JP2002281964A - Cell production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selectively producing an objective cell without using 3T3 cell having the function to inhibit adhesion of the other cells to a culture carrier. SOLUTION: This cell production method is characterized by comprising carrying out a selective culture of the objective cell C by contacting a peptide A having in one molecule at least one minimum amino acid sequence representing an adhesion signal with a sample including at least a cell B to be eliminated and the objective cell C, wherein it is preferable that the cell B to be eliminated is fibroblast, while the objective cell C is preferably an epidermal cell or an epithelial cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing cells. More specifically, the present invention relates to a method for producing cells in which specific cells are selectively adhered to a culture carrier and cultured.

[0002]

2. Description of the Related Art Conventionally, as a cell production method for selectively adhering cells to a culture carrier and culturing the cells, epidermal cells are selected from skin pieces (including fibroblasts and epidermal cells) using 3T3 cells. A cell production method of culturing the cells by adhering them to a culture carrier, that is, irradiating 3T3 cells with radiation and losing the cell growth ability after seeding the cells in a petri dish and allowing them to adhere.
A method in which a cell solution (including fibroblasts and epidermal cells) obtained from a skin piece is seeded and cultured in a 3T3 cell-adhesive petri dish (fibroblasts are inhibited from adhering to the petri dish by 3T3 cells, Epidermal cells adhere and displace 3T3 cells, so that every time the medium is replaced, 3T3 cells and fibroblasts are removed, and only epidermal cells are adhered to a Petri dish and cultured.) Is known as a typical method. (Tissue Engineering, published by Nagoya University Press, October 10, 1999).

[0003]

SUMMARY OF THE INVENTION A method for producing cells in which 3T3 cells are selectively adhered to a culture carrier using 3T3 cells and cultured is disclosed. Special equipment and special facilities for irradiation are required, and there is a problem that it takes a long time of 3 to 4 weeks to selectively adhere and grow target cells for co-culture with 3T3 cells. Was. That is, an object of the present invention is to provide a method for simply and selectively producing target cells without using 3T3 cells having a function of suppressing adhesion of non-target cells to a culture carrier. .

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the above objects can be achieved by using a specific peptide, and have reached the present invention.
That is, the feature of the cell production method of the present invention is that a sample containing at least the removed cells (B) and the target cells (C)
The objective is to selectively culture the target cell (C) by contacting a peptide (A) having at least one minimum amino acid sequence showing an adhesion signal in one molecule.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As the minimum amino acid sequence representing a cell adhesion signal, any one that functions as an adhesion signal can be used. For example, "Pathophysiology, Vol. 9,
No. 7, pp. 527-535, 1990 "and" Magazine for Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66. "
Page 1992 ". Among these, preferred are RGD sequences, LDV sequences, represented by one-letter amino acid notation, from the viewpoint of high ability to suppress the adhesion of specific cells to a culture carrier (hereinafter, cell adhesion suppressing power). REDV sequence (1), Y
EKPGSPPPREVPRPRPGV sequence (2), K
NNQKSEPLIGRKKY array (3), YIGSR
Sequence (4), PDSGR sequence (5), RYVVLPR sequence (6), LGTIPG sequence (7), RNIAEIIK
DI sequence (8), IKVAV sequence (9), LRE sequence,
HHLGGAKQAGDV sequence (10), CQEPGG
LVVPPTDAP sequence (11), VTXG sequence (1
2), DGEA sequence (13), GVKGDKGNPGW
These are the PGAP sequence (14), the GEFYFDLRLLKGDK sequence (15), the FTLCFR sequence (16), and the HAV sequence.

More preferred are RGD sequence, YIGSR sequence (4), PDSGR sequence (5), RYVVLPR sequence (6), and LGTIPG sequence (expressed in one-letter amino acid notation) from the viewpoint of high cell adhesion suppressing power. 7), RNIAEIIKDI sequence (8), IKVAV
Sequence (9), LRE sequence, GVKGDKGNPGGWPG
The AP sequence (14), the GEFYFDLRLKKGDK sequence (15) and the HAV sequence,
GD array.

[0007] The peptide (A) has at least one of the above-mentioned minimum amino acid sequences in one molecule, and preferably has two to 50 in the molecule, because the cell adhesion-suppressing power is further increased. When the minimum amino acid sequence is not contained, the cell adhesion inhibitory power is low or does not appear. Peptide (A)
Is G as an amino acid sequence other than the minimum amino acid sequence.
It is more preferable to have two or more AGAGS amino acid sequences (17) in one molecule, since heat resistance is strong and sterilization is easy.

