JPH03754A - Styrene polymer and carrier for adhesive cell culture - Google Patents

Abstract

PURPOSE:To improve adhesive properties, extension and propagation of a cell, to manifest sufficiently functions of the cultured cell and to make it useful as a polymer material for adhesive cell culture by incorporating specified two kinds of repeating units. CONSTITUTION:Coupling reaction of the aminomethylene group of an aminomethylstyrene with an aminoalkylcarboxylic acid (b) is performed to obtain an aminoacyl-modified monomer. Then, this monomer and, if necessary, styrene monomer are polymerized to obtain the title polymer with a wt.-average MW of 10,000-80,000, contg. repeating units A of formula I and repeating units B of formula II (wherein R is H or formula III; n is 1-10) wherein A+B=1, 0<B<=1 and at least one half of the whole repeating units B are those having a group of formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel styrenic polymer, a salt thereof, and an adhesive cell culture carrier made of the same.

H2HR (n is an integer of 1 to 10). ] Hundreds of types of cells that make up the organic rice technology living body are involved in various reactions such as metabolic regulation and immune response to exogenous and endogenous abnormalities, with ratios that satisfy A + B = 1 and O < B ≦ 1. It produces information transmitting substances and takes part in the production of many physiologically active substances, including hormones, and many of the currently known useful physiologically active substances are produced by culturing living cells. It is. However, in vitro
The above-mentioned cells that can be cultured in the culture vessel wall are: (1) those that adhere relatively strongly to the culture vessel wall and will die if the adhesion to the vessel wall is prevented, and (2) those that adhere relatively weakly to the vessel wall and have weak physical properties. They can be roughly divided into three types: those that detach from the vessel wall when manipulated but require the above-mentioned adhesion in order to survive, and those that can be weakly bonded to the vessel wall but can survive floating. Cells that are capable of producing physiologically active substances mainly belong to the above categories ■ and ■, which require adhesion to the organ wall for survival.
These cells are called adherent cells (anchorage dep.
dendent cells).

A typical example of the adhesive cells mentioned above is hepatocytes (hepatocytes)
can be mentioned. The hepatocytes are responsible for the important functions of the liver, which can be said to be the site of the largest exchange of substances in the body: synthesis and secretion of plasma proteins, gluconeogenesis, blood sugar regulation (glycogen metabolism), lipid synthesis, urea synthesis, bile synthesis and secretion, It is responsible for all functions such as detoxification, and has an amazing self-propagation ability compared to other living cells. However, the functions of secondary hepatocytes are expressed in vivo, are mainly regulated by substances in plasma, and are controlled by various structures and mechanisms of the liver itself. It is known that hepatocytes are extremely sensitive to environmental changes and easily lose their metabolic functions and become nonparenchymal even if they do not result in cell death. Conventionally, it has been extremely difficult to express this function.

The difficulties in in vitro culture of hepatocytes depend not only on the characteristics of the hepatocytes themselves, but also on the culture substrate (cell adhesion carrier) and the various substances and actions that exist between the cells and the carrier. Traditionally, petri dishes, test tubes, culture bottles, etc. made of glass or plastic have been used as carriers for culturing adherent cells such as hepatocytes, but in recent years, microcarriers, hollow fibers, etc. have been reported, but all of them are particularly effective in terms of adhesion (attack+menl), which is an absolutely necessary condition for the functional expression of adherent cells, and the subsequent cell spreading and proliferation. However, it did not have satisfactory performance in terms of adhesion and proliferation at the initial stage of culture, and was unsuitable for high-density and long-term cell culture.

The factors involved in the adhesion between the adherent cells and the polymeric material used as their culture carrier include direct action between the secondary cells and the carrier, and indirect action through the medium etc. that exists between them. Considering both, there are physicochemical (non-specific) interactions such as hydrophobicity, ionicity, and hydrogen bonding, and biochemical (specific) interactions such as specific receptor activity on the cell surface. It is thought that then. In fact, the adhesion of cells to carriers is determined by the fact that cells, which are highly fluid, higher-order molecular aggregates with a heterogeneous cell membrane in which the above factors are distributed in a mosaic pattern, It depends on how the carrier is recognized in the space in which it exists, and the action involved in adhesion between the cell and the carrier and the point of contact between the two are naturally not single, but may occur at the interface between the cell membrane and the carrier. The various interaction points mentioned above are distributed.

Recently, polystyrene having oligosaccharides, especially lactose, in its side chains [Kobunshi Ronshu, Mol.

42, No. 11.719-724 (1985), Journal of the Chemical Society of Japan, (3) 575-579 (1987)] and amino acid copolymers having specific glutamine derivative residues [JP-A-64-47373]. [Refer to the official bulletin], etc., was established as a reference room. However, all of these studies are based on research from the aspect of biological (specific) interactions mentioned above, and do not take into account multi-point interactions between cells and carriers, and in particular, the adhesiveness of hepatocytes and There is still room for improvement in the hydrophilicity and charge density of the carrier surface, which greatly affect proliferation, and the adhesiveness and proliferation of various adherent cells other than hepatocytes could not be expected. In place of these, there is a strong desire in the art for the development of new polymeric materials that are more universally effective against various types of cells and that can maintain culture and function of secondary cells over a long period of time.

