JP2010220611A - Medium for subculture and method for producing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem in conventional subculture with a microcarrier by preventing lowering of proliferation potency of the cell in passing a cultured cell to a new microcarrier. <P>SOLUTION: There are provided a subculture medium (B) for a subculturing process of a cell attached to a cell culture carrier containing a microcarrier (A) having a polypeptide (P) containing at least one cell adhesive minimum amino acid sequence (X) in one molecule in the culture of the adhesive cell, as the cell attached to a new cell culture carrier, wherein total concentration of calcium ion and magnesium ion in the medium is 0-2.3 mM, and a cell-producing method containing a washing step and a subculture step and using the medium. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a subculture medium. More specifically, when continuing cell culture over a long period of time in the field of cell culture, when the cell density becomes excessive, it is necessary to reduce the number of cells and transfer to a new cell culture carrier. The present invention relates to a medium used for this passage.

  A conventional procedure for passage of adherent cells is as follows. First, cells are cultured and grown on a cell culture carrier. When cells grow on the whole cell culture carrier, a protease such as trypsin or collagenase is allowed to act to detach the cells from the cell culture carrier, and a medium is added. After the cells are dispersed in the medium, the cell suspension is collected in a centrifuge tube. At this time, since the cell suspension contains enzymes such as trypsin, for the purpose of removing these enzymes, the cells are spun down by centrifugation and the supernatant medium is removed. Thereafter, a new medium is added to disperse the cells again, and the cell suspension is appropriately diluted and cultured with a new cell culture carrier.

In this method, there is a step of detaching cells, but the action of proteolytic enzymes such as trypsin and pipetting conditions in this step greatly affect the viability of the cells, and it takes a lot of time to recover the cell growth ability. Cost.
As a subculture method that does not allow trypsin or other proteolytic enzymes to act, a microcarrier is used as a cell culture carrier. As a subculture method between microcarriers, agitation is performed to promote the transfer of cells to a new microcarrier. There are an intermittent stirring culture method (Non-Patent Document 1) in which culture and stationary culture are alternately performed, and a subculture method (Non-Patent Document 2) in a medium in which the calcium ion concentration is lowered to 50% of the normal culture concentration. However, even in these methods, it is difficult to efficiently and uniformly pass new microcarriers, and the cell proliferation ability is reduced.

Bioprocess Engineering, 21, 211-213, 1999 Biotechnology and Bioengineering, 23, 983-993, 1981

  The object of the present invention is to solve the problems in the subculture with the above microcarriers. Specifically, it is intended to prevent a decrease in cell proliferation ability when subcultured cultured cells to a new microcarrier.

  In the method for culturing adherent cells, the subculture medium of the present invention is a cell culture comprising a microcarrier (A) having a polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. A subculture medium used in a step of subcultured cells attached to a carrier as cells attached to a new cell culture carrier, wherein the total concentration of calcium ions and magnesium ions is 0 to 2.3 mM. To do.

Further, the cell production method of the present invention is performed using a cell culture carrier including a microcarrier (A) having a polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule. A cell production method comprising a subculture step of culturing sex cells and then subcultured cells adhering to a cell culture carrier as cells adhering to a new cell culture carrier, comprising the following washing step and subculture step: The main point is that each step is performed in the order of the cleaning step and the subculture step.
Washing step: The cell culture support to which the cells obtained by cell culture are attached is taken out from the culture solution, and the subculture medium (B) according to claim 1 is added to the cell culture support to which the cells are attached. A step of washing the attached cell culture carrier and then removing the subculture medium (B).
Passage step: A new cell culture carrier soaked in the passage medium (B) is added to the cell culture carrier to which the cells obtained in the washing step are attached, and after passage, the passage medium (B) is used as the culture medium. The step of replacing with (C).

  When cells are produced using the subculture medium of the present invention (preferably, by the intermittent stirring culture method), the cells are easily transferred to new microcarriers efficiently and uniformly. In addition, subculture can be performed without reducing the cell growth ability, and subculture can be performed without reducing the cell growth ability.

  In addition, when cells are produced by the cell production method of the present invention (preferably by an intermittent stirring culture method), the cells are easily transferred to a new microcarrier efficiently and uniformly. In addition, subculture can be performed without reducing the cell growth ability, and subculture can be performed without reducing the cell growth ability.

  In the present invention, the adherent cell is not particularly limited as long as it adheres to a solid surface and proliferates. For example, epithelial cells (Vero cells, MDCK cells, CHO cells, HEK293 cells, COS cells, HmLu cells, etc.) Tumor cells (Hela cells, VACO cells, etc.), endothelial cells (HUVEC cells, DBAE cells, etc.), leukocytes (HIT-T15 cells, etc.), fibroblasts (WI38 cells, BHK21 cells, SFME cells, etc.), muscle cells ( HL1 cells, C2C12 cells, etc.), nerve / endocrine gland cells (ROC-1 cells, IMR-32 cells, etc.) and primary cells (chicken embryo primary cells, quail embryo primary cells, rabbit kidney primary cells, etc.) and the like. Among these, from the viewpoint of not reducing the cell proliferation ability, epithelial cells and primary cells are preferable, Vero cells, MDCK cells and primary cells are more preferable, and Vero cells, HmLu cells and MDCK cells are particularly preferable.

“Cell adhesion” means a property in which a specific minimum amino acid sequence is recognized by a cell integrin receptor, and the cell easily adheres to a substrate (Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66, 1992).
Examples of the cell adhesion minimal amino acid sequence (X) include “Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990” and “Osaka Prefectural Maternal and Child Medical Center, Vol. 8, No. 1, 58-66”. Page, 1992 ", etc. are used.

Among these minimal amino acid sequences (X), Arg Gly Asp sequence, Leu Asp Val sequence, Leu Arg Glu sequence, His Ala 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 Asn Ile Ala Glu Ile Ile Ile Ile L Lys Val Ala Val sequence (7), Asp Gly Glu Ala sequence (8), Gly Val Lys Gly Asp Lys Gly Asn Pro Gly Trp Pro Gly Ala Pro sequence (9), Gly Glu Plu Ph Tyr Phe Asp Leu Arg Leu Lys Gly Asp Lys sequence (10), Tyr Lys Leu Asn Val Asn Asr Ser sequence (11), Ala Lys Pro Ser Tyr Pro Pro Thr Pro Pro Thr Pro Pro Thr Pro Ile Thr Arg Phe Gly sequence (13), Thr Trp Tyr Lys Ile Ala Phe Gln Arg Asn Arg Lys sequence (14), Arg Lys Arg Leu Gln Val Le And at least one selected from the group consisting of Arg Gly Asp sequence and Tyr from the viewpoint of cell adhesion. Ile Gly Ser Arg sequence (2), Ile Lys Val Ala Val sequence (7), Arg Lys Arg Leu Gln Val Gln Leu Ser Ile Arg Thr (15) and Pro His Ser Arn group (16) One, particularly preferably Arg Gly Asp sequence.
At both ends of these minimum amino acid sequences (X), other amino acids {alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile) ), Cysteine (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), tryptophan (Trp), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid ( Glu), arginine (Arg), lysine (Lys), histidine (His) and the like} are preferably linked to the intervening amino acid sequence (Z).

  As the intervening amino acid sequence (Z), the Gly Ala Ala Val Thr Gly sequence (65), the Gly Leu Pro Gly Pro Lys Gly Asp sequence (66) are present at the N-terminus of the minimal amino acid sequence (X) from the viewpoint of cell adhesion. ), Gly Pro Ala Val Thr Gly sequence (67), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Thr Gly Sequence (68), Gly Ala Ala Val 69 Leu Cys Val Ser Glu Pro Gly sequence (70), Ser Pro Ala Ser Ala Ala Leu Cys Val Ser Glu Pro Gly sequence (71) , Ser Pro Ala Ser Ala Ala Val Cys Glu Pro Gly Sequence (72), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Cys Glu Pro Gly Sequence (73) Gly Pro Ala Leu Cys Val Ser Glu Pro Gly sequence (74), Gly Pro Ala Val Cys Glu Pro Gly sequence (75), Gly Pro Ala Leu Cys Val Ser Glu Pro Gly sequence (76) At least one intervening a selected from the group consisting of the sequence (77) It is preferable to bind the nonacid sequence (Z), and the Gly Ala Ala Val Thr Gly sequence (65), the Gly Leu Pro Gly Pro Lys Gly Asp sequence (66), the Gly Pro Ala Val Thr Gly sequence (67) and At least one selected from the group consisting of Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Val Thr Gly Sequence (68) is more preferable, and Gly Ala Ala Val Thr Gly Sequence (65) is particularly preferable.