[0008] The number average molecular weight of the peptide (A) is preferably from 300 to 3,000,000, more preferably from 300 to 300,000, from the viewpoint of the cell adhesion suppressing power. The number average molecular weight of the peptide (A) was determined by SDS-
The peptide (A) is separated in water by PAGE (SDS polyacrylamide gel electrophoresis), and the migration distance is determined by comparing with a standard substance.

[0009] The method for producing the peptide (A) includes, for example, a genetic recombination method such as an organic synthesis method (enzymatic method, solid phase synthesis method, liquid phase synthesis method, etc.) and a genetic recombination method (microorganisms (yeast and bacteria, etc.)). , Cell genetic recombination method, etc.) and biological material purification / extraction method. Among these, the organic synthesis method and the gene recombination method are preferred from the viewpoint that the amino acid sequence of the peptide (A) is clear and the amount of impurities is small. More preferably, the amino acid sequence of the peptide (A) is easily designed and manufactured. This is a genetic recombination method from the viewpoint that it can be performed.

Regarding the organic synthesis method, for example, see “Biochemical Experiment Course 1, Chemistry of Protein IV (July 1, 1981)
Japan Biochemical Society, edited by The Japan Biochemical Society, published by Tokyo Chemical Co., Ltd.) and "Seizoku Chemistry Laboratory Course 2, Protein Chemistry (below)" (May 20, 1987, edited by the Biochemical Society of Japan, published by Tokyo Chemical Co., Ltd.) )). The gene recombination method is exemplified in, for example, Japanese Patent Publication No. Hei 7-501443. The biological material purification / extraction method is exemplified in, for example, JP-A-4-305600.

[0011] Peptides prepared by the organic synthesis method include, for example, RGDS (18), GRGDS (19), GRGD
SP (20), RGDSPASSKP (21) and polymers of one or more of these peptides. The polymerization degree of the polymer is usually 2 to 50. Examples of the peptide produced by the gene recombination method include Pronectin F (trade name, manufactured by Sanyo Chemical Industries, Ltd .; cell adhesion signal RGD)
Sequence and GAGAGS sequence (17), each having about 13 in one molecule, a polypeptide produced by recombinant Escherichia coli having a molecular weight of about 110,000, Pronectin L (trade name, manufactured by Sanyo Chemical Industries, Ltd .; cell) Adhesion signal IKV
A polypeptide produced by genetically engineered Escherichia coli having a molecular weight of about 90,000 having about 7 AV sequence (9) and about 7 GAGAGS sequence (17) in one molecule.

Peptides obtained by the biological material purification / extraction method include, for example, type I collagen, type II collagen, II
Type I collagen, type IV collagen, type V collagen,
Examples include proteins such as fibronectin, laminin, vitronectin, fibroin, fibrinogen, entactin, and amyloid P, and degradation products thereof. The degraded product means a degraded product in which a minimal amino acid sequence representing a cell adhesion signal remains, treated with an enzyme (such as collagenase), treated with an acid (such as hydrochloric acid), treated with an alkali (such as sodium hydroxide) or hydrolyzed. (For example, a degradation product excluding telopeptide can be obtained by enzymatic treatment of type I collagen).

Although the peptide (A) can be used as it is, it can be used as an absorbent carrier or an aqueous solution to which (A) is immobilized. As a method for immobilizing the peptide (A) on the absorbent carrier, for example, after immersing the absorbent carrier in the aqueous solution of (A) to immobilize the peptide (A) on the absorbent carrier, the peptide (A) can be immobilized on distilled water or the like. A method of washing the immobilized absorbent carrier and the like can be mentioned. The concentration of the peptide (A) in the aqueous solution is preferably 1 μg / L to 100 g / L, more preferably 20 μg / L to 20 g / L, based on the volume of the aqueous solution, from the viewpoint of the cell adhesion suppressing power. That is,
The upper limit of the aqueous solution concentration of (A) is preferably 100 g / L,
More preferably, it is 20 g / L, and similarly, the lower limit is 1 μm.
g / L is preferred, and more preferably 20 μg / L.

