JP2003174869A - Base material for adhesive culture of multifunctional organ cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a base material for culture capable of growing multifunctional organ cells in adhering them to the base material and maintaining function inherent to the cells. <P>SOLUTION: The base material for adhesive culture of multifunctional organ cells (A) has a polypeptide (B) having at least one least amino acid sequence expressing a cell adhesion signal in one molecule. As (A), liver cells, pancreas cells, kidney cells, nerve cells, spleen cells, ovary cells and spermary cells are preferable. As the least amino acid sequence, Arg Gly Asp, Leu Asp Val, Arg Glu Asp Val, Tyr Ile Gly Ser Arg, Pro Asp Ser Gly Arg, Arg Tyr Val Val Leu Pro Arg, Leu Gly Thr Ile Pro Gly, Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile, Ile Lys Val Ala Val, Leu Arg Glu, Asp Gly Glu Ala and His Ala Val are preferable. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a culture substrate for adherent culture of multifunctional organ cells. More specifically, efficiently maintaining the original function of multi-functional organ cells,
The present invention relates to a culture substrate for growing in a state of being attached to a substrate.

[0002]

2. Description of the Related Art As a method for culturing multifunctional organ cells, a method is known in which a dish coated with type I collagen is used as a culture substrate (Non-patent Document 1).

[0003]

[Non-Patent Document 1] Uchino, J., American Society Artificial Internal Organs. 34, 1988, 97
Page 2-977

[0004]

However, even when a culture substrate coated with type I collagen is used, a multifunctional organ cell taken out of a living body is ex vivo and the original function of the organ (for example, the function of the cell) is obtained. , It is difficult to grow in the state of adhering to the base material while maintaining the ammonia metabolism function in the case of liver cells), and use it for devices such as drug metabolism simulator, drug production device and hybrid artificial liver. I couldn't. That is, an object of the present invention is to provide a culture substrate capable of growing a multi-functional organ cell in a state of being adhered to the substrate and maintaining the original function of the cell.

[0005]

Means for Solving the Problems As a result of intensive studies, the present inventor has found that the above-mentioned multi-functional organ cells are cultured by using a culture substrate containing a cell adhesion active substance. The present invention has been achieved by finding that the object can be achieved. That is, the feature of the culture substrate of the present invention is that it is a substrate for adherent culture of multifunctional organ cells (A), and at least one minimum amino acid sequence expressing a cell adhesion signal is present in one molecule. It has the polypeptide (B) which it has.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A multifunctional organ cell (A) is obtained from a multifunctional organ, and functions of the organ (for example, ammonia metabolism function in the case of liver, insulin secretion function in the case of pancreas, etc.). Cells having Examples of (A) include liver cells, pancreatic cells, kidney cells, nerve cells, spleen cells, ovary cells, testis cells, lung cells, prostate cells and mammary cells. Of these, liver cells, pancreatic cells, kidney cells, nerve cells, spleen cells, ovary cells and testis cells are preferred.

There is no particular limitation on the method for obtaining the multifunctional organ cells, and the method can be directly taken out from a normal multifunctional organ in the living body (primary cells can be obtained), or from a cancerous multifunctional organ in the living body. Examples of the method include a method of directly taking out (a cancer cell can be obtained), a method of obtaining a cell (cell line) transformed from a primary cell into a cell that can be passaged for many generations, etc. from a cell bank or a vendor. . Among these, the method of directly taking out from a normal multifunctional organ in a living body is preferable because it has the original function of the multifunctional organ as it is.

Next, the polypeptide (B) having at least one minimal amino acid sequence showing a cell adhesion signal in one molecule will be described. As the minimum amino acid sequence that expresses a cell adhesion signal, any amino acid sequence that functions as an adhesion signal can be used. For example, see “Pathophysiology” Vol. 9, No. 7 (1990)
Examples thereof include those described on page 527. Of these, Arg Gl has a large number of cell types that adhere.
y Asp sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser
Gly Arg sequence (3), Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg A
sn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile
Lys Val AlaVal sequence (7), Leu Arg Glu sequence, Asp Gly
At least one kind of sequence selected from the group consisting of Glu Ala sequence (8) and His Ala Val sequence is preferable, and more preferably Arg Gly Asp sequence, Ile Lys Val Ala Val sequence (7) and His Ala Val sequence. It is at least one kind of sequence selected from. The amino acid sequence is represented by the three-letter amino acid code, and the sequence number corresponding to the amino acid sequence listing is added in parentheses (the same applies hereinafter).