  As the intervening amino acid sequence (Z), Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78), Ser Pro Ala Ser Ala Ala Leu Cys Val are present at the C-terminus of the minimum amino acid sequence (X) from the viewpoint of cell adhesion. Ser sequence (79), Ser Pro Ala Ser Ala Ala Val Cys sequence (80), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro sequence (81), Ala Gly Pro Ly Aly Gly Aly Gly Aly A Cys sequence (82), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Leu Cys Val Ser Column (83), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro sequence (84), Ser Pro Ala Ser Ala Ala Gly Pro Val Gly Ser Pro sequence (85), Cys Asp Ala Gly Aly TyCy TyCy TyAy It is preferable to bind at least one intervening amino acid sequence (Z) selected from the group consisting of Gly Pro Val Gly Ser Pro sequence (87) and Ser Ala Gly Pro Ser Ala Gly Tyr sequence (88), Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78), Ser Pro Ala Ser Ala Ala Leu Cys Val Ser sequence (79), Ser Pro Ala Ser Ala Ala Val Cys sequence (80), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Pro sequence (81), Ala Gly Pro Lys Gly Ala Gly Ser Pro Gly Ser 82 Lys Gly Ala Asp Gly Ser Pro Gly Pro Ala Leu Cys Val Ser sequence (83), Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro sequence (84) and Ser Pro Ala Ser sequence More preferably, at least one selected from the group consisting of Ser Pro The Ala Ser Ala Ala Gly Tyr sequence (78) is particularly preferred.

  Polypeptide (P) may have at least one minimum amino acid sequence (X) in one molecule, but from the viewpoint of cell adhesiveness, one having 1 to 50 in one molecule is preferable, and more preferable. 2-50, then preferably 3-30, particularly preferably 4-20, most preferably 5-15. Two or more kinds of minimum amino acid sequences (X) may be included in one molecule.

  In addition to the minimum amino acid sequence (X), the polypeptide (P) preferably has an auxiliary amino acid sequence (Y) from the viewpoint of improving the thermal stability of the polypeptide (P).

  As the auxiliary amino acid sequence (Y), an amino acid sequence other than the minimum amino acid sequence (X) can be used. From the viewpoint of the thermal stability of the polypeptide (P), a sequence having Gly and / or Ala is preferable.

The auxiliary amino acid sequence (Y) includes (Gly Ala) _a sequence, (Gly Ala Gly Ala Gly Ser) _b sequence, (Gly Ala Gly Ala Gly Tyr) _c sequence, (Gly Ala Gly Val Gly Tyr) _d sequence Gly Ala Gly Tyr Gly Val) _e sequence, {Asp Gly Gly (Ala) _f Gly Gly Ala} _g sequence, (Gly Val Pro Gly Val) _h sequence, (Gly) _i sequence, (Ala) _j sequence, (yly Gl) Ala) _k sequence, (Gly Val Gly Val Pro) _m sequence, (Gly Pro Pro) _n sequence, (Gly Ala Gln Gly Pro Ala Gly Pro Gly) _o sequence, (Gly Ala Pro Gly Ala Pro Gly S Gly L eu Gln) _p sequence and / or (Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) sequence having _q sequence and the like. Of these, (Gly Ala) _a sequence, (Gly Ala Gly Ala Gly Ser ) b sequence, (Gly Ala Gly Ala Gly Tyr ) c sequence, (Gly Ala Gly Val Gly Tyr ) d sequence, (Gly Ala Gly Tyr Gly Val) _e , {Asp Gly Gly (Ala) _f Gly Gly Ala} _g sequence, (Gly Val Pro Gly Val) _h sequence, (Gly Val Gly Val Pro) _m sequence and / or (Gly Pro Pro) _n sequence More preferably, (Gly Ala Gly Ala Gly Ser) _b sequence, (Gly Val Pro Gly Val) _h sequence, (Gly Val Gly Val Pro) _m sequence and / or (Gly Pro Pro Pro) _n sequence , Especially preferred Those having a (Gly Ala Gly Ala Gly Ser) b sequence.

  A is an integer of 5 to 100, b is an integer of 1 to 33, c, d and e are integers of 2 to 33, f is an integer of 1 to 194, and g is {1} to {200 / (6 + f). } Is an integer obtained by rounding down the decimal point, h is an integer of 2 to 40, i and j are integers of 10 to 200, k is an integer of 3 to 66, m is an integer of 2 to 40, and n is an integer of 3 to 66. , O is an integer from 1 to 22, and p and q are integers from 1 to 13.

  The auxiliary amino acid sequence (Y) preferably contains glycine (Gly) and / or alanine (Ala). When glycine (Gly) and alanine (Ala) are included, the total content (%) of these is preferably 10 to 100, more preferably 20 to 95, based on the total number of amino acids in the auxiliary amino acid sequence (Y). Especially preferably, it is 30-90, Most preferably, it is 40-85. Within this range, the thermal stability is further improved.

  When both glycine (Gly) and alanine (Ala) are included, the content ratio (Gly / Ala) is preferably 0.03 to 40, more preferably 0.08 to 13, particularly preferably 0.2. ~ 5. Within this range, the thermal stability is further improved.

  In addition to the above examples, the auxiliary amino acid sequence (Y) includes other amino acids {alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), Isoleucine (Ile), Cysteine (Cys), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Proline (Pro), Tryptophan (Trp), Asparagine (Asn), Glutamine (Gln), Aspartic acid (Asp) , Glutamic acid (Glu), arginine (Arg), lysine (Lys), histidine (His) and the like}.

Examples of the auxiliary amino acid sequence having (Gly Ala) _a sequence include amino acid sequences represented by SEQ ID NOs: (17) to (19).
(Gly Ala Gly Ala Gly Ser) Examples of the auxiliary amino acid sequence having the _b sequence include amino acid sequences represented by SEQ ID NOs: (20) to (22).
(Gly Ala Gly Ala Gly Tyr) Examples of the auxiliary amino acid sequence having _c sequence include amino acid sequences represented by SEQ ID NOs: (23) to (25).
(Gly Ala Gly Val Gly Tyr) Examples of the auxiliary amino acid sequence having the _d sequence include amino acid sequences represented by SEQ ID NOs: (26) to (28).
(Gly Ala Gly Tyr Gly Val) Examples of the auxiliary amino acid sequence having the _e sequence include amino acid sequences represented by SEQ ID NOs: (29) to (31).
{Asp Gly Gly (Ala) _f Gly Gly Ala} Examples of the auxiliary amino acid sequence having the _g sequence include amino acid sequences represented by SEQ ID NOs: (32) to (34).
(Gly Val Pro Gly Val) Examples of the auxiliary amino acid sequence having the _h sequence include amino acid sequences represented by SEQ ID NOs: (35) to (38).
Examples of the auxiliary amino acid sequence having the (Gly) _i sequence include amino acid sequences represented by SEQ ID NOs: (39) to (41).
(Ala) _Examples of the auxiliary amino acid sequence having _j sequence include amino acid sequences represented by SEQ ID NOs: (42) to (44).
(Gly Gly Ala) Examples of the auxiliary amino acid sequence having _k sequence include amino acid sequences represented by SEQ ID NOs: (45) to (47).
(Gly Val Gly Val Pro) Examples of the auxiliary amino acid sequence having _m sequence include amino acid sequences represented by SEQ ID NOs: (48) to (50).
(Gly Pro Pro) Examples of the auxiliary amino acid sequence having _n sequence include amino acid sequences represented by SEQ ID NOs: (51) to (53).
(Gly Ala Gln Gly Pro Ala Gly Pro Gly) _Examples of the auxiliary amino acid sequence having the _o sequence include amino acid sequences represented by SEQ ID NOs: (54) to (56).
(Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln) Examples of the auxiliary amino acid sequence having the _p sequence include amino acid sequences represented by SEQ ID NOs: (57) to (59).
(Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Leu Pro Gly Ser Pro) _Examples of the auxiliary amino acid sequence having the _q sequence include amino acid sequences represented by SEQ ID NOs: (60) to (62).

  Among these auxiliary amino acid sequences, from the viewpoint of thermal stability and the like, SEQ ID NOs: (17), (18), (20), (21), (22), (23), (24), (26), (27), (29), (30), (32), (33), (34), (35), (36), (38), (39), (40), (42), (43 ), (45), (46), (48), (49), (51), (52), (54), (55), (57), (58), (60) or (61) The amino acid sequence represented is preferred, more preferably SEQ ID NO: (18), (20), (21), (22), (24), (27), (30), (34), (35), ( 36), (37), (38), (40), (43), (46), (49), (52), (55), (58) or amino represented by (61) Sequences, particularly preferably SEQ ID NO: 20 is the amino acid sequence represented by (21) or (38).