The absorption carrier on which the peptide (A) is immobilized,
Further, it is preferable to immobilize the blocking reagent from the viewpoint of cell adhesion suppressing power. Examples of the blocking reagent include serum albumin, plasma albumin, ovalbumin, conalbumin, lactalbumin, casein,
Skim milk, whey protein, collagen, gelatin, serum, plasma, blood, polyethylene glycol and the like.
Among these, serum albumin, plasma albumin, casein and serum are preferred, and serum albumin and serum are more preferred. As a method of immobilizing the blocking reagent on the absorbing carrier, for example, after the absorbing carrier is immersed in an aqueous solution of the blocking reagent to immobilize the blocking reagent on the surface of the absorbing carrier, the blocking reagent is immobilized with distilled water or the like. And a method for washing the absorbed carrier.

The concentration of the aqueous solution of the blocking reagent is based on the volume of the aqueous solution when the blocking reagent is serum albumin, plasma albumin, ovalbumin, conalbumin, lactalbumin, casein, skim milk, whey protein, collagen, gelatin, polyethylene glycol, or the like. The concentration is preferably 2 mg / L to 200 g / L, more preferably 20 mg / L to 20 g / L. That is, the upper limit of the concentration of the aqueous solution of the blocking reagent is preferably 200 g / L, more preferably 20 g / L, and similarly, the lower limit is preferably 2 mg / L, more preferably 20 mg / L.
/ L. When the blocking reagent is serum, plasma, blood, or the like, 10 μL /
L to 1 L / L (stock solution) is preferred, and more preferably 1 to 1 L / L.
mL / L to 500 mL / L. That is, the upper limit of the concentration of the aqueous solution of the blocking reagent is preferably 1 L / L (stock solution), more preferably 500 mL / L, and similarly, the lower limit is preferably 10 μL / L, and more preferably 1 m / L.
L / L.

As the absorbing carrier, for example, carriers such as petri dishes, tubes, flasks, plates, wells, beads and gels can be used. Petri dishes, tubes, flasks, plates, wells and beads can be used as general materials used for cell culture, for example, glass, polystyrene, polyethylene, polypropylene,
Examples include nylon, acrylic resin, and polyurethane. As a material of the gel, a general material used for cell culture can be used, and for example, a collagen gel or the like can be used.

When the peptide (A) is used as an aqueous solution, the concentration of (A) is preferably 1 μg / L to 100 g / L, more preferably 20 μg, based on the volume of the aqueous solution, from the viewpoint of the cell adhesion suppressing power. / L to 20 g / L. That is, in this case, the upper limit of the concentration of (A) is 10
It is preferably 0 g / L, more preferably 20 g / L, and likewise, the lower limit is preferably 1 μg / L, more preferably 20 μg / L. As a solvent for converting the peptide (A) into an aqueous solution, any solvent can be used as long as it is a solvent used for converting a protein into an aqueous solution, and examples thereof include a phosphate buffer and physiological saline.

When the solubility of the peptide (A) in water is 100 g / L or less based on the volume of water,
It is preferable to improve the solubility of (A) in water by modifying the amino group, carboxyl group, hydroxyl group, mercapto group and the like of (A) with a hydrophilic group. Examples of the hydrophilic group include an ammonium group (—N ⁺ H ₃ ) and a monoalkyl ammonium group (—N
⁺ H ₂ R), dialkyl ammonio group (-N ⁺ HR _2), trialkylammonio group (-N ⁺ R _3), carbonate carboxylate group (-CO ₂ ^-), sulfonate group (-SO ₃ ^-) and polyoxyethylene groups _{{- (CH 2 CH 2 O} ) n-H, n is an integer of 1 to 30} can be used and the like. R represents an alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl,
Propyl and isobutyl and the like. Among these hydrophilic groups, an ammonium group,
Monoalkylammonio group, dialkylammonio group,
Trialkylammonio groups and polyoxyethylene groups are preferred.

The method for modifying the hydrophilic group on the peptide (A) is not particularly limited, and known methods can be used.
The method described in “Tokuhyohei 10-500701” or “Chemical modification of proteins (by Motonori Ohno et al., Published by Gakkai Shuppan Co., Ltd.)” can be applied. For example, as a method for modifying the dialkylammonio group to the peptide (A), the following method can be mentioned. That is, a lithium perchlorate solution of Pronectin F (Sanyo Chemical Industries, Ltd.) and N, N-dimethylaminoethyl chloride hydrochloride are charged into a flask, and the inside of the flask is purged with nitrogen. Then, a solution of sodium hydroxide in lithium perchlorate is added thereto at a constant flow rate under stirring. 60 minutes after the start of stirring,
After adjusting the pH to 6.3 with acetic acid, the mixture is put into a dialysis tube, and dialyzed three times with distilled water. After dialysis, lyophilization is performed to obtain dimethylammonio group-modified Pronectin F in a powder state.