At least one of the above-mentioned minimum amino acid sequences must be contained in one molecule in the polypeptide (B). When the minimum amino acid sequence is present, cell adhesion activity is enhanced, and it becomes possible to promote the growth of multifunctional organ cells while maintaining the original function. On the other hand, when the above-mentioned minimum amino acid sequence is not contained, cell adhesion is reduced, resulting in insufficient cell growth, especially when a serum-free medium is used.

The content of this minimum amino acid sequence is preferably 3 to 50, more preferably 5 to 40, and particularly preferably 10 per molecule from the viewpoint of cell adhesion and proliferation.
~ 30. When the content is in this range, the cell adhesion activity is further increased, and it tends to be easy to promote the growth of the multifunctional organ cells while maintaining the original function.

The number average molecular weight (Mn) of (B) is 5,0 in terms of low toxicity to cells and high adhesiveness.
It is preferably from 0 to 5,000,000, more preferably from 10,000 to 1,000,000, and particularly preferably from 50,000 to 500,000. That is,
The number average molecular weight (Mn) of (B) is preferably 5,000 or more, more preferably 10,000 or more, particularly preferably 50,000 or more, and 5,000,00.
It is preferably 0 or less, more preferably 1,000,00.
It is 0 or less, particularly preferably 500,000 or less. In addition, the number average molecular weight (Mn) of (B) is SDS-PAG.
By method E (Na dodecyl sulfate-polyacrylamide gel electrophoresis), (B) was developed in water and the migration distance was compared with a standard substance.

(B) is an amino acid sequence that enhances the thermal stability of (B) in addition to the minimum amino acid sequence that exhibits a cell adhesion signal, for example, Gly Ala Gly derived from silk fibroin.
It is preferable to have at least two AlaGly Ser sequences (9), more preferably 5 or more amino acid sequences, and particularly preferably 3 to 30 amino acid sequences.

As (B), for example, (Gly Ala Gly
Ala Gly Ser) ₉ sequences (10) and ArgGly Asp sequence and a polypeptide having a polypeptide having the (Gly Ala Gly Ala Gly Ser) 9 sequences (10) and Tyr Ile Gly Ser Arg sequence (2), (Gly A polypeptide having an Ala Pro (Gly Pro Pro) ₄ ) ₂ sequence (11) and an Arg Gly Asp sequence,
(Gly Ala Pro (Gly Pro Pro) ₄ ) ₂ array (11) and Tyr I
a polypeptide having the le Gly Ser Arg sequence (2),
(Gly Ala Gly Ala Gly Ser) ₉ sequence (10) and IleLys V
Examples include a polypeptide having the al Ala Val sequence (7) (Japanese Patent Laid-Open No. 3-502935).

The commercially available product (B) is, for example, Pronectin F manufactured by Sanyo Chemical Industry Co., Ltd.
(Manufactured by genetically modified Escherichia coli, which contains Arg Gl in one molecule.
y Asp sequence and (Gly Ala Gly Ala Gly Ser) ₉ sequence (1
0) and about 13 each of number average molecular weight (Mn) of about 1
10,000 polypeptides), Pronectin F plus (pronectin F made water-soluble by reacting with dimeraminoethyl chloride), Pronectin L (manufactured by genetically modified E. coli, and Ile Lys Val in one molecule) Ala Va
l sequence (7) and (Gly Ala Gly Ala Gly Ser) ₉ sequence (1
0) and a number-average molecular weight (Mn) of about 90,000 each having about 7) and the like. In addition, Takara Shuzo Co., Ltd.
Manufactured by RetroNectin (recombinant human fibronectin CH-296) {polypeptide having a number average molecular weight (Mn) of about 60,000 having one CS1 signal which is a human fibronectin cell adhesion signal and one cell adhesion domain Type III and heparin binding domain II}, RGDS-Protein A {polypeptide having a number average molecular weight (Mn) of about 30,000 in which an Arg Gly Asp sequence is inserted in Protein A (IgG binding domain)} can also be used as (B).

