JPH05260957A - Hepatic cell spheroid and its preparation - Google Patents

Abstract

PURPOSE:To readily peel and recover spheroidal hepatic cells from a cell culture support in a high yield without damaging the cells and without being mixed or contaminated with the third component such as a chemical by culturing the hepatic cells in the spheroidal state and subsequently peeling and recovering the hepatic cells cultured in the spheroid state only by changing the circumferential temperature. CONSTITUTION:Hepatic cells are cultured at <= an upper limit critical dissolution temperature or at >= a lower limit critical dissolution temperature on a cell culture support produced by coating a polymer having the critical dissolution temperature of 0-80 deg.C in water on the surface of a substrate, and subsequently subjected to >= the upper limit critical dissolution temperature or <= the lower limit critical dissolution temperature.

Description

Detailed Description of the Invention

[0001]

The present invention relates to hepatocyte spheroids,
And its preparation method. More specifically, the present invention cultivates spheroids, and in order to recover the spheroids, it is only necessary to change the ambient temperature, and as a result, the third component such as a drug is mixed with high yield without damaging the cultured cells. Alternatively, it relates to a method for preparing hepatocyte spheroids that can be easily separated and collected from a cell culture support without contamination. The present invention also relates to hepatocyte spheroids obtained by such a preparation method.

[0002]

2. Description of the Related Art In recent years, cell culture has been widely performed in which cells obtained from a living body are grown in a medium. In particular, hepatocyte cell aggregates and spheroids (spherical aggregates) are examined in vitro.
Culturing in o) is a study that has recently been drawing attention from the viewpoint of development of hybrid artificial liver and biosimulator.

However, unlike other cells, these hepatocytes are susceptible to external factors. Therefore, conventional culture methods in which hepatocytes are cultivated on a collagen-coated substrate of biological origin and forcibly detached after culturing can damage the hepatocytes during the detachment operation and kill the cells. is there. In addition, such a culture method has a problem that hepatocytes are cultured in a monolayer and it is difficult to form spheroids.

Therefore, recently, a method of three-dimensionally culturing hepatocytes by forming a cell-adhesive substance (eg, collagen, fibronectin, laminin) into a sponge form, or culturing on a cell-non-adhesive substance such as proteoglycan Method,
Furthermore, poly-, which is known as an artificial matrix material for hepatocyte adhesion,
A method of culturing on N-vinylbenzyl-D-lactonamide (PVLA) and the like have been proposed as a method for forming a multi-layered aggregate having high biochemical functions of hepatocytes themselves.

However, in the case of a multilayer aggregate obtained by any of these methods, it is necessary to peel it off from the base material and to use it in a higher order (for example, a hybrid artificial liver) by using trypsin or the like. You must use proteolytic enzymes and chemicals. As a result, cells that formed a multi-layered assembly were separated, cells were denatured and the original functions of the cells were impaired, and various artificial substrates such as proteolytic enzymes and chemicals were contaminated and contaminated. , Etc. have problems.

[0006] Therefore, the present inventors previously investigated cells grown in culture simply by changing the ambient temperature without treatment with a protease such as trypsin or ethylenediaminetetraacetic acid (EDTA) which is a chelating agent. A cell culture support material has been proposed which can be separated and collected from the culture support as it is as a multi-layered assembly (JP-A-2-211865).

[0007]

DISCLOSURE OF THE INVENTION The present invention is a further development of the cell culture support material of the above publication so that it can be applied to the preparation of hepatocyte spheroids. That is, one object of the present invention is to provide a method for preparing hepatocyte spheroids which is cultured in a spheroid form and can be easily exfoliated and recovered in a high yield without damaging cells and without contamination or contamination with a third component such as a drug. It is to be. Another object of the present invention is to provide hepatocyte spheroids obtained by such a preparation method.

[0008]

In order to achieve the above object, when a cell culture support having a base material coated with an appropriate amount of a polymer is used, a normal culture temperature is 37.
It is possible to culture hepatic cells in a spheroid form while keeping them alive even at ℃, and the spheroids can be exfoliated and recovered simply by changing the temperature, and the recovered cells have their original function. The present invention has been accomplished by discovering that the value is maintained without any deterioration.

That is, according to the present invention, on a cell culture support comprising a base material surface coated with a polymer having a critical dissolution temperature in water of 0 to 80 ° C., hepatocytes are below the upper critical solution temperature or below the lower critical solution temperature. Provided is a method for preparing a hepatocyte spheroid, which comprises culturing at a melting temperature or higher and then exfoliating and recovering at or above an upper critical melting temperature or below a lower critical melting temperature. The present invention also provides a hepatocyte spheroid prepared by such a preparation method.