Any of the removed cells (B) and the target cells (C) can be applied to the cell production method of the present invention as long as they have adhesive properties. It is necessary that the adhesion to the peptide (A) is different. By utilizing this difference in adhesiveness, the removed cells (B)
Is eliminated, and the target cell (C) can be selectively cultured. The removed cell (B) is preferably a cell strongly adhering to a peptide having a pronectin F, fibronectin or RGD sequence, and examples thereof include fibroblasts. The target cells (C) include Pronectin L, type IV collagen or GVGKGDK.
Cells that strongly adhere to the peptide having the GNPGWPGAP sequence (14) are preferable, and include, for example, epidermal cells and epithelial cells.

The sample used in the cell production method of the present invention contains at least two types of (B) and two or more types of (C) if they contain at least the removed cells (B) and the target cells (C).
May be contained, but (C) preferably contains only one type. As the form of the sample, for example, a cell solution,
It can be in the form of known cell-containing samples such as tissue fluids and tissue fragments. As a method of contacting the peptide (A) with a sample containing at least the removed cells (B) and the target cells (C), any method can be applied as long as the peptide (A) can be brought into contact with the removed cells (B). And a method in which a sample is brought into contact with a peptide (A) solution, or a method in which a sample is brought into contact with an absorbent carrier on which peptide (A) is immobilized.

(A) uses a material in which the adhesion between (B) and (A) is higher than the adhesion between (C) and (A). For cells,
(A) is preferably a peptide having an RGD sequence (for example, Pronectin F, fibronectin, etc.), and (C) is preferably an epidermal cell or an epithelial cell. When (B) is an epidermal cell, (A) is preferably Pronectin L or type IV collagen, and (C) is preferably a fibroblast. When (B) is an epithelial cell, (A) is pronectin L
Alternatively, type IV collagen is preferable, and (C) is preferably fibroblasts.

The selective cultivation of the target cell (C) is carried out by contacting a sample containing (B) and (C) with the peptide (A) and then culturing it by transferring it onto a culture carrier, or Sample containing (B) and (C) above and peptide (A)
And culturing the cells as they are in contact.

The culture carrier used for culturing cells is not particularly limited as long as the target cell (C) adheres, and any carrier used for cell culture can be used. For example, petri dishes, tubes, flasks , Plates, wells, carriers such as beads and gels (the materials are the same as above),
Examples thereof include those obtained by subjecting a carrier surface to a hydrophilic treatment and those obtained by immobilizing the peptide (D) on the carrier surface. Among these, those in which the peptide (D) is immobilized on the carrier surface are preferred. The material of the culture carrier is the same as the material of the above-mentioned absorption carrier. Further, similarly to the above-mentioned “absorbent carrier on which peptide (A) is immobilized”, it is possible to further immobilize a blocking reagent on “culture carrier” or “culture carrier on which peptide (D) is immobilized”. It is preferable from the viewpoint of suppressing power. As the peptide (D), the same as the peptide (A) can be used. Among them, pronectin L, laminin, type IV collagen, and degradation of type IV collagen in which the minimum amino acid sequence representing a cell adhesion signal remains And GVKGDKGNPGWPGAP sequence (14)
Are preferred. The method for immobilizing the peptide (D) on the culture carrier is the same as the method for immobilizing (A) on the absorbent carrier.

[0025] (D) uses a material whose adhesion between (C) and (D) is higher than the adhesion between (B) and (D). When (D)
Is preferably Pronectin L or type IV collagen,
(B) is preferably a fibroblast. When (C) is an epithelial cell, (D) is preferably Pronectin L or type IV collagen, and (B) is preferably a fibroblast. Also,
When (C) is a fibroblast, (D) is preferably a peptide having an RGD sequence (eg, Pronectin F, fibronectin), and (B) is preferably an epidermal cell or an epithelial cell.

When the peptide (D) is used, the target cell (C) using the culture carrier on which the peptide (D) is immobilized is used.
Is selectively contacted with a sample containing (B) and (C) and a peptide (A), and then transferred onto a culture carrier having immobilized peptide (D) and cultured. The sample containing (B) and (C) is brought into contact with the peptide (A) on a culture carrier on which D) is immobilized, and the peptide (A) is cultured as it is. Are not the same. That is, for example,
Pronectin F cannot be used simultaneously as (A) and (D).