The method for producing (B) is not particularly limited, and it can be produced in the same manner as a conventionally known method for peptide synthesis. For example, organic synthesis methods (solid phase synthesis method, liquid phase synthesis method, etc.) And biochemical synthetic methods [genetically modified microorganisms (yeast,
Bacteria, Escherichia coli, etc.)] and the like.
Regarding the organic synthesis method, for example, “Biological Chemistry Laboratory Lecture 2, Protein Chemistry (2)”, 641 edited by the Biochemical Society of Japan.
˜Page 694 (May 20, 1987; issued by Tokyo Kagaku Doujin Co., Ltd.). Regarding the biochemical synthesis method, for example, Japanese Patent Publication No. 3-50293
The method described in Japanese Patent No. 5 is used. From the viewpoint of easily synthesizing a high molecular weight (B), a biochemical synthesis method using a genetically engineered microorganism is preferable, and a method in which genetically engineered E. coli is used is particularly preferable.

The content of (B) is preferably 0.1 μg to 100 mg, more preferably 1 μg to 10 mg, particularly preferably 10 μg to 1 mg, and most preferably 100 to ⁵ per 100 cm ² of culture surface area of the culture substrate.
It is 00 μg. That is, as the content of (B),
0.1 μg per 100 cm ² of culture surface area of the culture substrate
The above is preferable, more preferably 1 μg or more, particularly preferably 10 μg or more, most preferably 100 μg or more and 100 mg or less, further preferably 10 mg or less, particularly preferably 1 mg or less, most preferably 500 μg or less. The culture surface area means the surface area of the surface of the culture substrate to which the multifunctional living organ cells (A) can adhere. Here, fine irregularities (for example, 0.1 mm or less) that do not allow cells to enter are treated as flat surfaces, but for those with ribs (ridges) provided to increase the culture surface area, the surface area of the ribs Is included in the culture surface area. Further, as the culture surface area of the culture substrate, the surface area of the culture container described in the catalog by the substrate manufacturer can be applied as it is.

The culture substrate of the present invention need only have (B) in the culture container, may be kneaded into the culture container, or may be attached to the surface thereof. The culture vessel is not particularly limited, and those generally used for cell culture can be used, and for example, plates, petri dishes, T-flasks, roller bottles, microcarrier beads, hollow fibers and the like can be used. As a material for these culture vessels, any material can be used as long as it can be used for cell culture, and examples thereof include plastic, glass, naturally derived materials (dextran, cellulose, etc.), inorganic materials (hydroxyapatite, etc.), and the like. .

As a method of obtaining the culture substrate of the present invention, for example, a method of coating a culture container with (B),
And (B) are kneaded into the raw material of the culture vessel and then molded into the culture vessel. As a method of coating (B) on the culture substrate, a solution in which (B) is dissolved in a solvent is prepared in advance, added to the culture container, left for a predetermined coating time, and then the excess solution is discarded and dried, or A method of drying without discarding the excess solution, or adding the solution of (B) to the roller bottle and rotating the roller bottle for a predetermined coating time, then discarding the excess solution and drying,
Method to dry while rotating without discarding excess solution,
A method in which microcarrier beads are put in the solution of (B) and a predetermined coating time is agitated as necessary, and then taken out of the solution and dried as necessary, and a solution of (B) is coated in a hollow fiber for a predetermined coating time. Examples thereof include a method of circulating and drying if necessary.

There are no particular restrictions on the solvent used to prepare the solution (B), but an inorganic salt, organic acid salt, amino acid, vitamin, alcohol, lipid / sugar, acid and / or base may be used. An aqueous solution containing 50% by weight (preferably 1% to 30% by weight), water or the like can be used. Examples of the inorganic salt include metal halide salts, metal sulfate salts, metal phosphate salts,
Metal nitrates, metal carbonates, metal perhalogenates and the like can be used, and examples thereof include sodium chloride, sodium sulfate, sodium phosphate, calcium chloride, iron nitrate, potassium chloride, magnesium sulfate, sodium carbonate, sodium hydrogen phosphate and phosphorus. Examples thereof include potassium acid salt, potassium hydrogen phosphate, copper sulfate, iron sulfate, lithium chloride, sodium bromide, lithium bromide sodium perchlorate and lithium perchlorate.