The cell culture support used in the method for preparing hepatocyte spheroids of the present invention has the surface of the substrate coated with a suitable polymer. Suitable polymers have a critical dissolution temperature in water of 0 to 80 ° C., preferably 0 to 50 ° [note that the “critical dissolution temperature” means that the two substances separating the two layers are brought into contact with each other at a certain temperature. It refers to the temperature at which the solution completely dissolves to form a uniform layer. In particular, the temperature when increasing the temperature to reach complete dissolution may be referred to as the "upper limit critical melting temperature", and the temperature when decreasing the temperature to complete dissolution may be referred to as the "lower limit critical dissolution temperature". If the critical lysis temperature exceeds 80 ° C, cells may be killed, which is not preferable. The critical melting temperature is 0
If the temperature is lower than 0 ° C, generally the cell growth rate is extremely decreased or the cells are killed, which is not preferable. Examples of such a polymer include, for example, JP-A-2-2118.
Examples thereof include the polymers described in Japanese Patent Publication No. 65 (which is incorporated herein by reference and is its disclosure). That is, such polymers are prepared, for example, by homo- or copolymerization of monomers such that the homo-polymer of the monomer has a critical solution temperature of 0-80 ° C. Examples of such a monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. One or more of these may be used. In addition, when it is necessary to adjust the critical lysis temperature depending on the type of proliferating cells, when it becomes necessary to enhance the interaction between the coating substance and the cell culture support, or when the hydrophilic / hydrophobic balance of the cell support is balanced. When adjustment is necessary, other monomers other than the above may be further added for copolymerization. Further, a graft or block copolymer of the above polymer used in the present invention and another polymer,
Alternatively, a mixture of the polymer of the present invention and another polymer may be used. Further, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

Examples of the material of the base material to be coated include glass, modified glass, polymer compounds such as polystyrene and polymethylmethacrylate, which are generally used for cell culture, and ceramics and metals. The surface of the base material may be hydrophilized using a processing technique such as ozone treatment, plasma treatment, or sputtering. The shape is not limited to a petri dish, and plates, fibers, (porous) particles, or the shape of a container (flask, etc.) generally used for cell culture and the like may be provided.

The support for cell culture of the present invention is obtained by coating the above-mentioned substrate with the polymer used in the present invention.
The coating amount of the polymer is 20 to 80 μg / cm ² , particularly 30 to
70 μg / cm ² is preferred. Polymer coating amount is 80μg / c
When it exceeds m ² , hepatocytes do not adhere to the surface of the cell culture support. Conversely, when the coating amount is less than 20 μg / cm ² , hepatocytes maintain a monolayer state and do not form spheroids. Such a polymer coating amount is, for example, a Fourier transform infrared spectrometer total reflection method (FT-IR-ATR method), an analysis by dyeing a coated part or an uncoated part or a fluorescent substance, and a surface by contact angle measurement or the like. Analysis can be performed alone or in combination.

The polymer may be coated on the substrate by a chemical method or a physical method, which will be described later, alone or in combination. When the monomer is used for coating, the monomer may be in a gas, liquid or solid state. Further, even when a polymer is used, the polymer may be in a liquid state or a solid state. When these are combined by a chemical reaction, electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation,
Plasma treatment, corona treatment, and generally used organic reactions such as radical and ionic reactions when the substrate and the coating material have appropriate reactive functional groups can be used. Examples of the physical interaction method include, but are not limited to, a method in which a coating material alone or a matrix having a good compatibility with a base material is used as a medium, and physical adsorption such as coating or kneading is used. ..

The hepatocyte spheroids of the present invention are prepared by culturing hepatocytes on the cell culture support obtained as described above. Examples of hepatocytes include rat hepatocytes, human hepatocytes, bovine hepatocytes and the like. Such hepatocytes can be obtained by methods well known to those skilled in the art. In the culture conditions of the hepatocytes, the culture temperature is not more than the upper limit critical temperature of the polymer coated on the substrate, and is not less than the lower limit critical dissolution temperature of the polymer. The culture period is appropriately selected depending on the type of hepatocytes. Other culture conditions are not particularly limited,
Culturing may be carried out under the conditions commonly used in the art. For example, the medium may be one to which serum such as fetal calf (FCS) is added, or may be a serum-free medium. After the above culture, hepatocyte spheroids are formed on the cell culture support. In order to separate and recover the hepatocyte spheroids thus formed from the cell culture support, it suffices to change the ambient temperature to not less than the upper critical solution temperature or not more than the lower critical solution temperature of the polymer,
The culture medium in which the cells are cultivated or other isotonic solution can be used and can be selected according to the purpose. At that time, for the purpose of efficiently and easily exfoliating the spheroids, the cell culture support may be lightly tapped or shaken, or the medium may be stirred using a pipette or the like.