The medium used for culturing cells is not particularly limited, and any known medium used for cell culture can be used. For example, MEM medium, DME medium, Ham F-12
Medium, mixed medium of DME and ham F-12, EpiLif
e-KG2 and EpiLife.

These media include cell growth factors (IG)
Known additives such as F, EGF, and FGF, antibacterial agents (amphotericin B, gentamicin, penicillin, streptomycin, and the like), and serum (human serum, bovine serum, horse serum, sheep serum, and the like) can be contained.
When a cell growth factor is used, the concentration of the cell growth factor in the medium is preferably from 10 pg / L to 1000 μg / L,
More preferably, it is 100 pg / L to 100 μg / L. That is, in this case, the upper limit of the concentration of the cell growth factor in the medium is preferably 1000 μg / L, more preferably 100 μg / L, and similarly, the lower limit is 10 pg / L.
And more preferably 100 pg / L.

When an antibacterial agent is used, the concentration of the antibacterial agent in the medium is preferably 1 ng / L to 100 g / L, and more preferably 10 ng / L to 10 g / L. That is, in this case, the upper limit of the concentration of the antibacterial agent in the medium is 100
g / L is preferred, and more preferably 10 g / L. Similarly, the lower limit is preferably 1 ng / L, more preferably 10 g / L. When serum is used, the concentration of serum in the medium may be 1 × 10 ^{−6 to} 5 × 10 ⁻⁶ , based on the total volume of the medium, in order to reduce the risk of infectious diseases such as mad cow disease derived from the serum.
0% (v / v) is preferable, and 1 × 10 is more preferable.
^-5 to 10% (v / v), particularly preferably 1 × 10
^{-4 to} 2% (v / v). That is, in this case, the upper limit of the serum concentration in the medium is preferably 50% (v / v), more preferably 10% (v / v), and particularly preferably 2% (v / v). The lower limit is 1 × 10 ^-6 %
(V / v) is preferred, and more preferably 1 × 10 ⁻⁵ %
(V / v), particularly preferably 1 × 10 ⁻⁴ % (v / v).

The culture temperature is not particularly limited as long as the cells can grow well, and is usually about 15 to 45 ° C. The culture period is not particularly limited as long as the cells can grow well, and is usually about 1 to 30 days. The cell production method of the present invention can easily and selectively produce only the target cell (C), so that cells close to human tissue can be easily produced. Therefore, it can be used for research use such as cell line conversion of cells and medical use such as cultured tissue. The cultured tissue can be used for producing cultured skin, cultured cornea, cultured bone, cultured cartilage, artificial blood vessel, artificial liver, artificial kidney, artificial pancreas and the like.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Preparation of culture carrier: 100 μg / m of type IV collagen (manufactured by Nippon Becton, Dickinson and Company)
L is dissolved in 0.05N HCl at a concentration of L, and 3 mL of the type IV collagen solution is charged into a 60 mm diameter petri dish and reacted at room temperature for 1 hour. After 1 hour reaction, the remaining type IV collagen solution is discarded from the petri dish. Then, the Petri dish was washed three times with 5 mL of distilled water to prepare a culture carrier. The prepared type IV collagen coated petri dish is used up to 4
Stored at ° C.

Example 1 <Selective culture of epidermal cells from a mixed sample of fibroblasts and epidermal cells 1> 20 mg of peptide (A1) (trade name: Pronectin F, Sanyo Chemical Industry Co., Ltd.) was added at 4.5 M. Dissolve in 1 mL of lithium chlorate aqueous solution and further add Epi
100 in Life-KG2 medium (Kurashiki Boseki Co., Ltd.)
A double dilution was performed to prepare a Pronectin F-containing medium (M1) [a medium having an RGD concentration of 0.02 mM]. Into 5 mL of this Pronectin F-containing medium (M1), 2 million normal human skin fibroblasts (manufactured by Kurashiki Spinning Co., Ltd.) and 8 million normal human keratinocytes (manufactured by Kurashiki Spinning Co., Ltd.) were added. did. After leaving at room temperature for 1 hour, centrifugation (room temperature,
(1,000 rpm, 10 minutes), and the supernatant was removed by suction. Next, 5 mL of EpiLife-KG2 medium was added and stirred to obtain a cell solution (C1). Cell liquid (C1)
Cell number was measured with a hemocytometer.