Examples of the organic acid salt include sodium formate, sodium acetate, lithium acetate and sodium tartrate. Examples of amino acids include arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan,
Examples thereof include tyrosine, valine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine and glycine. Examples of vitamins include choline,
Examples include inositol, nicotinamide, glutamine, vitamin A and vitamin B ₁₂ . Examples of alcohols include methanol, ethanol, isopropyl alcohol and butanol. Examples of lipids / sugars include lipids, monosaccharides, disaccharides, oligosaccharides, amino sugars and acidic sugars. As the acid, an inorganic acid, an organic acid having 1 to 6 carbon atoms and the like can be used, and examples thereof include hydrochloric acid, phosphoric acid, acetic acid, formic acid, phenol and sulfuric acid.
As the base, an inorganic base and an organic base having 2 to 6 carbon atoms can be used, and examples thereof include sodium hydroxide, potassium hydroxide, ammonia and triethylamine.
Examples of water include distilled water, ion-exchanged water, tap water, and ion-exchanged distilled water. Among these solvents, an aqueous solution and water containing an inorganic salt, an acid and / or a base are preferable, and an aqueous solution and water containing an inorganic salt are more preferable,
Particularly preferred is an aqueous solution containing an inorganic salt.

The concentration of the solution (B) is 1 ml of the solvent,
0.01 μg to 100 mg is preferable, more preferably 0.1 μg to 10 mg, and particularly preferably 1 μg to 1 m.
It is g. That is, the concentration of the solution of (B) is 1 m of the solvent.
It is preferably 0.01 μg or more, more preferably 0.1 μg or more, and particularly preferably 1 μg or more, per 1
Further, it is preferably 100 mg or less, more preferably 10 mg.
mg or less, particularly preferably 1 mg or less. The coating time varies depending on the culture substrate used, but is usually 30 seconds to 48 hours, preferably 1 minute to 24 hours, more preferably 3 minutes to 12 hours. That is, the coating time varies depending on the culture substrate used, but is usually 30 seconds or longer, preferably 1 minute or longer,
It is more preferably 3 minutes or longer, and usually 48 hours or shorter, preferably 24 hours or shorter, and more preferably 12 hours or shorter. There is also no particular limitation on the drying conditions performed as necessary, and ordinary methods can be applied, for example,
If necessary, use a normal air dryer or a vacuum dryer to reduce the
~ 200 ° C, 0.001 Pa ~ under atmospheric pressure, 1 ~
It can be done by drying for 100 hours.

Further, before or after the drying, which is carried out as the case requires, it is possible to wash with an aqueous solution containing an inorganic salt or water by a usual method. Moreover, you may sterilize after coating as needed. The sterilization method is not particularly limited, and examples thereof include radiation (gamma ray and the like) sterilization, ethylene oxide gas sterilization, autoclave sterilization and dry heat sterilization.

The method for culturing the multifunctional organ cell (A) using the substrate of the present invention is not particularly limited, and a usual method can be used. As the medium, depending on the type of (A) used, MEM medium, BME medium, DM
E medium, αMEM medium, IMEM medium, ES medium, DM
-160 medium, Fisher medium, F12 medium, WE medium, RPMI medium and the like, basal medium described in "Technology of Tissue Culture, Third Edition, edited by The Tissue Culture Society of Japan, 3rd Edition", page 581, serum components (bovine Fetal serum and the like) and commercially available serum-free medium [serum-free medium ASF103, ASF104, ASF3 manufactured by Ajinomoto Co., Inc.]
01, Gibco serum-free medium CHO-SFM, same VP-
SFM, etc.] are used. The amount of the medium used varies depending on the type of culture substrate used and the type of (A), but is usually 0.0001 to 50 per cm ^{2 of} the culture substrate surface area.
mL, preferably 0.001 to 5 mL, more preferably 0.01 to 0.5 mL. That is, the usage amount (mL) of the medium is usually 1 cm ^{2 of the} surface area of the culture substrate,
It is 0.0001 or more, preferably 0.001 or more, more preferably 0.01 or more, and usually 50 or less, preferably 5 or less, more preferably 0.5 or less.

Further, if necessary, cell growth factor (C)
By including the compound (A) in the medium, the proliferation rate of (A) can be further increased, the cell activity can be enhanced, and the function originally possessed by the cell can be expressed. The cell growth factor (C) is a substance having an activity of growing cells, and includes, for example, FGF, VEGF, HGF, EGF, PDGF, I.
Examples thereof include GF and BMP, and in addition to them, they are described, for example, in "Minami Ueda Tissue Engineering (1999)", pages 43 to 51, published by the Foundation of Nagoya University Press, and in the references cited therein. Things etc. are also used. Among these, FGF, VEGF, HGF and EGF are preferable, and HGF and EGF are more preferable, as those having high proliferation activity of multifunctional organ cells.