[0015]

The action of the present invention will be described more concretely by taking as an example the case where N-isopropylacrylamide is used as the monomer for the coating polymer of the base material and polystyrene which is generally used as the petri dish material for cell culture is used as the support base material. To explain.

The cell culture support is obtained, for example, by coating the above polystyrene substrate with N-isopropylacrylamide monomer at a coating amount of ²⁰ to 80 μg / cm ² , and then polymerizing by electron beam irradiation (EB). As a result, the resulting polymer, poly-N-isopropylacrylamide, is coated on the polystyrene substrate. It should be noted that such poly-N-isopropylacrylamide is about 32 ° C in an aqueous solution.
Has a lower critical solution temperature. At 32 ° C. or higher, which is the lower critical solution temperature, this polymer occupies a small volume and eliminates water molecules in the polymer, and the surface of the support exhibits hydrophobicity. However, conversely, at 32 ° C. or less, the volume occupied by the polymer becomes large, the volume fraction occupied by water molecules in the polymer rises, and the surface of the support becomes hydrophilic.
Therefore, only by controlling the temperature, the hydrophilicity / hydrophobicity of the surface of the cell culture support is adjusted, and the adhesion of the cells to the support is changed. Therefore, it is considered that it is possible to easily peel and collect cells from the cell culture support without destroying the cells after culturing and proliferation simply by changing the temperature.

When normal hepatocytes are seeded on a cell culture support having the above-mentioned properties and cultured at a lower critical temperature of poly-N-isopropylacrylamide of 32 ° C. or higher, normal hepatocytes are generally adhered. It forms spheroids, rather than extending in a monolayer over the support found in sex cells. In order to exfoliate the spheroids thus obtained from the support, the spheroids can be maintained in a high yield in a high yield simply by setting the temperature of the culture solution to 32 ° C. or lower as the lower critical temperature of the above-mentioned polymer. Without impairing the function,
It can be recovered without mixing the third component.

[0018]

EFFECTS OF THE INVENTION According to the present invention, a cell culture support can be easily formed in a spheroidal form at a high yield without changing the ambient temperature, without damaging cells and without contamination or contamination with a third component such as a drug. Hepatocyte spheroids can be peeled off and collected.

[0019]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Examples 1, 2, 3, 4, 5 Becton Dickinson Loveware (Be
Falcon (FALC) manufactured by cton Dickinson Labware
ON) 3002 Petri dish was used, and rat hepatocytes were adopted as the cells to be cultured. N-isopropylacrylamide was dissolved in isopropyl alcohol at the concentration shown in Table-1, and 0.1 ml of the solution was placed on a Petri dish.
After the addition, the surface of the Petri dish was covered with poly-N-isopropylacrylamide by irradiating each with an electron beam at a dose shown in Table 1. After the electron beam irradiation, the Petri dish was thoroughly washed with ion-exchanged water to remove residual monomers and free poly-N-isopropylacrylamide not bound to the surface of the Petri dish, dried in a clean bench, and further dried with ethylene oxide ( E
O) After gas sterilization, deaeration was further performed to obtain a cell culture support material.

The amount of coating on the surface of the obtained cell culture support was determined by the Fourier-exchange infrared spectrometer total reflection method (FT-IR-A).
TR method) to calculate using the ratio of the absorption intensity of amide II (1540 cm ^-1 ) derived from the coating poly-N-isopropylacrylamide to the absorption intensity of 1028 cm ^-1 derived from the base material. Sought by. At that time, a calibration curve was prepared by using a sample in which a known amount of N-isopropylacrylamide homopolymer was coated on the surface of a substrate having a known area. The results are shown in Table-5.