The cell solution (C1) was diluted with EpiLife-KG2 medium so that the cell concentration became 200,000 cells / mL.
The 10mL of diluted solution was poured into type IV collagen-coated Petri dish, after stirring gently with the lid in a petri dish, and allowed to stand in a CO ₂ incubator, CO ₂ concentration 5
% (V / v) at 37 ° C. for 2 weeks. One day after the start of culture, 10 mL of EpiLife-KG2 medium
The medium was changed at the end of the day, and until the 7th day, EpiLife-K
The medium was changed every day using the G2 medium. Day 8-14
On the day, the medium was replaced every day with a medium prepared so that the calcium chloride concentration in the EpiLife-KG2 medium was 3 mM. On the fifteenth day, the medium was removed, and 5 mL of dispase (trade name, manufactured by Becton Dickinson Co., Ltd.) was added to perform cell detachment treatment.

Example 2 <Selective culture 2 of epidermal cells from a mixed sample of fibroblasts and epidermal cells 2> 1 mg of peptide (A2) (RGDS, Peptide Research Institute Co., Ltd.) was added to EpiLife-KG2 medium (Kurashiki Boseki Co., Ltd.) Dissolved in 100 mL with RGD
An S-containing medium (M2) was prepared [RGD concentration of 0.02
mM medium]. Subsequently, a cell solution (C2) was prepared in the same manner as in Example 1 except that the RGDS-containing medium (M2) was used instead of the pronectin F-containing medium (M1). Cell culture was performed in the same manner as in 1.

Example 3 <Modification of Peptide (A) with Hydrophilic Group> Peptide (A1)
(Pronectin F, Sanyo Chemical Industry Co., Ltd.) 30 g of a 4.5 M lithium perchlorate solution in which 1 g is dissolved and N, N-dimethylaminoethyl chloride hydrochloride 3 g are charged into the flask, and the inside of the flask is purged with nitrogen. . Next, stirring with a magnetic stirrer was started under room temperature conditions, and 26.5 mL of a 4.5 M lithium perchlorate solution in which 2 g of sodium hydroxide was dissolved was added at a flow rate of 1.68 mL / min. 60 minutes after the start of stirring, the content liquid is adjusted with acetic acid to pH
After adjusting to 6.3, the mixture was put into a dialysis tube, and dialyzed three times with 5 L of distilled water. After dialysis, lyophilization was performed to obtain dimethylammonio group-modified Pronectin F (peptide (A3)) in a powder state.

<Selective culture 3 of epidermal cells from a mixed sample of fibroblasts and epidermal cells 3> 20 mg of peptide (A3) (dimethylammonio group-modified Pronectin F) was added to Epi.
100 in Life-KG2 medium (Kurashiki Boseki Co., Ltd.)
and modified Pronectin F-containing medium (M1 +)
[A medium having an RGD concentration of 0.02 mM] was prepared. Subsequently, a cell solution (C1 +) was prepared in the same manner as in Example 1 except that the modified pronectin F-containing medium (M1 +) was used instead of the pronectin F-containing medium (M1), and then the cell solution (C1 +) was used. Cell culture was performed in the same manner as in Example 1.

Example 4 <Selective culturing of epidermal cells 1 from skin pieces 1> The culture medium containing Pronectin F (M1) prepared in the same manner as in Example 1
To the mL, an epidermis piece (5 mm × 5 mm) obtained by dispase treatment from a rat skin piece was put, left at room temperature for 1 hour, and the medium was removed by suction. Next, EpiLife-KG
2 mL of the medium was added, and the epidermis pieces were loosened with forceps to obtain a cell solution (C3). Cell liquid (C3)
Cell number was measured with a hemocytometer. Subsequently, cell culture was performed in the same manner as in Example 1 except that the cell solution (C3) was used instead of the cell solution (C1).

Example 5 <Selective culture 2 of epidermal cells from skin pieces> A RGDS-containing medium (M1) was used instead of the pronectin-F-containing medium (M1).
Cell solution (C) was prepared in the same manner as in Example 4 except that 2) was used.
After preparing 4), cell culture was performed in the same manner as in Example 1 using the cell solution (C4).

Example 6 <Selective cultivation 3 of epidermal cells from skin pieces> Cell culture was performed in the same manner as in Example 4 except that a modified Pronectin F-containing medium (M1 +) was used instead of the Pronectin F-containing medium (M1). After preparing the solution (C3 +), cell culture was performed in the same manner as in Example 1 using the cell solution (C3 +).