When cell growth factor (C) is used,
The content of (C) is 0.0 with respect to 1000 mL of the medium.
The amount is preferably 01 to 50,000 μg, more preferably 1 to 100 μg, and particularly preferably 10 to 90 μg. That is, in this case, the content (μg) of (C) is
The amount is preferably 0.001 or more, more preferably 1 or more, particularly preferably 10 or more, and preferably 50,000 or less, more preferably 100 or less, and particularly preferably 90 or less, relative to 1000 mL of the medium.
When the content of (C) is within this range, cell proliferation is further promoted. Examples of the method of adding (C) to the medium include a method of directly adding (C) to the medium, a method of adding (C) previously dissolved or dispersed in the solvent to the medium, and the like.

Further, in the medium, if necessary, other components (D) such as stabilizers (eg antioxidants and antibacterial agents) and pH adjusters (eg calcium carbonate, calcium phosphate etc.), etc. Can be included.

The amount of multifunctional organ cells (A) varies depending on the type of culture substrate used and the type of (A), but is usually 0.1 to 1 per 1 cm ^{2 of} culture substrate surface area.
The number is preferably 10,000,000, more preferably 1 to 5,000,000, and particularly preferably 1 to 1,000,000. The culture conditions are not particularly limited, and the CO ₂ concentration is 1 to 20% by volume, the temperature is 5 to 45 ° C., and the time is 1 to 100 days.
If the medium is replaced every 7 days, it may be cultured. A particularly preferable example is, for example, culturing under conditions of CO ₂ concentration of 5% by volume and 37 ° C. for 1 to 36 days while changing the medium every 2 to 3 days.

The culture substrate of the present invention is capable of proliferating multi-functional organ cells while maintaining their original functions, and can be applied to the following devices, etc.
It can be usefully used in researches using cells such as in drug discovery and regenerative medicine. A drug metabolism simulator for measuring the metabolic rate of drugs and determining the metabolic mechanism (drug discovery research field), which was previously only possible in animal experiments. For example, the concentration of the drug or metabolite in the medium can be measured by culturing the medium by adding the drug to the medium in a state where the primary liver cells are grown in a monolayer on a 96-well microplate coated with the polypeptide (B). , How the drug changes is required, and the drug metabolism in the living body can be simulated.

A pharmaceutical production apparatus for producing cytokines and hormones produced by multifunctional organ cells. For example, the cells produce insulin in the medium by adding glucose to the medium in a state in which primary pancreatic Langerhans islet cells are grown in a monolayer on the surface of microcarrier beads coated with (B). A drug can be produced by collecting and isolating this insulin.

Hybrid artificial liver (using liver cells) that temporarily or semi-permanently replaces the function of the liver, hybrid artificial pancreas (using pancreatic cells) that secretes insulin temporarily or semi-permanently, and temporary Alternatively, a hybrid artificial dialyzer (using kidney cells) that semi-permanently dialyzes blood. For example, on the surface of a hollow fiber coated with the polypeptide (B), primary liver cells, primary pancreatic Langerhans islet cells or primary kidney cells grown in a monolayer, by circulating blood, respectively,
It can be used as a hybrid artificial liver, a hybrid artificial pancreas or a hybrid artificial kidney.

[0031]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. <Example 1><Preparation of cell culture plate> According to the method described in Example in JP-A-3-502935, (Gly Ala Gly Al
a Gly Ser) ₉ sequences (10) and 12 Arg Gly Asp sequences
A recombinant protein E. coli produced protein "SLPF" having an Mn of about 110,000 was prepared. Next, a 4.5 normal lithium perchlorate solution of SLPF (concentration of SLPF; 1 mg /
ml) was diluted with a phosphate buffer solution (PBS) so that the concentration of SLPF was 50 μg / ml to prepare an SLPF solution. 0.5 ml of the SLPF solution was added to a 35 mmφ cell culture dish manufactured by Asahi Techno Glass, and left at room temperature for 1 week to dry (SLPF coating amount = 25 μg / dish). Further, the plate was washed twice with 2 ml of PBS to obtain the culture substrate (dish 1) of the present invention.