  When the auxiliary amino acid sequence (Y) is included, the content of (Y) is preferably 2 to 50, more preferably 3 to 30, particularly preferably 1 molecule of the polypeptide (P) from the viewpoint of thermal stability. Preferably it is 4-20, Most preferably, it is 5-15. The polypeptide (P) may also contain two or more auxiliary amino acid sequences (Y).

  The polypeptide (P) may contain a branched chain, may be partially crosslinked, or may contain a cyclic structure. However, the polypeptide (P) is preferably not cross-linked from the viewpoint of easy recognition of the cellular polypeptide, more preferably a non-cross-linked linear structure, particularly preferably a cyclic structure without a cyclic structure. It is a straight chain structure that is not made. The linear structure also includes a β structure (secondary structure in which linear peptides are bent and the portions are arranged in parallel and a hydrogen bond is formed therebetween).

  Polypeptide (P) has a minimum amino acid sequence (X) and an auxiliary amino acid sequence (Y), and, if necessary, other amino acids between (X) and (Y) from the viewpoints of cell adhesion and thermal stability. It is preferable that the structure is formed by alternately chemically bonding through the sequence, and the structure in which the amino acid sequence including the intervening amino acid sequence (Z) at both ends of (X) and (Y) are alternately chemically bonded. More preferably it is. In these cases, the number (units) of repeating units (XY) of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) is preferably 1 to 50, more preferably from the viewpoint of cell adhesion. It is 2-40, Most preferably, it is 3-30, Most preferably, it is 4-20.

  Further, the number of the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) may be the same or different. When they are different from each other, it is preferable that the content of one of them is one less than the content of the other {in this case, the auxiliary amino acid sequence (Y) is preferably small}. The content ratio (X / Y) between the minimum amino acid sequence (X) and the auxiliary amino acid sequence (Y) in the polypeptide (P) is preferably 0.5 to 2, and more preferably 0.9 to 1.4. Especially preferably, it is 1-1.3.

  Moreover, you may contain another amino acid in the terminal part (From the minimum amino acid sequence (X) or auxiliary amino acid sequence (Y) to a peptide terminal) of polypeptide (P). When other amino acids are included, the content is preferably from 1 to 1000, more preferably from 3 to 300, particularly preferably from the viewpoint of easy recognition of the polypeptide of the cell per polypeptide (P). Is 10-100.

  The polypeptide (P) may be further modified with a compound (AM) containing an amino group and / or an ammonio group. When it is modified with (AM), the cell adhesiveness of the polypeptide (P) of the present invention is further improved.

Examples of the compound (AM) containing an amino group and / or an ammonio group include polyamines, amino alcohols, halides having an amino group, amino group-containing monomers, polymers having amino group-containing monomers as constituent monomers, and these Can be used.
As the polyamine, a polyamine having at least one primary amino group or secondary amino group (having 2 to 56 carbon atoms) is used, such as an aliphatic polyamine, an alicyclic polyamine, a heterocyclic polyamine, and an aromatic polyamine. Is used.

  Aliphatic polyamines include alkylene diamines (ethylene diamine, propylene diamine, trimethylene diamine, tetramethylene diamine, hexamethylene diamine, etc.), polyalkylene polyamines having 2 to 6 carbon atoms in the alkylene group (diethylene triamine, iminobispropylamine, Triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, etc.), and alkyl (C1-C18) substitution products thereof (dimethylaminopropylamine, diethylaminopropylamine, dipropylaminopropylamine, methylethylaminopropylamine, Trimethylhexamethylenediamine, N, N-dioctadecylethylenediamine, trioctadecylethylenediamine, methyliminobispropylamine, etc.) And the like.

  Examples of alicyclic polyamines include 1,3-diaminocyclohexane, 1,3-bis (methylamino) cyclohexane, 1,3-bis (dihydroxyamino) cyclohexane, isophorone diamine, menthane diamine, and 4,4′-methylene dicyclohexane. Examples include diamines.

  Examples of the heterocyclic polyamine include piperazine, N-methylpiperazine, N-aminoethylpiperazine and 1,4-diaminoethylpiperazine.

  Aromatic polyamines include phenylenediamine, N, N′-dimethylphenylenediamine, N, N, N′-trimethylphenylenediamine, diphenylmethanediamine and 2,6-diaminopyridine, tolylenediamine, diethyltolylenediamine, 4, Examples include 4′-bis (methylamino) diphenylmethane and 1-methyl-2-methylamino-4-aminobenzene.

  As the amino alcohol, an amino alcohol having 2 to 58 carbon atoms is used, and an alkanolamine having 2 to 10 carbon atoms [monoethanolamine, diethanolamine, triethanolamine, monoisopropanolamine, monobutanolamine, triethanolamine, triethanolamine, Propanolamine, tributanolamine, N, N-bis (hydroxyethyl) ethylenediamine and N, N, N ′, N′-tetrakis (hydroxyethyl) ethylenediamine, etc.], alkyl (C 1-18) substituted products thereof [ N, N-dimethylethanolamine, N, N-diethylethanolamine, N-ethyldiethanolamine, N-octadecyldiethanolamine, N, N-diethyl-N ′, N′-bis (hydroxyethyl) ethylenediamine, N, - dioctadecyl -N ', N'-bis (hydroxyethyl) ethylenediamine and N, N, N'trioctadecyl -N'- hydroxyethyl ethylenediamine] and the like.

  As the halide having an amino group, halogenated (such as chlorine and bromine) of alkylamine having 2 to 17 carbon atoms is used, and aminoethyl chloride, N-methylaminopropyl chloride, dimethylaminoethyl chloride, diethylaminoethyl chloride are used. , Dibenzylaminoethyl bromide, dimethylaminopropyl bromide, diethylaminopropyl chloride, dibenzylaminopropyl chloride and the like.

As the amino group-containing monomer, an amino group-containing vinyl compound having 5 to 21 carbon atoms, ethyleneimine, an amino acid having 2 to 20 carbon atoms, or the like is used.
Examples of the amino group-containing vinyl compound include amino group-containing (meth) acrylates, amino group-containing (meth) acrylamides, amino group-containing aromatic vinyl hydrocarbons, amino group-containing allyl ethers, and the like. In addition, (meth) acryl ... means acryl ... and / or methacryl ...

  As the amino group-containing (meth) acrylate, aminoethyl (meth) acrylate, N-methylaminoethyl (meth) acrylate, N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminopropyl (meth) acrylate, N, N-dipropylaminoethyl (meth) acrylate, N-benzyl-N-methylaminoethyl (meth) acrylate, N, N-dibenzylaminoethyl (meth) acrylate, N, N-dibenzylaminopropyl (meth) ) Acrylate, morpholinoethyl (meth) acrylate, N-methylpipetidinoethyl (meth) acrylate, and the like.

  Examples of amino group-containing (meth) acrylamides include aminoethyl acrylamide, N-methylaminopropyl acrylamide, N, N-dimethylaminoethyl (meth) acrylamide, N, N-diethylaminopropyl (meth) acrylamide, and N, N-dipropyl. Examples include aminoethyl (meth) acrylamide, N-benzyl-N-methylaminoethyl (meth) acrylamide, morpholinoethyl (meth) acrylamide, and N-methylpipetidinoethyl (meth) acrylamide.

  Examples of amino group-containing aromatic vinyl hydrocarbons include aminoethylstyrene, N-methylaminoethylstyrene, N, N-dimethylaminostyrene, N, N-dipropylaminostyrene, and N-benzyl-N-methylaminostyrene. Can be mentioned.

  Examples of the amino group-containing allyl ether include aminoethyl allyl ether, N-methylaminoethyl allyl ether, N, N-dimethylaminoethyl allyl ether, and N, N-diethylaminoethyl allyl ether.

  As amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, proline, cysteine, lysine, serine, glycine, 3-aminopropionic acid , 8-aminoactanoic acid and 20-aminoeicosanoic acid.

Examples of the polymer of the amino group-containing monomer include a vinyl polymer composed of an amino group-containing vinyl compound, polyethyleneimine, and a polypeptide (not including a linear polypeptide (P)).
The weight average molecular weight of the polymer of amino group-containing monomers is preferably 500 to 1,000,000, more preferably 1,000 to 800,000, and particularly preferably 2,000 to 500,000. The weight average molecular weight can be measured by gel permeation chromatography (GPC) {reference substance: polystyrene standard having a molecular weight of 420 to 20,600,000 (manufactured by Tosoh Corp.)}.

  Examples of these quaternized products include those obtained by quaternizing these amino groups with a quaternizing agent (such as methyl chloride, ethyl chloride, benzyl chloride, dimethyl carbonate, dimethyl sulfate and ethylene oxide).