Example 7 <Selective culture of corneal epithelial cells 1 from a mixed sample of fibroblasts and corneal epithelial cells 1> In 5 mL of Pronectin F-containing medium (M1) prepared in the same manner as in Example 1, normal human Two million dermal fibroblasts (manufactured by Kurashiki Spinning Co., Ltd.) and eight million normal human corneal epithelial cells (manufactured by Kurashiki Spinning Co., Ltd.) were charged. After standing at room temperature for 1 hour, centrifugation (room temperature, 1000 rpm, 10 minutes) was performed, and the supernatant was removed by suction. Next, 5 mL of EpiLife-KG2 medium was added and stirred to obtain a cell solution (C5). Cell fluid (C
The cell number of 5) was measured with a hemocytometer.

The cell solution (C5) was diluted with EpiLife-KG2 medium so that the cell concentration became 200,000 cells / mL.
The 10mL of diluted solution was poured into type IV collagen-coated Petri dish, after stirring gently with the lid in a petri dish, and allowed to stand in a CO ₂ incubator, CO ₂ concentration 5
% (V / v) at 37 ° C. for 2 weeks. One day after the start of culture, 10 mL of EpiLife-KG2 medium
The medium was replaced with the above, and until the fourth day, EpiLife-KG
Medium exchange using two mediums was performed every day.

Example 8 <Selective culture of corneal epithelial cells 2 from a mixed sample of fibroblasts and corneal epithelial cells 2> Pronectin F-containing medium (M1)
After preparing a cell solution (C6) in the same manner as in Example 7 except that the RGDS-containing medium (M2) was used instead of the above, cell culture was performed in the same manner as in Example 7 using the cell solution (C6). Was.

Example 9 <Selective culture of corneal epithelial cells 3 from a mixed sample of fibroblasts and corneal epithelial cells 3> Pronectin F-containing medium (M1)
Instead of using the modified Pronectin F-containing medium (M1 +), a cell solution (C5 +) was prepared in the same manner as in Example 7, and then the cell culture was performed using the cell solution (C5 +) in the same manner as in Example 7. Was done.

Comparative Example 1 Cell culture was carried out in the same manner as in Example 1 except that “EpiLife-KG2 medium” was used instead of “Pronectin F-containing medium (M1)”.

Comparative Example 2 Cell culture was carried out in the same manner as in Example 4 except that “EpiLife-KG2 medium” was used instead of “Pronectin F-containing medium (M1)”.

Comparative Example 3 Cell culture was performed in the same manner as in Example 7, except that “EpiLife-KG2 medium” was used instead of “Pronectin F-containing medium (M1)”.

Evaluation 1 <Evaluation of the Number of Epidermal Cells> The number of fibroblasts and the number of epidermal cells on the Petri dishes of Examples 1 to 6 and Comparative Examples 1 and 2 were observed on the fourth day of culture by morphological observation using an optical microscope. It was measured and the selective culture rate of epidermal cells was calculated from this. Table 1 shows the results. (Selective culture rate of epidermal cells) = (number of epidermal cells) × 100 /
(Fibroblast count + epidermal count)

[0048]

[Table 1]

Evaluation 2 <Evaluation of skin sheet formation> The formation of the skin sheet on the Petri dishes of Examples 1 to 4 and Comparative Examples 1 and 2 was observed on the 14th day of the culture and evaluated according to the following criteria. Table 2 shows the results. ：: Cells after peeling treatment were in sheet form ×: Cells after peeling treatment were in granular form

[0050]

[Table 2]

Evaluation 3 <Evaluation of the number of corneal epithelial cells> The number of corneal epithelial cells on the Petri dishes of Examples 5 to 6 and Comparative Example 3 was counted on the fourth day of culture by morphological observation using an optical microscope. The selective culture rate of the cells was calculated. Table 3 shows the results. (Selective culture rate of corneal epithelial cells) = (number of corneal epithelial cells) x
100 / (number of fibroblasts + number of corneal epithelial cells)

[0052]

[Table 3]

When the method for producing cells of the present invention is used, it can be seen that the adhesion of fibroblasts to the culture carrier can be suppressed, and the desired cells (epidermal cells or corneal epithelial cells) can be selectively adhered. Furthermore, a sheet of the epidermis can be formed by the cell production method of the present invention.

[0054]

According to the production method of the present invention, target cells can be selectively produced without using 3T3 cells. Therefore, when cells (human) are produced and clinically applied, they are derived from 3T3 cells (mouse). There is no risk of immune rejection. Furthermore, when producing a skin sheet, it can be prepared in two weeks as compared with the conventional production period of three to four weeks, so that the burden on the patient can be dramatically reduced.