<Cell culture; primary rat hepatocytes> Primary rat hepatocytes were isolated from 7-week-old male Wistar rats by the two-step collagenase perfusion method. This dispersion was added to a medium (Williams' medium E manufactured by Sigma; 1000 mL, E
GF; 50 μg, insulin; 10 mg, CuSO ₄ ;
0.1 μmol, H ₂ SeO ₃ ; 3 μg, ZnSO ₄ ; 50 p
Mol, linoleic acid; 50 μg, penicillin; 58.5
mg, streptomycin; 100 mg, NaHC
O ₃ ; 1.05 g, HEPES; 1.19 g) was suspended to obtain a cell suspension having a cell concentration of 2.5 × 10 ⁵ cells / ml. 2 ml of this cell suspension was added to the culture substrate (dish 1) (cell number: 500,000 cells / dish), and 37
Culturing was carried out at 5 ° C. and 5 vol% CO ₂ . 4 hours after cell seeding, 1
The medium was exchanged after 3 days.

On the 1st, 2nd, 3rd and 5th days after the cell seeding, the number of cell nuclei was counted by the cell nuclei counting method using crystal violet by Wezel (nuclei-counting method) to pass through the culture period. The immobilization density of cells per one dish (arithmetic mean value at 1, 2, 3 and 5 days) was determined. At the same time, the morphology of cells [whether they are adhered to the substrate or in a floating state (spheroid)] on the 4th, 2nd and 3rd days was observed with a phase contrast microscope.
The same culture was performed using a medium containing 1 mM-NH ₄ Cl, and ammonia was left in the medium according to the prescription using an ammonia test Wako kit manufactured by Wako Pure Chemical Industries, Ltd. on the first, second and third days. By measuring the amount of urea and the amount of urea at the same time as counting the number of cell nuclei, the ratio of ammonia metabolism and the ratio of nitrogen metabolism [urea amount (mol)]
/ Ammonia amount (mol); the same applies hereinafter. The results are shown in Table 1 and FIGS.

<Comparative Example 1> The same as Example 1 except that instead of the culture substrate (dish 1), a 35 mmφ cell culture dish made by Asahi Techno Glass was used as it was as a culture substrate for comparison (dish 2). Then, the cells were cultured, and the immobilization density, cell morphology, ammonia metabolism activity and nitrogen metabolism ratio were determined. These results are shown in Table 1 and FIG.
~ 6.

Comparative Example 2 0.3 manufactured by Nitta Gelatin Co., Ltd.
Culture medium for comparison (dish 3) was prepared in the same manner as in Example 1 except that a solution of a weight% type I collagen A hydrochloric acid solution (pH 3) diluted with PBS to 200 μg / ml was used instead of the SLPF solution. Got Then, cell culture was performed in the same manner as in Example 1 using the culture substrate (dish 3), and the immobilization density, cell morphology, ammonia metabolism activity and nitrogen metabolism ratio were determined. The results are shown in Table 1 and FIGS.

[0036]

[Table 1]

As shown in Table 1, the immobilization density in Example 1 and Comparative Example 2 in the form of a monolayer was the initial immobilization density (about 45% of hepatocytes had two nuclei in one cell). Therefore, the initial immobilization density determined by the number of nuclei was higher than the initial number of cells, which is 1.45 times that of 750,000 cells / dish, and cell proliferation was observed. In addition, as a result of observing the morphology of the cells, it can be seen that cell spreading has started at 4 hours except for Comparative Example 1 (Table 1, FIGS. 1, 4, and 7).
Further, on the second day, cells are aggregated in Comparative Example 1, but spreading and proliferation proceed in Example 1 and Comparative Example 2, and it is understood that they are proliferating in a monolayer (Table 1, FIG. 2). 5 and 8). On the 3rd day, Comparative Example 1 was completely spheroidal and formed into floating clumps, but in Example 1, cell boundaries and nuclei were proliferated in a single layer with clear states. On the other hand, in Comparative Example 2, it is considered that the cell boundaries and nuclei are unclear, the occupied area of the cells is increased, and the cell activity is low (Table 1, FIGS. 3, 6, and 9).

Regarding the ammonia metabolism activity, as shown in Table 1, on the first day, all the Examples and Comparative Examples show high metabolic activity. However, it can be seen that in Comparative Example 2 only, the metabolic activity started to decrease from the second day, and the ammonia metabolic activity was hardly seen on the third day. Further, in Example 1 and Comparative Example 1, the nitrogen metabolism ratio was almost constant during the culture period, and it can be seen that the normal cell activity is maintained. In Comparative Example 2, it can be seen that the nitrogen metabolism ratio increases and the cell activity decreases depending on the culture period. That is, it is shown that only the culture substrate of the present invention (Example 1) can be grown in a state in which the cells of the multifunctional manufacturer are adhered to the substrate while maintaining the function of the cells.