As a method of modifying with a compound (AM) containing an amino group and / or an ammonio group, (1) a chemical bond between a compound (AM) containing an amino group and / or an ammonio group and a polypeptide before modification { A covalent bond, an ionic bond and / or a hydrogen bond}, and (2) physical adsorption of a compound (AM) containing an amino group and / or an ammonio group to a polypeptide before modification (adsorption by van der Waals force) ) Can be applied.
Among these, from the viewpoint of bond strength and the like, the method (1) of chemical bonding is preferable, and covalent bonding is more preferable.

  As a specific example of covalently bonding a compound (AM) containing an amino group and / or an ammonio group and a peptide (P) before modification, an amino acid containing a hydroxyl group in the side chain in the peptide (P) (for example, Ser And Tyr) are alkyl etherified with an alkyl halide having an amino group and / or an ammonio group, and amino acids (eg, Asp and Glu) containing a carboxyl group in the side chain have an amino group and / or an ammonio group. The method of esterifying with alcohol is mentioned.

  The weight average molecular weight (hereinafter, Mw) of the polypeptide (P) is preferably 1,000 to 1,000,000, more preferably 2,000 to 700,000, particularly preferably 3,000 to 400,000. Most preferably, it is 4,000-200,000.

  The Mw of the polypeptide (P) is a known method such as a method in which a measurement sample {polypeptide etc.} is separated by SDS-PAGE (SDS polyacrylamide gel electrophoresis) and the migration distance is compared with a standard substance. (Hereinafter the same).

A part of preferable polypeptide (P) is illustrated below.
(1) When the minimum amino acid sequence (X) is Arg Gly Asp sequence (x1) 13 Arg Gly Asp sequences (x1) and 12 (Gly Ala Gly Ala Gly Ser) ₉ sequences (21) (y1) (X1) has a Gly Ala Ala Val Thr Gly sequence (65) at the N-terminus and a Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78) at the C-terminus of (x1). Polypeptide having a structure of about 110,000 Mw {“pronectin F”, pronectin is a registered trademark (Japan and USA) of Sanyo Chemical Industries, Ltd. Sanyo Chemical Industries Co., Ltd. <hereinafter the same>}; (x1) and (Gly Ala Gly Ala Gly Ser) ₃ sequence (20) (y2) 5 Mw 20,000 polypeptide having made structure Te ( "ProNectin F2"); (x1) 3 pieces and the _{(Gly Val Pro Gly Val) 2} Gly Gly (Gly Ala Gly Ala Gly Ser) 3 sequences (38) ( a polypeptide having a structure of about 10,000 Mw (“pronectin F3”) and the like having a structure in which three of y3) and these are alternately chemically bonded.

(2) When the minimum amino acid sequence (X) is the Ile Lys Val Ala Val sequence (x2) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is changed to the Ile Lys Val Ala Val sequence (7) (x2) Gly Ala Ala Val Thr Gly sequence (65) that binds to the N-terminus of the minimal amino acid sequence (X) is bound to Gly Ala Ala Pro Gly Ala Ser sequence (77) and the C-terminus of the minimal amino acid sequence (X) Serpro Ala Ser Ala Ala Gly Tyr sequence (78) is changed to Ser Ala Gly Pro Ser Ala Gly Tyr sequence (88), etc. “Pronectin L”, “Pronectin L2”, “Pronectin L3” and the like.

(3) When the minimum amino acid sequence (X) is a Tyr Ile Gly Ser Arg sequence (x3) The Arg Gly Asp sequence (x1) of pronectin F, pronectin F2 or pronectin F3 is reduced to the Tyr Ile Gly Ser Arg sequence (x3). Gly Ala Ala Val Thr Gly sequence (65) binding to the N-terminus of the amino acid sequence (X) is linked to the Gly Ala Ala Val Cys Glu Pro Gly sequence (69), Ser Pro binding to the C-terminus of the minimum amino acid sequence (X) “Pronectin Y”, “Pronectin Y2”, “Pronectin Y3”, etc., in which the Ala Ser Ala Ala Gly Tyr sequence (78) is changed to the Cys Asp Ala Gly Tyr sequence (86).

  Specific examples of the peptide (P) modified with (AM) include a peptide obtained by etherifying the above “pronectin F” with N, N-dimethylaminoethyl chloride hydrochloride.

  In addition to the polypeptides of (1) to (3), RetroNectin (recombinant human fibronectin CH-296) manufactured by Takara Shuzo Co., Ltd. {Arg Gly Asp sequence (x1) and Leu Asp Val sequence as the minimum amino acid sequence (X) Mw containing about 60,000 polypeptides} and RGDS-Protein A {polypeptides having Mw of about 30,000 containing Arg Gly Asp sequence (x1) as the minimum amino acid sequence (X)} can be preferably used. This polypeptide is derived from nature and does not contain the auxiliary amino acid sequence (Y). Therefore, thermal stability etc. are inferior to said (1)-(3). The amino acid sequences of these polypeptides are disclosed in JP-A-2-31498. }.

The polypeptide (P) may be an artificial polypeptide that is artificially synthesized. In the case of an artificial polypeptide, for example, organic synthesis methods (solid phase synthesis method, liquid phase synthesis method, etc.) and biochemical methods are used. It is produced by a synthetic method [genetically modified microorganism (yeast, bacteria, E. coli, etc.)] or the like. That is, the artificial polypeptide does not contain cell-adhesive proteins such as animal-derived collagen and fibronectin.
The polypeptide (P) is preferably an artificial polypeptide from the viewpoint that it does not contain animal-derived components.

  The organic synthesis method is described in, for example, “Sequel Biochemistry Experiment Course 2, Protein Chemistry (Part 2)” pages 641-694 (May 20, 1987; published by Tokyo Chemical Co., Ltd.) edited by the Japanese Biochemical Society. The method etc. currently used are used. As for the biochemical synthesis method, for example, the method described in JP-T-3-502935 is used. A biochemical synthesis method using a genetically modified microorganism is preferable in that an artificial polypeptide can be easily synthesized, and a method using a genetically modified Escherichia coli is particularly preferable.

The material of the microcarrier (A) is not particularly limited as long as it is a material capable of adhering adherent cells, but the following substances are preferably the main components from the viewpoint of cytotoxicity. The main component means that the content of the following substances is 90% by weight or more based on the weight of the material.
(1) Synthetic polymer: poly (meth) acrylic acid (salt), vinyl resin, polyester, polyurethane, epoxy resin, nylon, polycarbonate, and the like.
(2) Natural polymers: cellulose, cellulose derivatives (such as cellulose diacetate and cellulose triacetate) and dextran.
(3) Inorganic substances: aluminum oxide, hydroxyapatite, titanium oxide, silica, glass and the like.

  Of these, from the viewpoint of cell affinity, synthetic polymers, natural polymers and hydroxyapatite are preferable, more preferably synthetic polymers and natural polymers, particularly preferably synthetic polymers, and most preferably poly (meth) acrylic. Acid (salt).

Examples of the poly (meth) acrylic acid (salt) include the following polymers (1) to (3).
(1) Crosslinked polyacrylic acid (salt) obtained by aqueous solution polymerization (adiabatic polymerization, thin film polymerization, spray polymerization, etc.) described in JP-A No. 55-133413.
(2) A crosslinked polyacrylic acid (salt) obtained by reverse phase suspension polymerization described in JP-B-54-30710, JP-A-56-26909, or JP-A-11-5808.
(3) JP 2001-2935 A, JP 2003-054242 A, JP 2003-082250 A, JP 2003-165883 A, JP 2003-176421 A, JP 2003-183528 A. JP, 2003-192732, JP 2003-225565, JP 2003-238696, JP 2003-335970, JP 2004-009673, JP 2004-121400, JP 2004-123835, JP 2005-075982, JP 2005-095759, JP 2005-097569, JP 2005-186015, JP 2005-186016, JP 2005. No. 247931, etc. (Meth) acrylic acid (salt).

Of these, (2) and (3) are preferred from the viewpoint of adhesion of cells to the carrier. That is, a cross-linked polymer obtained by polymerizing a monomer having a carboxyl group is preferable, and cross-linked poly (meth) acrylic acid (salt) is more preferable.
In addition, (meth) acrylic acid means acrylic acid and / or methacrylic acid, and an acid (salt) means an acid and / or an acid salt.

  The form of the microcarrier (A) includes a solid type and a porous type, and any of them can be used. From the viewpoint of nutrition to cells, oxygen supply efficiency, and cell recovery rate, the solid type is preferable. Moreover, as the shape of the microcarrier, any of spherical shape, flat shape (ellipse) and the like can be used.