[Sequence List] <110> SANYO CHEMICAL INDUSTRIES, LTD .; SANYO CHEMICAL INDUSTRIES, LTD. <120> Cell production method <160> 21 <210> 1 <211> 4 <212> PRT <213> Homo sapiens <400> 1 Arg Glu Asp Val 1 <210> 2 <211> 19 <212> PRT <213> Homo sapiens <400> 2 Tyr Glu Lys Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg 1 5 10 15 Pro Gly Val < 210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly Arg Lys Lys Tyr 1 5 10 15 <210> 4 <211> 5 <212> PRT <213> Homo sapiens <400> 4 Tyr Ile Gly Ser Arg 1 5 <210> 5 <211> 5 <212> PRT <213> Homo sapiens <400> 5 Pro Asp Ser Gly Arg 1 5 <210> 6 < 211> 7 <212> PRT <213> Homo sapiens <400> 6 Arg Tyr Val Val Leu Pro Arg 1 5 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Leu Gly Thr Ile Pro Gly 1 5 <210> 8 <211> 10 <212> PRT <213> Homo sapiens <400> 8 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile 1 5 10 <210> 9 <211> 5 <212> PRT <213> Homo sapiens <400> 9 Ile Lys Val Ala Val 1 5 <210> 10 <211> 12 <212> PRT <213> Homo sapiens <400> 10 His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 <210> 11 <211> 15 <212> PRT <213> Homo sapiens <400> 11 Cys Gln Glu Glu Gly Gly Leu Val Val Pro Pro Thr Asp Ala Pro 1 5 10 15 <210> 12 <211 > 4 <212> PRT <213> Homo sapiens <400> 12 Val Thr Xaa Gly 1 <210> 13 <211> 4 <212> PRT <213> Homo sapiens <400> 13 Asp Gly Glu Ala 1 <210> 14 <211> 15 <212> PRT <213> Homo sapiens <400> 14 Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro 1 5 10 15 <210> 15 <211> 13 <212> PRT <213 > Homo sapiens <400> 15 Gly Glu Phe Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys 1 5 10 <210> 16 <211> 6 <212> PRT <213> Homo sapiens <400> 16 Phe Thr Leu Cys Phe Arg 1 5 <210> 17 <211> 6 <212> PRT <213> Bombyx mori <400> 17 Gly Ala Gly Ala Gly Ser 1 5 <210> 18 <211> 4 <212> PRT <213> Homo sapiens <400 > 18 Arg Gly Asp Ser 1 <210> 19 <211> 5 <212> PRT <213> Homo sapiens <400> 19 Gly Arg Gly Asp Ser 1 5 <210> 20 <211> 6 <212> PRT <213> Homo sapiens <400> 20 Gly Arg Gly Asp Ser Pro 1 5 <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <400> 21 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro 1 5 10

Claims (5)

[Claims] 1. A peptide containing at least one minimal amino acid sequence showing an adhesion signal in one molecule (A) is brought into contact with a sample containing at least the removed cells (B) and the target cells (C). A cell production method characterized by selectively culturing C). 2. The RGD sequence, the LDV sequence, the REDV sequence (1), wherein the minimum amino acid sequence representing the cell adhesion signal of the peptide (A) is represented by one-letter amino acid code.
YEKPGSPPPREVVPPRPGV sequence (2),
KNNQKSEPLIGRKKY sequence (3), YIGS
R sequence (4), PDSGR sequence (5), RYVVLPR
Sequence (6), LGTIPG sequence (7), RNIAEII
KDI sequence (8), IKVAV sequence (9), LRE sequence, HHLGGAKQAGDV sequence (10), CQEP
GGLVVPPTDAP sequence (11), VTXG sequence (12), DGEA sequence (13), GVKGDKKGNP
GWPGAP sequence (14), GEFYFDLRLKGD
K sequence (15), FTLCFR sequence (16) and HAV
The cell production method according to claim 1, wherein the cell production method is at least one kind of sequence selected from the group consisting of sequences. 3. The method for producing a cell according to claim 1, wherein the method for producing the peptide (A) is an organic synthesis method or a gene recombination method. 4. The cell production method according to claim 1, wherein the removed cells (B) are fibroblasts. 5. The cell production method according to claim 1, wherein the target cell (C) is an epidermal cell or an epithelial cell.