Rat hepatocytes were obtained by the commonly known collagenase perfusion method of Berry and Friend et al. Culture of rat liver parenchymal cells was carried out on the obtained cell culture support with 5% fetal calf serum (FCS),
10 ^-8 M dexamethasone, 10 ^-7 M insulin, 10 m
M nicotinamide 10 ng / ml epidermal growth factor
Williams E medium containing (FGF) was used as a medium in 5% carbon dioxide at a temperature of 37 ° C. After 3 days of culturing, the Petri dish containing the proliferated cells is kept at 5 ° C.
The cells were exfoliated by cooling and leaving to stand, and the proliferative cell exfoliation recovery rate was calculated according to the following formula.

Proliferated cell detachment recovery rate (%) = 100 × (total number of detached and recovered cells) / (total number of proliferated cells)

At this time, in order to measure the total number of cells recovered by peeling and the total number of cells grown, the cells must be in individual states. Therefore, the total number of cells recovered by peeling was
The cells were treated with trypsin-EDTA to give individual cells. In addition, the total number of grown cells is the total number of cells detached and collected by the above-described method, cells that have not been detached even if left to cool to 5 ° C. and left to stand by trypsin-EDTA treatment to detach cells into individual states. It was calculated by adding the total number. The results are shown in Table 5.

[0025]

[Table 1]

Examples 6, 7 and 8 Falcon 3002 Petri dish manufactured by Becton Dickinson Labware was used as a base material, and the coating amount of the N-isopropylacrylamide isopropyl alcohol solution on the Petri dish is shown in Table 2. A cell culture support material was obtained in the same manner as in Example 2 except that the amounts shown were obtained, the coating amount was determined, the cells were cultured, and the cells were detached and recovered, and the proliferating cell detachment recovery rate was determined. .. Table of results
5 shows.

[0027]

[Table 2]

Examples 9, 10 and 11 Falcon 3002 Petri dishes manufactured by Becton Dickinson Labware were used as the base material, and the monomers shown in Table 3 were used in place of N-isopropylacrylamide. A cell culture support material was obtained in the same manner as in Example 4 except that the surface was coated, the coating amount was determined, and the cells were further cultured, followed by peeling.
The collected cells were collected and the recovery rate of exfoliated proliferating cells was calculated. The results are shown in Table-5.

[0029]

[Table 3]

Comparative Example 1 Falcon 3002 Petri dishes manufactured by Becton Dickinson Labware Co., Ltd. were used as a substrate for cell culture, and Examples 1, 2, 3 were used without surface treatment.
Experiments similar to 4, 5 were performed. For the culture of rat hepatocytes, the same method as in Examples 1, 2, 3, 4, 5 was adopted. Then, after 3 days of culturing, the cells that had adhered and proliferated were cooled by cooling to 5 ° C. and left to isolate the proliferating cell detachment recovery rate.
The results are shown in Table-5.

Comparative Example 2 A Falcon 3002 Petri dish manufactured by Becton Dickinson Labware was used as a support base material for cell culture, and the monomer solution was not applied under the same conditions as in Examples 2 and 3. Hepatocytes were cultivated in the same manner as in Examples 1, 2, 3, 4, and 5 by using only the material subjected to electron beam irradiation as a cell culture support material, and after 3 days of culturing, the cells were cooled to 5 ° C. and left to stand. The cells that had adhered and proliferated were exfoliated, and the exfoliated cell recovery rate was determined. The results are shown in Table-5.

Comparative Examples 3 and 4 Falcon 3002 Petri dish manufactured by Becton Dickinson Labware was used as a support substrate for cell culture, and N-isopropylacrylamide was dissolved in isopropyl alcohol at a concentration shown in Table 4. After adding 0.1 ml onto a Petri dish, the surface of the Petri dish was coated with poly-N-isopropylacrylamide by irradiating with an electron beam at a dose shown in Table-4, and Examples 1, 2, 3, 4 were used. The same experiment as 5 was performed. For the culture of rat hepatocytes, the same method as in Examples 1, 2, 3, 4, 5 was adopted. Then, after 3 days of culturing, the cells that had adhered and proliferated were detached by cooling to 5 ° C. and leaving them to determine the detachment recovery of proliferating cells. The results are shown in Table-5.

[0033]

[Table 4]

[0034]

[Table 5]

According to the results of the above Examples and Comparative Examples, in Examples 1, 2, 3, 4, 5, 6, 7, and 8, the concentration of the monomer solution applied to the surface of the base material and the application amount were changed. Thus, a coating amount of the cell culture support material shown in the present invention was obtained.
By culturing at 37 ° C., which is higher than the lower critical melting temperature of N-isopropylacrylamide, hepatocytes form spheroids, and the medium is heated to 5 ° C. or lower than the lower critical melting temperature.
By setting the temperature to ℃, it was possible to peel and collect in a high yield while maintaining the spheroid state. This was the same even when the monomer species used were changed as shown in Examples 9, 10, and 11.