In the case of a solid type, the particle size (μm) of the microcarrier (A) is preferably 20 to 2,000, more preferably 40 to 1,000, and particularly preferably 80 to 500. On the other hand, in the case of a porous type, the particle diameter (μm) is preferably 30 to 25,000, more preferably 60 to 12,000, and particularly preferably 120 to 6,000. In the case of a flat shape, the maximum diameter (μm) is preferably 20 to 20,000, more preferably 50 to 10,000, and particularly preferably 100 to 5,000. Within these ranges, the amount of cell proliferation is further increased.
From the viewpoint of uniformity of cell growth, the distribution of the particle diameter or maximum diameter in (A) is the weight of the microcarrier (A) in which the weight of particles having the particle diameter or the maximum diameter in the range of the above-mentioned particle diameter or maximum diameter is the same. Is preferably 90 to 100% by weight, more preferably 95 to 100% by weight.

The true specific gravity of the microcarrier (A) is not particularly limited, but in a general method of culturing (A) with stirring with a medium, it is preferable that (A) floats during stirring and settles when stirring is stopped. . From such a viewpoint, the true specific gravity (g / cm ³ ) of (A) is preferably 1.00 to 1.10, more preferably 1.01 to 1.08, and particularly preferably 1.01 to 1. 05.

The microcarrier having the polypeptide (P) or the microcarrier support not having the polypeptide can be easily obtained from the market, and the following products can be used.
(1) Made of polystyrene: Biosilon manufactured by Nargenunk, Plastic beads manufactured by Solohill, Cytosphere manufactured by Lux, and the like.
(2) Made of polyacrylamide: Biorad, Biocarrier, etc.
(3) Made of polyurethane: PUF manufactured by INOAC CORPORATION.
(4) Cellulose: Biomaterials Co., Ltd. Cell Snow, etc.
(5) Dextran: Cytodex manufactured by Amsham Pharmacia.
(6) Glass: Silane (SIRAN) manufactured by Scott.

  In order to contain the polypeptide (P) in the microcarrier, (P) may be contained on the surface of the support, and (P) is a chemical bond (ionic bond, hydrogen bond and / or covalent bond, etc.). And / or bonded to the surface of the support by physical adsorption (adsorption by van der Waals forces). Among these, a chemical bond is preferable from the viewpoint that the polypeptide does not desorb from the microcarrier, and a covalent bond is more preferable.

  The reaction for covalently bonding the polypeptide (P) to the microcarrier can be performed by a known method. For example, the method described in “Basics and Experiments of Peptide Synthesis, October 5, 1997, published by Maruzen Co., Ltd.” can be mentioned, and specifically, the methods are as follows (1) to (3).

(1) a polypeptide having a primary amino group or a secondary amino group and a microcarrier support not containing a polypeptide (P) (hereinafter referred to as P-free support) having a carboxyl group; , The carboxyl group of the P-free support is previously reacted with a carbodiimide compound, and acylisourea {R′—N═C (OCOR) —NH—R ′ (the portion where —OCOR is derived from the support)} Then, a polypeptide having a primary amino group or a secondary amino group among the polypeptides can be added to the acylisourea to amide bond the P-free support to the polypeptide.

  Examples of the carbodiimide compound include N, N′-dicyclohexylcarbodiimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.

(2) When reacting a polypeptide having a primary amino group or a secondary amino group with a P-free support having a hydroxyl group, the hydroxyl group of the P-free support is previously converted to carbonyl di- After reacting with an imidazole compound to obtain an imidazole derivative {R-Im, Im is an imidazoline ring, R is derived from a support}, a polypeptide having a primary amino group or a secondary amino group is obtained from this imidazole derivative. In addition, the P-free support and the polypeptide can be N—C bonded.

  Examples of the carbonyldiimidazole compound include N, N′-carbonyldiimidazole.

(3) When reacting a polypeptide having a hydroxyl group with a P-free support having a carboxyl group, the carboxyl group of the P-free support is reacted with a carbodiimide compound in advance, After being obtained, by adding a polypeptide having a hydroxyl group to this acylisourea, the P-free support and the polypeptide can be ester-bonded.

As a method of physically adsorbing, ionic bonding and / or hydrogen bonding of the polypeptide (P) to the P-free support, the polypeptide (P) and the P-free support are put into a solvent and mixed. Examples of the method are as follows. Although there is no restriction | limiting in particular as a solvent, An inorganic salt, organic acid salt, an acid, and / or a base are 0.001 to 50 weight% (preferably 0.005 to 30 weight%, More preferably, 0.01 to 10 weight% ) Containing aqueous solution (described in JP-A No. 2003-189848, etc.) can be used.
Among these solvents, from the viewpoint of polypeptide binding efficiency, an aqueous solution containing an inorganic salt, an acid and / or a base, and water are preferable, and an aqueous solution containing an inorganic salt, an acid and / or a base is more preferable. Also, ion exchange distilled water, particularly preferably an aqueous solution containing an inorganic salt, an acid and / or a base.

  The cell culture carrier is not particularly limited as long as it comprises the microcarrier (A).

  The content of the polypeptide (P) is preferably 5 ng / g to 500 mg / g, more preferably 50 ng / g to 50 mg / g, and most preferably 500 ng per gram of cell culture carrier from the viewpoint of cell adhesion and the like. / G to 50 mg / g, then preferably 500 ng / g to 20 mg / g, particularly preferably 500 ng / g to 5 mg / g, most preferably 5 μg to 5 mg / g.

  In addition, when the content of the polypeptide (P) exceeds 500 μg / g, the Biret method (for example, “Biochemistry Experiment Course 1, Protein Chemistry I,” 45-55 pages (December 11, 1979; issued by Tokyo Chemical Co., Ltd.).

  On the other hand, when the content of the polypeptide (P) is 500 μg / g or less, the Kjeldahl method (for example, Biochemical Experiment Course 1, Protein Chemistry I, pages 45-55 (December 11, 1979) Japan; issued by Tokyo Chemical Co., Ltd.).

It can also be measured using an immunological measurement method (described in JP 2004-049921 A). Specifically, (1) Cell culture carrier whose polypeptide (P) content is known {Cell culture carrier whose polypeptide (P) content is known by Biuret method, Kjeldahl method, etc.} Is immersed in physiological saline, the antibody that binds to the polypeptide (P) is reacted with an enzyme labeled (hereinafter referred to as an enzyme-labeled antibody), the amount of the enzyme of the reacted enzyme-labeled antibody is measured for absorbance, and a calibration curve is obtained. (Polypeptide (P) content and absorbance relative thereto) are prepared. (2) Similarly, the absorbance of a specimen (a cell culture carrier whose polypeptide (P) content is unknown) is measured. The content of the polypeptide (P) in the sample can be determined from the calibration curve obtained in (1) and the absorbance obtained in (2).
In addition, as a measurement sample, what was dried for 1 hour with the vacuum dryer {120 degreeC, 0.1 kPa or less} is used.

  The subculture medium (B) means a medium used for culturing cells using a cell culture carrier and then substituting cells attached to the cell culture carrier as cells attached to a new cell culture carrier. Usually, the subculture medium (B) is the same as the culture medium (C), but the characteristics of the subculture medium of the present invention are the cells in which the cells attached to the cell culture carrier are attached to the new cell culture carrier. From the viewpoint of passage, the total concentration of calcium ions and magnesium ions is 0 to 2.3 mM, preferably 0 to 1.5 mM, particularly preferably 0 to 0.1 mM.

The concentration of calcium ions can be adjusted by adding a substance soluble in the subculture medium, for example, calcium chloride (CaCl ₂ ) or calcium nitrate (Ca (NO ₃ ) ₂ ). From the viewpoint of cytotoxicity, calcium chloride (CaCl ₂ ) is preferably used.
The concentration of magnesium ions can be adjusted by adding magnesium sulfate (MgSO ₄ ) or magnesium chloride hexahydrate (MgCl ₂ .6H ₂ O) to the subculture medium. From the viewpoint of cytotoxicity, it is preferable to use magnesium sulfate (MgSO ₄ ).
The concentration (mM) of calcium ions is preferably 0 to 2.0 mM, more preferably 0 to 0.5 mM, from the viewpoint of passage of cells attached to the cell culture carrier as cells attached to a new cell culture carrier. is there.
The concentration of magnesium ion (mM) is preferably 0 to 2.0 mM, more preferably 0 to 0.5 mM, from the viewpoint of substituting cells attached to the cell culture carrier as cells attached to a new cell culture carrier. is there.
The ratio between the calcium ion concentration and the magnesium ion concentration (calcium ion concentration / magnesium ion concentration) is 1/1 from the viewpoint of subculture of cells attached to the cell culture carrier as cells attached to a new cell culture carrier. ˜1 / 3 is preferable, and more preferably 1/1 to 1 / 2.5.

