. Human dermis-derived fibroblasts were suspended in the same medium as used in Example 1 and preheated to 37 ° C. to give an initial seeding concentration of about 4.0 on the above type I substrate.
Seed at 10 ⁴ / cm ² . The cells were cultured in a humidified incubator containing 5% carbon dioxide and 95% air for 4 days at 37 ° C. until they became confluent. Here, confirmation of the confluent state and measurement of the density of the fibroblasts in the confluent state were performed by an inverted phase contrast microscope. Then, the above-mentioned 2-3 islets of Langerhans suspended in a medium heated to 37 ° C. were seeded on the above-mentioned fibroblast monolayer. 1
After 0 hours, it was confirmed by an inverted phase contrast microscope that the seeded Langerhans islets adhered to the monolayer of fibroblasts. The Type I substrate was then removed from the 37 ° C incubator and left at room temperature (about 25 ° C). By this operation, it was confirmed by an inverted phase-contrast microscope observation that the fibroblast monolayer to which the Langerhans islets were adhered was gradually detached from the type I substrate and wrapped around the Langerhans islets. No separation from the fibroblast layer of Langerhans islets was observed during this detachment process. The cell aggregate consisting of fibroblasts and islets of Langerhans thus obtained was washed with PBS for 2 to 3 times, and then transferred to a pre-warmed medium in the above-mentioned type II substrate, and every 4 days. Suspension culture was performed while exchanging the medium. During this culturing process, cell aggregates were transformed into complete cell aggregates. After culturing on a type II substrate for 14 days, the cell aggregates were fixed with 10% formalin neutral buffer, dehydrated and embedded in paraffin wax. About 2μ thick from the center of the cell mass
A section layer of m was cut out. After removing the wax, the section layer was HE-stained according to the usual method. It was confirmed by HE staining that the seeded Langerhans islets were completely encapsulated by fibroblasts, and the cell mass consisted of Langerhans islets and fibroblasts.

Example 3 In the same manner as in Example 2, a cell mass composed of human dermal-derived fibroblasts containing one Langerhans islet having a diameter of about 250 μm was prepared. At 37 ° C. in a humidified incubator with 5% carbon dioxide and 95% air, the cell mass was treated with DMEM containing 10% (W / V) FBS while changing the medium every 4 days. Cultured on wood. The amount of insulin secreted from the cell aggregates into the medium was measured by conventional immunoassay. Even after 40 days of culturing, the amount of secreted insulin was about 33 μU / Langerhans island / day. Further, after culturing for 2 months, the cell aggregate was observed by the above-mentioned HE staining, and no necrosis was found inside the islets of Langerhans encapsulated by fibroblasts. As a comparative experiment, the diameter is about 350 μm
Of Langerhans islets of the same type were cultured on the type II substrate under the same conditions as above, and the amount of insulin secretion was reduced by about 4 μU / Langerhans / day after 10 days of culturing.
By staining, necrosis was observed inside the islets of Langerhans after 2 weeks of culture. These facts indicate that the formation of cell aggregates by fibroblasts is effective for maintaining the activity and function of islets of Langerhans for a long period of time.

Example 4 O from Langerhans Island similar to that used in Example 2
Langerhans islet cells were prepared by the method of No. et al. (Ono, J., et al., Endocrinol. Japan (1977) 24: 26.
Five). By using Langerhans islet cells instead of Langerhans islets, cell aggregates composed of fibroblasts were prepared in the same manner as in Example 2. As a result of HE staining, it was found that the Langerhans islet cells were completely encapsulated by fibroblasts.

Example 5 The same medium and type I substrate as used in Example 1 were preheated to 37 ° C. and human dermis-derived fibroblasts were initially seeded at a density of about 4.0 × 10 ⁴ / cm ² . Sowed. 37
The cells were cultured in a humidified incubator of 5% carbon dioxide gas and 95% air at 3 ° C for 3 days to prepare a confluent fibroblast monolayer. Then, the human umbilical artery-derived vascular endothelial cells suspended in the above-mentioned medium were seeded at a density of about 5.0 × 10 ⁴ /
The cells were seeded at cm ² on the fibroblast monolayer described above. 60 minutes later,
Most of the seeded vascular endothelial cells adhered on the fibroblast monolayer. The Type I substrate was then removed from the 37 ° C. incubator and left at room temperature (about 25 ° C.). It was confirmed by an inverted phase-contrast microscope that the fibroblast monolayer to which the vascular endothelial cells adhered was gradually detached from the type I substrate by this operation and wrap the vascular endothelial cells. Almost no dissociation of vascular endothelial cells and fibroblasts was observed during this desorption process.

The cell aggregates thus obtained from fibroblasts and vascular endothelial cells were washed 2-3 times with PBS and then transferred into a preheated medium at 37 ° C. in a type II substrate. Suspension culture was performed while changing the medium every 4 days. During this culture process, the cell aggregates changed into complete cell aggregates. After culturing on a type II substrate for 14 days, the culture mass was fixed in 10% formalin neutral buffer, dehydrated, and embedded in paraffin wax. A section layer was cut out with a layer thickness of 2 μm from the vicinity of the center of the cell aggregate. After removing the wax, the sections were HE-stained according to the usual method. It was confirmed by HE staining that the seeded vascular endothelial cells were completely encapsulated by fibroblasts, and that the cell mass consisted of vascular endothelial cells and fibroblasts.

[0051]

EFFECTS OF THE INVENTION According to the method of the present invention, a cell aggregate comprising a plurality of cell types having a desired structure and a desired cell composition can be easily obtained. Therefore, the method of the present invention is
It can be widely applied to the formation of various cell aggregates, and its technical value is extremely high.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location // C12M 3/00 Z 9050-4B (72) Inventor Yuichi Mori 1642-21 Kamariya-cho, Kanazawa-ku, Yokohama-shi, Kanagawa B-4 (72) Inventor Manabu Yamazaki 1200-1 Tsurumaki, Hadano City, Kanagawa Corp. Tsurumaki Corp. A-405 (72) Inventor, Kubota Ya 21-3-24 Higashi 3-chome, Kunitachi, Tokyo

Claims (4)

[Claims] 1. A cell aggregate or sheet comprising a plurality of cell types and having a desired size and a desired cell composition. 2. (i) Seeding and culturing a plurality of cell types on a cell culture substrate composed of a temperature-sensitive polymer compound having an LCST lower than the cell culture temperature and a cell adhesion / growth factor, and ii) detaching the cell aggregate consisting of a plurality of cell types grown on the cell culture substrate from the cell culture substrate by lowering the temperature below LCST, and (iii) removing the detached cell aggregate. A method for producing a cell mass or sheet comprising a plurality of cell types, which comprises each step of suspension culture on a cell non-adhesive substrate to form a cell mass or sheet. 3. A cell mass or sheet produced by the production method of claim 2. 4. A prosthesis for bioremediation, which comprises the cell aggregate or sheet of claim 3.