<Relationship between SLPF application amount, average immobilization density of cells, cell morphology, ammonia metabolic activity and nitrogen metabolism ratio> In Example 1, the concentration of SLPF was 50 μm.
From g / ml (0.5 ml input to the dish), 1
0 μg / ml (0.5 ml input into dish), 1
μg / ml (1ml input to dish), 0.1μ
g / ml (2 ml input into dish) or 0.01
The amount of SLPF applied was changed from 25 μg / dish to 5 μg / dish, 1 μg / dish, 0.2 μg / dish or 0.02 μg / dish by changing to μg / ml (2 ml input into the dish) respectively. (Dish Nos. 4 to 7) were prepared and the average immobilized density of cells, cell morphology, ammonia metabolism activity and nitrogen metabolism ratio were measured in the same manner as in Example 1. The results are shown in Table 2 and FIGS.

[0040]

[Table 2]

In this experiment using primary rat hepatocytes, the amount of SLPF applied had little effect on the immobilization of hepatocytes, but the morphology (adhesion morphology) of the cells was greatly affected. In addition, both the rapid formation of characteristic morphologies such as monolayers and hemispheroids exhibited good ammonia metabolism activity, but urea synthesis activity was not so high. That is, it can be seen that the morphology of hepatocytes can be easily controlled by the application amount of SLPF (RGD immobilization density), and cells exhibit functions such as ammonia metabolism in correlation with the morphology.

[0042]

EFFECTS OF THE INVENTION By using the substrate for culturing a multifunctional organ cell of the present invention, the function of the original organ possessed by the cell (for example, in the case of liver cell, ammonia metabolism function,
In the case of the pancreas, it can be proliferated while maintaining the insulin secretory function). This makes it easier to feed and discharge the medium and allows direct observation of cells with a microscope, which makes it useful for research using cells such as in drug discovery and regenerative medicine. It can contribute to the practical application of production equipment and equipment such as hybrid artificial liver and artificial kidney.

[0043]

[Brief description of drawings]

FIG. 1 shows that, in Example 1, the cells at 4 hours of culture were magnified 1 times.
It is the figure which took a micrograph at 00 times.

FIG. 2 shows that the cells on the second day of culture in Example 1 were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 3 shows that the cells on the third day of culture in Example 1 were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 4 shows that, in Example 2, the cells at 4 hours of culture were magnified 1 times.
It is the figure which took a micrograph at 00 times.

FIG. 5: For Example 2, the cells on the second day of culture were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 6: For Example 2, cells on day 3 of culture were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 7: In Example 3, cells at 4 hours of culture were magnified 1 times.
It is the figure which took a micrograph at 00 times.

FIG. 8: For Example 3, cells on day 2 of culture were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 9: For Example 3, cells on day 3 of culture were magnified 10 times.
It is the figure which took the micrograph at 0 times.

FIG. 10 is a photomicrograph of cells at 4 hours of culture when the amount of SLPF applied is 5 μg / dish (magnification 1
(00 times).

FIG. 11 is a photomicrograph of cells on the second day of culture when the amount of SLPF applied is 5 μg / dish (magnification: 10
It is the figure which is 0 times).

FIG. 12 is a photomicrograph of cells on day 3 of culture when the amount of SLPF applied is 5 μg / dish (magnification: 10
It is the figure which is 0 times).

FIG. 13 is a photomicrograph of cells at 4 hours of culture when the application amount of SLPF is 1 μg / dish (magnification: 1
(00 times).

FIG. 14 is a photomicrograph of cells on the second day of culture when the amount of SLPF applied is 1 μg / dish (magnification: 10
It is the figure which is 0 times).

FIG. 15 is a photomicrograph of cells on day 3 of culture when the amount of SLPF applied is 1 μg / dish (magnification: 10
It is the figure which is 0 times).

FIG. 16 is a micrograph (100 × magnification) of cells at 4 hours of culture when the amount of SLPF applied is 0.2 μg / dish.

FIG. 17 is a drawing of micrographs (magnification: 100) of cells on the second day of culture when the amount of SLPF applied was 0.2 μg / dish.