The medium used for the subculture medium (B) and the culture medium (C) is not limited other than the total concentration of calcium ions and magnesium ions described above, and any of a serum medium and a serum-free medium can be used. . As the serum medium, a general medium (DMEM medium, DME medium, RPMI medium, MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher, depending on the type of cells to be used. Medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium, Drosophila medium, and a mixed medium thereof) And the like. Serum includes human serum and animal serum (bovine serum, horse serum, goat serum, sheep serum, pig serum, rabbit serum, chicken serum, rat serum, mouse serum, etc.).
The serum-free medium includes Grace medium, IPL-41 medium, Schneider's medium, OPTI PRO ^™ SFM medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle ^™. 293 medium, CD-CHO ATG ^™ medium, and mixed media thereof.
Among these mediums, DMEM medium and MEM medium are preferable from the viewpoint of preparation of the medium.

The culture conditions are not particularly limited, and include conditions for culturing while changing the medium every 1 to 10 days as necessary at a carbon dioxide (CO ₂ ) concentration of 1 to 29% by volume, 5 to 45 ° C. for 1 hour to 100 days, etc. Is applicable. Preferable conditions are conditions of culturing while changing the medium every 1 to 3 days at a CO ₂ concentration of 3 to 10% by volume, 30 to 40 ° C., for 1 to 20 days.

The amount of cells to be seeded varies depending on the type of cell culture carrier used, the type of cells, and the like, but is preferably 10 to 10 million cells per 1 cm ² of the culture surface area of the cell culture carrier, more preferably 100 to 100. 10,000, particularly preferably 1,000 to 100,000.

  The subculture medium (B) of the present invention is preferably used in the cell production method of the present invention. The method for producing cells, which includes the washing step and the passage step of the present invention and performs each step in the order of the washing step and the passage step, takes out the cell culture carrier to which the cells obtained by cell culture are attached from the culture solution, A washing step of adding the subculture medium (B) according to claim 1 to the cell culture carrier to which the cells are adhered, washing the cell culture carrier to which the cells are adhered, and then removing the subculture medium (B). Then, a new cell culture carrier soaked in the subculture medium (B) is added to the cell culture carrier to which the cells obtained in the washing step are attached, and after the subculture, the subculture medium (B) is used as the culture medium (C ). Hereinafter, each process will be described sequentially.

<Washing process>
A subculture medium (B) is added to a container containing a cell culture carrier (hereinafter, sometimes referred to as a cell attachment carrier) containing a microcarrier (A) to which cells are attached, separated from the culture solution, Let stand or gently stir. Next, the cell adhesion carrier is allowed to settle in the container by allowing to stand, and the supernatant (B) is removed and discarded. The temperature at the time of standing or stirring is preferably 25 to 40 ° C., more preferably 30 to 38 ° C., from the viewpoint of the living environment of the cells. The standing time or stirring time is preferably 10 to 60 minutes, more preferably 10 to 30 minutes, from the viewpoint of loading on the cells. The method for removing the supernatant (B) is not particularly limited as long as it can be separated from the cell adhesion carrier, such as removal by decantation or suction removal with a pipette.

The washing step can be repeated a plurality of times as necessary. From the viewpoint of damage to the cells, the number of washing steps is preferably 1 to 10 times, more preferably 1 to 3 times. Within the range of the number of washing steps, the culture solution component remaining in (A) can be more reliably removed.
The weight ratio (g / L) between the cell adhesion carrier and the washing solution during the washing step is preferably 0.01 to 10 (g / L), more preferably 0.1 to 5 (g / L), particularly preferably 0. .1-3 (g / L).

<Passage process>
It is preferable that the washing step and the passage step are carried out in succession, and a new cell culture carrier soaked in the passage medium (B) is added to the container containing the cell attachment carrier after the washing step, and gently stirred. To do. The weight ratio (g / L) between the cell culture carrier and the subculture medium (B) of a new cell culture carrier immersed in the subculture medium (B) is 0.01 to 10 (g / L) is preferable, more preferably 0.1 to 5 (g / L), and particularly preferably 0.1 to 3 (g / L). The immersion time is preferably 10 to 60 minutes, more preferably 10 to 30 minutes from the viewpoint of cell growth. The immersion temperature is preferably 25 to 40 ° C., more preferably 30 to 38 ° C., from the viewpoint of maintaining the performance of the cell culture carrier and the deterioration of the medium.
As needed, you may implement the intermittent stirring method which repeats stirring and stationary in this process. The stirring time of the intermittent stirring method is preferably 1 to 30 minutes, more preferably 1 to 20 minutes, and particularly preferably 1 to 10 minutes from the viewpoint of passage. The stationary time of the intermittent stirring method is preferably 5 to 120 minutes, more preferably 5 to 90 minutes, and particularly preferably 5 to 60 minutes from the viewpoint of passage. The intermittent stirring time is preferably 3 to 96 hours, more preferably 5 to 72 hours, and particularly preferably 8 to 48 hours from the viewpoint of passage. The temperature during intermittent stirring is preferably 5 to 50 ° C., more preferably 15 to 45, and particularly preferably 25 to 40 from the viewpoint of damage to cells.
In addition, the process temperature in a washing | cleaning process and a subculture process may be the same, or may differ.

  The passaging step is carried out until the cells are uniformly transferred to a new cell culture carrier. From the viewpoint of damage to the cells, it is preferably 3 to 96 hours, more preferably 5 to 72 hours, and particularly preferably 8 to 48 hours.

  The ratio (weight ratio) between the cell attachment carrier and the new cell culture carrier during the passage step is preferably 1: 1 to 100, more preferably 1: 1 to 30, particularly preferably 1: 1 from the viewpoint of cell proliferation. -10.

  After the passage, the passage medium (B) is replaced with the culture medium (C). The replacement method for the subculture medium (B) is not particularly limited as long as it can be separated from the cell adhesion carrier, such as removal by decantation or suction removal with a pipette.

  After the passaging step, normal cell culture is performed. When the confluence rate of the cell culture carrier reaches 80 to 100%, if the required total number of cells has not been reached, the above passage method is performed again.

  Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited to these examples. In the following, unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.

<Production Example 1>
<Preparation of microcarrier (A)>
A reaction vessel equipped with a stirrer, a monomer supply pipe, a nitrogen gas introduction pipe, a thermometer, and a reflux condenser was charged with 624 parts of cyclohexane and 3.1 parts of sorbitan monostearate as a polymerization dispersant, and nitrogen bubbling was performed for 30 minutes or more. The dissolved air was driven out and the temperature was raised to 75 ° C.
A separate reactor was charged with 173 parts of an 80% aqueous acrylic acid solution, and neutralized by adding 207 parts of a 28% aqueous sodium hydroxide solution while cooling. To this aqueous solution was added 0.90 part of a crosslinkable monomer {ethylene glycol diglycidyl ether}, 0.278 part of a polymerization initiator {potassium persulfate}, and 0.053 part of a chain transfer agent {sodium hypophosphite}. Thereafter, nitrogen bubbling was performed to drive out dissolved air to obtain an aqueous monomer solution.
The obtained monomer aqueous solution was continuously fed at a rate of 6.5 parts / min from the monomer supply pipe of the above polymerization reactor into the cyclohexane solution being stirred in the polymerization reactor (stirring speed was 500 rpm) for about 1 hour. The polymerization was carried out under reflux of cyclohexane.
Next, 160 parts of water was extracted by azeotropic dehydration, and then the hydrogel polymer was taken out and further dried at 120 ° C. for 2 hours to obtain a dried crosslinked polyacrylic acid sodium salt.
The dried crosslinked polyacrylic acid sodium salt is classified with a sieve having an opening of 75 μm and a sieve having a sieve opening of 106 μm (JIS Z8801-1: 2000), and particles {crosslinked polyacrylate sodium salt particles} having a particle diameter of 75 to 106 μm (G ) 0.85% of sodium chloride was added to 1 g of water-absorbing resin (G), water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide / hydrochloride 3.83 g and aminoethanol / hydrochloride 3.90 g. After adding 25 mL of 0.02 M phosphate buffer (pH 5.2, PBS) contained in the above, and stirring at 25 ° C. for 4 hours, the reaction solution was removed by suction. After adding 100 mL of water and stirring for 1 hour, the washing operation of removing by suction was performed 5 times, and subsequently, drying under reduced pressure for 4 hours with a vacuum dryer {40 ° C., 0.1 kPa or less} to obtain the water absorbent resin (H). To 1 g of water-absorbing resin (H), 8.6 g of N-diethylaminoethyl chloride / hydrochloride, and 4.35 g of 48% aqueous sodium hydroxide solution, 10 mL of ion-exchanged water was added, and the mixture was heated at 60 ° C. After stirring and reacting for 5 hours, the reaction solution was removed by suction, 100 mL of ion-exchanged water was added, the mixture was stirred for 1 hour, and then the suction operation was performed 5 times, followed by a vacuum dryer {40 ° C., 0 .1 kPa or less} was dried under reduced pressure for 4 hours to obtain a water-absorbent resin (I-1), and the water retention amount of (I-1) was 10 g / g.