On the other hand, as shown in Comparative Example 1, when a material which was not subjected to any surface treatment was used, or as shown in Comparative Example 2, the surface of the Petri dish was not coated with the monomer solution, In the case of the material irradiated only with the rays, the cells in culture were in a monolayer state, did not form spheroids, and even when the temperature was lowered, no detachment phenomenon was observed.

Further, as shown in Comparative Example 3, when the coating amount on the substrate is as small as 13 μg / cm ² , the cells on the cell culture support material can be exfoliated by lowering the temperature. Although it was possible, the cells were in a monolayer state and spheroids were not formed.

Further, as shown in Comparative Example 4, when the coating amount on the substrate was as large as 95 μg / cm ² , it was no longer sufficient.
No cell attachment was observed.

Example 12 In order to confirm the degree of damage of the exfoliated cells obtained in Example 2, this was recovered by centrifugation (600 G, 5 minutes), and the entire amount was Falcon 3002 manufactured by Becton Dickinson Labware. The cells were cultured again on the Petri dish. For cell culture, the same method as in Examples 1, 2, 3, 4, and 5 was adopted. The degree of cell damage was judged by determining the albumin secretory ability using the conventional enzyme immunoassay (EIA) method after 1 day of culture. The results are shown in Table-6.

Example 13 Albumin-secreting ability of the exfoliated cells obtained in Example 4 was determined in the same manner as in Example 12. The results are shown in Table-6.

Comparative Example 5 The hepatocytes cultured in Comparative Example 1 were detached using a 0.05% trypsin solution-0.02% EDTA solution, and the subsequent operation was performed in the same manner as in Example 12 to detach the hepatocytes. The albumin secretory capacity of the cells was determined. The results are shown in Table-6.

Comparative Example 6 In order to examine the albumin secretory ability of hepatocytes before the exfoliation procedure of Examples 12 and 13 and Comparative Example 5, hepatocytes cultured in Comparative Example 1 were not exfoliated, and albumin was prepared by the above EIA method. I asked for secretory capacity. The results are shown in Table-6.

[0043]

[Table 6]

From the results of Examples 12 and 13 and Comparative Examples 5 and 6, the hepatocytes used this time originally had the albumin secreting ability of the value shown in Comparative Example 6, and Example 12 of the present invention The cultured cells obtained in No. 13 exhibited an albumin-secreting ability which is considered to be almost the same as the original secretory ability. On the other hand, the cultured cells obtained in Comparative Example 5 showed only about 1/5 of the secretory ability. This means that the exfoliated cells of the present invention have less damage to the cells themselves than conventional cells.

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[Procedure amendment]

[Submission date] February 19, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction content]

However, in the case of a multilayer aggregate obtained by any of these methods, it is necessary to peel it off from the base material and to use it in a higher order (for example, a hybrid artificial liver) by using trypsin or the like. You must use proteolytic enzymes and chemicals. Therefore, the cells that formed a multi-layered assembly are separated from each other, the cells are denatured and the original functions of the cells are impaired, and various artificial substrates such as proteolytic enzymes and chemicals are mixed and contaminated. , Etc. have problems. ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] April 21, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction content]

However, in the case of a multilayer aggregate obtained by any of these methods, it is necessary to peel it off from the base material and to use it in a higher order (for example, a hybrid artificial liver) by using trypsin or the like. You must use proteolytic enzymes and chemicals. Therefore, cells that had formed a multi-layered assembly are individually separated or the cells are denatured and the original functions of the cells are impaired, or various artificial substrates such as proteolytic enzymes and chemicals are contaminated and contaminated. Have problems such as.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0008

[Correction method] Change

[Correction content]

[0008]

To achieve the above object, when a cell culture support having a substrate coated with an appropriate amount of a polymer is used, hepatocytes can be kept alive.
In addition, it can be cultured in the form of spheroids, and the spheroids can be exfoliated and recovered simply by changing the temperature. Furthermore, the recovered cells retain their original functions without any deterioration. Heading out, the present invention has been accomplished.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