FIG. 18 is a drawing of micrographs (magnification of 100) of cells on the 3rd day of culture when the applied amount of SLPF was 0.2 μg / dish.

FIG. 19 is a microphotograph (magnification: 100) of cells at 4 hours of culture when the amount of SLPF applied is 0.02 μg / dish.

FIG. 20 is a micrograph (magnification: 100) of cells on the second day of culture when the amount of SLPF applied is 0.02 μg / dish.

FIG. 21 is a micrograph (magnification: 100) of cells on the 3rd day of culture when the amount of SLPF applied is 0.02 μg / dish.

[0044]

[Sequence list] <110> SANYO CHEMICAL INDUSTRIES, LTD. <120> Substrate for adherent culture of multifunctional organ cells <130> P5751 <150> Japanese Patent Application 2001-287886 <151> 2001-9-20 <160> 11 <210> 1 <211> 4 <212> PRT <213> Homo sapiens <400> 1 Arg Glu Asp Val   1 <210> 2 <211> 5 <212> PRT <213> Homo sapiens <400> 2 Tyr Ile Gly Ser Arg   1 5 <210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Pro Asp Ser Gly Arg   1 5 <210> 4 <211> 7 <212> PRT <213> Homo sapiens <400> 4 Arg Tyr Val Val Leu Pro Arg   1 5 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5 Leu Gly Thr Ile Pro Gly   1 5 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile   1 5 10 <210> 7 <211> 5 <212> PRT <213> Homo sapiens <400> 7 Ile Lys Val Ala Val   1 5 <210> 8 <211> 4 <212> PRT <213> Homo sapiens <400> 8 Asp Gly Glu Ala   1 <210> 9 <211> 6 <212> PRT <213> Bombyx mori <400> 9 Gly Ala Gly Ala Gly Ser   1 5 <210> 10 <211> 54 <212> PRT <213> Bombyx mori <400> 10 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala   1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala              20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser          35 40 45 Gly Ala Gly Ala Gly Ser      50 <210> 11 <211> 30 <212> PRT <213> Artificial Sequence <400> 11 Gly Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly   1 5 10 15 Ala Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro              20 25 30

Front page continuation (51) Int.Cl. ⁷ identification code FI theme code (reference) C07K 7/06 C07K 14/475 14/475 C12N 5/00 ZNAE F term (reference) 4B065 AA90 AC20 BB34 BC41 BD42 CA44 4H045 AA30 BA12 BA13 BA14 BA15 EA60

Claims (8)

[Claims] 1. A culture substrate for adherent culture of a multifunctional organ cell (A), which comprises a polypeptide (B) having at least one minimum amino acid sequence which expresses a cell adhesion signal in one molecule. A culture substrate comprising. 2. The (A) is at least one multifunctional organ cell selected from the group consisting of liver cells, pancreatic cells, kidney cells, nerve cells, spleen cells, ovary cells and testis cells. The described substrate. 3. The substrate according to claim 1 or 2, wherein the number of the minimum amino acid sequence that exhibits a cell adhesion signal is 3 to 50. 4. Arg Gly As, which is a three-letter amino acid code for the minimal amino acid sequence that expresses a cell adhesion signal.
p sequence, Leu Asp Val sequence, Arg Glu Asp Val sequence (1), Tyr Ile Gly Ser Arg sequence (2), Pro Asp Ser
Gly Arg sequence (3), Arg Tyr Val Val Leu Pro Arg sequence (4), Leu Gly Thr Ile Pro Gly sequence (5), Arg A
sn Ile Ala Glu Ile Ile Lys Asp Ile sequence (6), Ile
Lys Val Ala Val sequence (7), Leu Arg Glu sequence, Asp Gl
The base material according to any one of claims 1 to 3, which is at least one kind of sequence selected from the group consisting of y Glu Ala sequence (8) and His Ala Val sequence. 5. The base material according to any one of claims 1 to 4, wherein (B) further has at least two Gly Ala Gly Ala Gly Ser sequences (9) represented by a three-letter amino acid code. 6. The culture surface area of the substrate (B) is 100 cm.
The base material according to any one of claims 1 to 5, which is contained in a ratio of 0.1 µg to 100 mg per ² parts. 7. The substrate according to claim 1, further comprising a cell growth factor (C). 8. The substrate according to claim 7, wherein (C) is at least one cell growth factor selected from the group consisting of FGF, VEGF, HGF and EGF.