<Preparation of polypeptide (P1)>
In accordance with the method described in the examples in JP-T-3-502935, it has 13 Arg Gly Asp sequences (x1) and (Gly Ala Gly Ala Gly Ser) ₉ sequences (21), a structure having a Gly Ala Ala Val Thr Gly sequence (65) at the N-terminus of x1) and a Ser Pro Ala Ser Ala Ala Gly Tyr sequence (78) at the C-terminus of (x1), which are alternately chemically bonded A peptide “pronectin F” having an Mw of about 110,000 was produced as a polypeptide (P1).

<Preparation of polypeptide (P1-1)>
Polypeptide (P1) 50 mg and N, N-dimethylaminoethyl chloride hydrochloride (special grade reagent) 150 mg were dissolved in 1.5 mL of 4.5 M aqueous lithium perchlorate solution at 20-40 ° C. While stirring at ° C., 1.325 mL of 4.5 M aqueous lithium perchlorate solution in which 100 mg of sodium hydroxide (special grade reagent) was dissolved was added dropwise at a constant rate over 45 to 50 seconds. After stirring at room temperature (25 ° C.) for 1 hour, the reaction solution was dialyzed against 10 L of deionized water for 48 hours using a dialysis membrane having a molecular weight cut off of 12,000 to 14,000 to remove by-products. . In the first 12 hours, deionized water was changed every 4 hours. The obtained aqueous solution was lyophilized for 24 hours at −20 ° C. and 0.1 kPa or less, and the polypeptide (P1) was etherified with N, N-dimethylaminoethyl chloride hydrochloride. Peptide (P1-1) was obtained.

<Preparation of water absorbent resin to which polypeptide (P1) is bound>
A 0.02M, pH 5.2 PBS solution containing 1 g of water-absorbing resin (I-1) with water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) at a concentration of 60 mM. 15 mL of was added, stirred at 25 ° C., and reacted for 0.5 hour to obtain a modified water absorbent resin.
Next, 1 mL of 0.2 M, pH 7.2 PBS solution containing the polypeptide (P1) at a concentration of 2.4 mg / mL is added to the modified water-absorbent resin, and the mixture is stirred at 25 ° C. and allowed to react for 2 hours. After that, the reaction solution is removed by suction, and 40 ml of PBS is added and removed by suction 5 times. Further, 40 ml of ion-exchanged water is added and the washing operation is removed by suction 2 times, followed by a vacuum dryer. It was dried under reduced pressure at {40 ° C., 0.1 kPa or less} for 4 hours to obtain a microcarrier (polypeptide-bonded water absorbent resin) (A-1). The true specific gravity of A-1 was 1.03.

<Preparation of water absorbent resin to which polypeptide (P1-1) is bound>
A 0.02M, pH 5.2 PBS solution containing 1 g of water-absorbing resin (I-1) with water-soluble carbodiimide (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) at a concentration of 60 mM. 15 mL of was added, stirred at 25 ° C., and reacted for 0.5 hour to obtain a modified water absorbent resin.
Next, 1 mL of a 0.2 M, pH 7.2 PBS solution containing the polypeptide (P1-1) at a concentration of 2.4 mg / mL is added to the modified water-absorbent resin, and the mixture is stirred at 25 ° C. for 2 hours. After the reaction, the reaction solution is removed by suction, and 40 ml of PBS is added and removed by suction five times. Further, 40 ml of ion-exchanged water is added and removed by suction and then washed twice. Drying under reduced pressure with a dryer {40 ° C., 0.1 kPa or less} for 4 hours gave a microcarrier (polypeptide-bonded water-absorbing resin) (A-2). The true specific gravity of A-1 was 1.03.

<Passage evaluation using Vero cells>
<Example 1>
To a spinner flask, 0.03 g of microcarrier (A-1) and a phosphate buffer prepared at pH = 7.2 (0.02% disodium hydrogen phosphate, 0.076% potassium dihydrogen phosphate and sodium chloride) (Contains 0.85%, hereinafter abbreviated as PBS.) 10 mL was charged and sterilized by autoclave (121 ° C., 20 minutes). After autoclave sterilization, PBS was removed by suction with an aspirator, and 2 mL of a medium containing 0.2% by weight of an antibacterial agent (trade name: gentamicin / amphotericin B, manufactured by Cascade) was added to the MEM medium. Then, the medium was removed by suction, and 10 mL of the same serum-free medium was added again. The spinner flask was allowed to stand in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume for 1 hour, and then pre-cultured VERO cells were serum-free so that the cell concentration became 200,000 cells / mL. Seeded in culture medium. In a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume, while stirring at 60 rpm, the cells were adhered by culturing for 7 days until almost the entire surface of (A-1) was covered with cells (A -1) was obtained. A MEM medium (B-1) with a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 is placed in a container containing (A-1) to which cells are attached, separated from the culture solution. 10 ml was added and allowed to stand at 37 ° C. for 10 minutes. Subsequently, the cell adhesion carrier was allowed to settle in the container by allowing to stand, and the supernatant (B-1) was removed with a micropipette. The cell attachment carrier was put into a spinner flask containing 0.3 g of microcarrier (A-1) previously immersed in 37 ml of MEM medium (B-1) for 1 hour at 37 ° C. Thereafter, intermittent stirring (stirring for 2 minutes, standing for 28 minutes) was carried out for 30 hours at 37 ° C. in a carbon dioxide gas concentration of 5 vol% CO ₂ incubator. After completion of intermittent stirring, the cell adhesion carrier is settled in the container, and the supernatant (B-1) is removed with a micropipette, and the culture medium (C-1) {MEM medium is added with an antibacterial agent (trade name: gentamicin / amphotericin B the medium} which contains the manufactured cascade Co.) at 0.2% by weight 100ml turned, 37 ° C., in a carbon dioxide gas concentration of 5 vol% CO ₂ incubator with agitation of 60 rpm. Sampling was performed on the seventh day of culture, and the adhesion rate to the microcarrier was measured by the method described in “Evaluation of cell adhesion rate to microcarrier” described later. On the 4th, 5th, and 6th days of culture, half of the medium was replaced with a new culture medium (C-1).

<Evaluation of cell adhesion rate to microcarriers>
The cell adhesion rate to the microcarrier was evaluated as follows. Take 0.5 mL of the cell culture carrier-containing culture solution after completion of the passage step, and measure the number of microcarriers to which one or more cells are adhered and the number of microcarriers to which the cells are not adhered with a microscope. After the measurement, the cell adhesion rate to the microcarrier was calculated from the following formula, and the results are shown in Table 1.

Cell adhesion rate to microcarriers (%) = {(number of microcarriers to which one or more cells are adhered) / (number of microcarriers to which one or more cells are adhered + micros to which cells are not adhered) Number of carriers)} × 100

<Evaluation of cell proliferation>
Take 0.5 mL of the cell culture solution 7 days after culturing at the end of the subculture step, remove the medium, add 0.5 mL of an aqueous solution containing 0.1% crystal violet and 1.92% citric acid, and add 60 mL at 37 ° C. After maintaining for a minute, the number of cell nuclei was counted using an erythrocyte counter, and the cell concentration (10,000 cells / mL) in the medium was measured. The results are shown in Table 1.

<Example 2>
In Example 1, the total concentration of calcium ions and magnesium ions is 2.3 mM and the ratio is 1 in the MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25. : Evaluation was performed in the same manner as in Example 1 except that the MEM medium (B-2) was changed to 2.25.

<Example 3>
In Example 1, a MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 was changed to a MEM medium (B-) having a total concentration of calcium ions and magnesium ions of 0 mM. Evaluation was performed in the same manner as in Example 1 except that the procedure was changed to 3).

<Example 4>
In Example 1, MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 was used, and the total concentration of calcium ions and magnesium ions was 1.15 mM and the ratio was 1. : Evaluation was carried out in the same manner as in Example 1 except that the medium was changed to 5 MEM medium (B-4).

<Example 5>
In Example 1, evaluation was performed in the same manner as in Example 1 except that the number of times of washing in the washing step was set to 2.

<Example 6>
In Example 1, evaluation was performed in the same manner as in Example 1 except that the microcarrier (A-1) was changed to dextran beads (trade name Cytodex 1 manufactured by Amersham).