The polymer may be coated on the substrate by a chemical method or a physical method, which will be described later, alone or in combination. When the monomer is used for coating, the monomer may be in a gas, liquid or solid state. Further, even when a polymer is used, the polymer may be in a liquid state or a solid state. When these are combined by a chemical reaction, electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation,
Plasma treatment, corona treatment, and generally used organic reactions such as radical and ionic reactions when the substrate and the coating material have appropriate reactive functional groups can be used. Examples of the method based on physical interaction include a method in which a coating material is used alone or a matrix having a good compatibility with a substrate is used as a medium, and physical adsorption such as coating and kneading is used, but the method is not limited thereto. Absent.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

The hepatocyte spheroids of the present invention are prepared by culturing hepatocytes on the cell culture support obtained as described above. Examples of hepatocytes include rat hepatocytes, human hepatocytes, bovine hepatocytes and the like. Such hepatocytes can be obtained by methods well known to those skilled in the art. In the culture conditions of the hepatocytes, the culture temperature is not more than the upper limit critical temperature of the polymer coated on the substrate, and is not less than the lower limit critical dissolution temperature of the polymer. The culture period is appropriately selected depending on the type of hepatocytes. Other culture conditions are not particularly limited,
Culturing may be carried out under the conditions commonly used in the art. For example, the medium may be one to which serum such as fetal calf serum (FCS) has been added, or a serum-free medium. After the above culture, hepatocyte spheroids are formed on the cell culture support. In order to separate and recover the hepatocyte spheroids thus formed from the cell culture support, it suffices to change the ambient temperature to not less than the upper critical solution temperature or not more than the lower critical solution temperature of the polymer,
The culture medium in which the cells are cultivated or other isotonic solution can be used and can be selected according to the purpose. At that time, for the purpose of efficiently and easily exfoliating the spheroids, the cell culture support may be lightly tapped or shaken, or the medium may be stirred using a pipette or the like.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Examples 1, 2, 3, 4, 5 Becton Dickinson Loveware (Be
Falcon (FALC) manufactured by cton Dickinson Labware
ON) 3002 Petri dish was used, and rat hepatocytes were adopted as the cells to be cultured. N-isopropylacrylamide was dissolved in isopropyl alcohol at a concentration shown in Table 1, and N, N'-methylenebisacrylamide was used as a cross-linking agent in an amount of 0.
8% by weight was added, and the solution was placed on a Petri dish at 0.1 m.
After the addition, the surface of the Petri dish was coated with poly-N-isopropylacrylamide by irradiating each with an electron beam at a dose shown in Table-1. After the electron beam irradiation, the Petri dish was thoroughly washed with ion-exchanged water to remove residual monomers and free poly-N-isopropylacrylamide not bound to the surface of the Petri dish,
Dry in a clean bench and add ethylene oxide
After (EO) gas sterilization, deaeration was further performed to obtain a cell culture support material.

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

[0027]

[Table 2]

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

Comparative Examples 3 and 4 Falcon 3002 Petri dish manufactured by Becton Dickinson Labware was used as a support base material for cell culture, and N-isopropylacrylamide was used at the concentrations shown in Table 4 and electron beams were used. Experiments similar to those in Examples 1, 2, 3, 4, 5 were performed except that irradiation was performed at the dose shown in FIG.
Culture of rat liver parenchymal cells was carried out according to Examples 1, 2, 3, 4, and 5.
The same method was adopted. Then, after 3 days of culturing, the cells that had adhered and proliferated were detached by cooling to 5 ° C. and leaving them to determine the detachment recovery of proliferating cells. The results are shown in Table-5.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Koichi Nakamura 1030, Sonobe, Wakayama City, Wakayama Prefecture 15 (72) Inventor Mitsuo Okano 6-12-12 Kokufudai, Ichikawa City, Chiba Prefecture (72) Noriko Yamada Itabashi Ward, Tokyo Prefecture 6-10 Maenocho Heights 1-601 (72) Inventor Yasuhisa Sakurai 3-17-6 Eifuku, Suginami-ku, Tokyo

Claims (2)

[Claims] 1. An upper limit of hepatocytes on a cell culture support comprising a base material surface coated with a polymer having a critical dissolution temperature in water of 0 to 80 ° C. in a coating amount of ²⁰ to 80 μg / cm ^2. Culture at or below the critical melting temperature or above the lower critical melting temperature,
Then, a method for preparing hepatocyte spheroids, which comprises exfoliating and recovering at an upper critical melting temperature or higher or a lower critical melting temperature or lower. 2. A hepatocyte spheroid prepared by the preparation method according to claim 1.