<Passage evaluation using HmLu cells>
<Example 7>
A spinner flask was charged with 0.03 g of microcarrier (A-1) and 10 mL of PBS adjusted to pH = 7.2, and autoclaved (121 ° C., 20 minutes). After autoclave sterilization, PBS was removed by suction with an aspirator, and 2% fetal bovine serum (FBS) -containing Eagle-MEM medium contained 0.2% by weight of an antibacterial agent (trade name: gentamicin / amphotericin B, manufactured by Cascade) After adding 2 mL of the prepared medium and allowing to stand for 1 hour, the medium was removed by suction, and 10 mL of the same serum-free medium was added again. After leaving the spinner flask in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume for 1 hour, pre-cultured HmLu cells in the medium so that the cell concentration becomes 200,000 cells / mL. Sowing. In a CO ₂ incubator with a temperature of 37 ° C. and a carbon dioxide gas concentration of 5% by volume, while stirring at 60 rpm, the cells were attached by culturing for 4 days until almost the entire surface of (A-1) was covered with cells (A -1) was obtained. Eagle-MEM medium (B-1) in which the total concentration of calcium ions and magnesium ions is 1.15 mM and the ratio is 1: 2.25 in a container containing (A-1) to which cells are attached, separated from the culture solution. 10 ml) was added and allowed to stand at 37 ° C. for 10 minutes. Subsequently, the cell adhesion carrier was allowed to settle in the container by allowing to stand, and the supernatant (B-1) was removed with a micropipette. The cell attachment carrier was put into a spinner flask containing 0.3 g of microcarrier (A-1) previously immersed in 37 ml of MEM medium (B-1) for 1 hour at 37 ° C. Thereafter, intermittent stirring (stirring for 2 minutes, standing for 28 minutes) was carried out for 30 hours at 37 ° C. in a carbon dioxide gas concentration of 5 vol% CO ₂ incubator. After intermittent stirring, the cell adhesion carrier is allowed to settle in the container, and the supernatant (B-1) is removed with a micropipette, and the culture medium (C-1) {2% FBS-containing Eagle-MEM medium is added with an antibacterial agent (product) Name: gentamicin / amphotericin B, manufactured by Cascade Co., Ltd.) at a concentration of 0.2% by weight, and then stirred at 60 rpm in a CO ₂ incubator at 37 ° C. and a carbon dioxide gas concentration of 5% by volume. Incubated while incubating. Sampling was performed on the 6th day of culture, and the cell adhesion rate to the microcarrier and the cell proliferation were measured in the same manner as in Example 1.

<Passage evaluation using MDCK cells>
<Example 8>
A spinner flask was charged with 0.03 g of microcarrier (A-1) and 10 mL of PBS adjusted to pH = 7.2, and autoclaved (121 ° C., 20 minutes). After autoclave sterilization, PBS was removed by suction with an aspirator, and 2 mL of a medium containing 0.2% by weight of an antibacterial agent (trade name: gentamicin / amphotericin B, manufactured by Cascade) was added to the MEM medium. Then, the medium was removed by suction, and 10 mL of the same serum-free medium was added again. The spinner flask is allowed to stand in a CO ₂ incubator with a carbon dioxide gas concentration of 5% by volume at 37 ° C. for 1 hour, and then pre-cultured MDCK cells are added to the medium so that the cell concentration becomes 200,000 cells / mL. Sowing. In a CO ₂ incubator with a temperature of 37 ° C. and a carbon dioxide gas concentration of 5% by volume, while stirring at 60 rpm, the cells were attached by culturing for 4 days until almost the entire surface of (A-1) was covered with cells (A -1) was obtained. A MEM medium (B-1) with a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 is placed in a container containing (A-1) to which cells are attached, separated from the culture solution. 10 ml was added and allowed to stand at 37 ° C. for 10 minutes. Subsequently, the cell adhesion carrier was allowed to settle in the container by allowing to stand, and the supernatant (B-1) was removed with a micropipette. The cell attachment carrier was put into a spinner flask containing 0.3 g of microcarrier (A-1) previously immersed in 37 ml of MEM medium (B-1) for 1 hour at 37 ° C. Thereafter, intermittent stirring (stirring for 2 minutes, standing for 28 minutes) was carried out for 30 hours at 37 ° C. in a carbon dioxide gas concentration of 5 vol% CO ₂ incubator. After completion of intermittent stirring, the cell adhesion carrier is settled in the container, and the supernatant (B-1) is removed with a micropipette, and the culture medium (C-1) {MEM medium is added with an antibacterial agent (trade name: gentamicin / amphotericin B 100 ml of a medium containing 0.2% by weight of Cascade Co., Ltd.} was added and cultured in a CO ₂ incubator with a carbon dioxide gas concentration of 5% by volume at 37 ° C. with stirring at 60 rpm. Sampling was performed on the 6th day of culture, and the cell adhesion rate to the microcarrier and the cell proliferation were measured in the same manner as in Example 1.

<Example 9>
In Example 1, it evaluated similarly to Example 1 except having changed the microcarrier (A-1) into the microcarrier (A-2).

<Comparative Example 1>
A MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 has a total concentration of calcium ions and magnesium ions of 2.4 mM and a ratio of 1: 2.25. Evaluation was performed in the same manner as in Example 1 except that the medium was changed to MEM medium (B′-1).

<Comparative example 2>
In Example 1, it evaluated similarly to Example 1 except having changed microcarrier (A-1) into the water absorbing resin (I-1) which does not contain (P1).

<Comparative Example 3>
A MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 has a total concentration of calcium ions and magnesium ions of 2.4 mM and a ratio of 1: 2.25. Evaluation was performed in the same manner as in Example 7 except that the medium was changed to MEM medium (B′-1).

<Comparative example 4>
A MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 has a total concentration of calcium ions and magnesium ions of 2.4 mM and a ratio of 1: 2.25. Evaluation was carried out in the same manner as in Example 8 except that the medium was changed to MEM medium (B′-1).

<Comparative Example 5>
A MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 has a total concentration of calcium ions and magnesium ions of 2.4 mM and a ratio of 1: 2.25. Evaluation was carried out in the same manner as in Example 9 except that the medium was changed to MEM medium (B′-1).

<Comparative Example 6>
A MEM medium (B-1) having a total concentration of calcium ions and magnesium ions of 1.15 mM and a ratio of 1: 2.25 has a total concentration of calcium ions and magnesium ions of 2.4 mM and a ratio of 1: 2.25. Evaluation was carried out in the same manner as in Example 1 except that the medium was changed to MEM medium (B′-1) and the microcarrier (A-1) was changed to dextran beads (trade name Cytodex 1 manufactured by Amersham).

  From Table 1, it can be seen that the cell adhesion rate and cell proliferation of Examples 1 to 6 of the present invention are far superior to the results of Comparative Examples 1 and 2. By using a microcarrier having a polypeptide and using the subculture medium of the present invention, the cell adhesion rate and the cell proliferation property are dramatically improved as compared with the comparative example, and the cell proliferation ability is reduced. It can be seen that they can be subcultured. In addition, by carrying out the passage method of the present invention, cells can be passaged uniformly on microcarriers, cell proliferation is improved compared to comparative examples, and without reducing cell proliferation ability It can be seen that subculture can be performed without reducing the cell growth ability.

When cells are produced using the subculture medium of the present invention (preferably, by the intermittent stirring culture method), the cells are easily transferred to new microcarriers efficiently and uniformly. In addition, subculture can be performed without reducing the cell growth ability, and subculture can be performed without reducing the cell growth ability.
In addition, when cells are produced by the cell production method of the present invention (preferably by an intermittent stirring culture method), the cells are easily transferred to a new microcarrier efficiently and uniformly. In addition, subculture can be performed without reducing the cell growth ability, and subculture can be performed without reducing the cell growth ability.
Therefore, the present invention is effective for the culture production of all adherent cells such as production of cells for virus culture and production of useful protein-expressing cells.

Claims (2)

In the method for culturing adherent cells, cells attached to a cell culture carrier including a microcarrier (A) having a polypeptide (P) having at least one amino acid sequence (X) having a minimum cell adhesiveness in one molecule are newly added. A subculture medium used in the step of subcultured as cells attached to a cell culture carrier, wherein the total concentration of calcium ions and magnesium ions is 0 to 2.3 mM (B). After culturing adhesive cells using a cell culture carrier comprising a microcarrier (A) having a polypeptide (P) having at least one cell adhesion minimal amino acid sequence (X) in one molecule, A cell production method comprising a subculture step of substituting attached cells as cells attached to a new cell culture carrier, comprising the following washing step and subculturing step, and in the order of washing step and subculturing step A method for producing cells in which each step is performed.
Washing step: The cell culture support to which the cells obtained by cell culture are attached is taken out from the culture solution, and the subculture medium (B) according to claim 1 is added to the cell culture support to which the cells are attached. A step of washing the attached cell culture carrier and then removing the subculture medium (B).
Passage step: A new cell culture carrier soaked in the passage medium (B) is added to the cell culture carrier to which the cells obtained in the washing step are attached, and after passage, the passage medium (B) is used as the culture medium. The step of replacing with (C).