Furthermore, even when using the various known polymeric materials mentioned above, when culturing adherent cells, serum components such as fetal calf serum (FCS) are generally added to the culture medium at a concentration of about 10 to 2
It is said that it is necessary to add 0% of serum components, but the serum components themselves are expensive, and since they are natural products, their quality is inconsistent, and there are no useful substances. Removal after production requires complicated operations, and therefore, there is a need in the art for the development of the above-mentioned new polymeric materials that are effective for adhesion and proliferation of adherent cells, especially in serum-free media. In the current situation.

Problems to be Solved by the Invention The object of the present invention is to provide a material suitable for culturing the above-mentioned adherent cells, particularly in a serum-free medium, and also to eliminate all the drawbacks of this kind of polymeric material known in the art. The object of the present invention is to provide a new adhesive polymeric material for cell culture, which has particularly excellent cell adhesion, spreading and proliferation properties, and is capable of fully expressing the functions of cultured cells.

Means for Solving the Problems According to the present invention, repeating unit A: and repeating unit B: HR (n is an integer of 1 to 10) are shown. ] in a ratio that satisfies A + B = 1 and ORB≦1, has a weight average molecular weight in the range of 10,000 to 80,000, and has a small number (both 1/2 of the above R groups other than hydrogen atoms) in the total repeating unit B. Provided are a styrenic polymer and its salt, which are units having the following: and an adhesive cell culture carrier made from the styrenic polymer and/or its salt.

As a result of extensive research, the present inventors found that among the interactions between adherent cells and carriers, the hydrophilicity and charge density state of the carrier surface have a significant influence on the adhesion and proliferation of adherent cells. We found that by appropriately adjusting the isosurface charge and the degree of hydration, adherent cells can be successfully attached and proliferated without using biochemical effects such as cell surface receptor activity. They discovered that it is possible for the cells to express their functions over a long period of time, and succeeded in synthesizing a new polymeric material suitable as a carrier having such properties, thereby completing the present invention.

By using the polymer of the present invention as a carrier for culturing adherent cells, it is possible to impart excellent adhesion and proliferation to adherent cells solely through the above-mentioned physicochemical effects, and this property is also achieved in the absence of serum. It is expressed by culturing adherent cells in a medium, and thus the cells can be cultured, proliferated, maintained, and functionally expressed over a long period of time. In addition, the adhesive cell culture carrier of the present invention is particularly suitable for the growth and maintenance of hepatic parenchymal cells in a serum-free medium, and its use will also enable the development of hybrid artificial livers and hexioreactors. There are advantages.

The styrenic polymer of the present invention and the adhesive cell culture carrier of the present invention using the polymer will be described in detail below.

The polymer of the present invention is soluble in various organic solvents and has good moldability.
The base material is polystyrene, which can be easily prepared as a plate-shaped culture carrier and has been commercially available as a culture dish for this kind of cell culture.Basically, after aminomethylating the aromatic ring of the polystyrene, aminoacyl It can be obtained by converting

As a more preferable method for producing the polymer of the present invention, for example, first, a styrene monomer that provides the repeating unit A and an aminomethylstyrene monomer that provides the same repeating unit B (where R represents a hydrogen atom) are mixed in a predetermined ratio ( (including the case where a monomer giving repeating unit B is used alone) is polymerized,
An example of a method in which the aminomethylene group of the resulting polymer is then subjected to a coupling reaction with an aminoalkylcarboxylic acid that provides a group R other than a hydrogen atom to modify it with an aminoacyl group can be exemplified.

Another preferred method for producing the polymer of the present invention is to first add a group R other than a hydrogen atom to an aminomethylene group of an aminomethylstyrene monomer that provides the repeating unit B (where R represents a hydrogen atom). A method is exemplified in which a desired monomer modified with an aminoacyl group is synthesized by a coupling reaction of an alkyl carboxylic acid, and this monomer is polymerized alone or in a predetermined ratio of the monomer and a styrene monomer providing the repeating unit A. can.

In the above method, the aminomethylstyrene monomer that provides the repeating unit B (R represents a hydrogen atom) can be produced according to a conventional method. For example, chloromethylstyrene is phthalimidated according to the Gabriel method (Gsbrtel) to obtain vinylbenzyl phthalimide,
Then, it can be produced by decomposing it with hydrazine [for example, RID].

A11en el it,, J, Ce1l Biol,
, 83.126 (1979)].

Modification of the aminomethylene group of the aminomethylstyrene monomer obtained as described above or the aminoacyl group present in the polymer obtained by polymerizing this with a styrene monomer is to modify the aminomethylstyrene group with an aminoacyl group. This can be carried out by coupling an aminoalkylcarboxylic acid that provides a group R other than a hydrogen atom.

The reaction is carried out using the aminoalkylcarboxylic acid whose N-terminus has been protected in advance as a spacer reagent, and a suitable condensing agent such as dicyclohexylcarbodiimide (DCC) to form a simple amide bond. After modification by the coupling reaction, desired monomers and polymers can be produced by removing the N-terminal protecting group according to a usual deprotecting group reaction. Specific examples of the spacer reagent used in the modified coupling reaction include aminobutyric acid, aminocaproic acid, aminocaprylic acid, aminoundecanoic acid, etc. Among these, aminobutyric acid is particularly preferred.

Further, as the N-terminal protecting group of the secondary aminoalkylcarboxylic acid, common amino group protecting groups, typically tert-butoxycarbonyl (Boc) group, benzyloxycarbonyl, 0-chlorobenzyloxycarbonyl group, etc. can be exemplified. . The above coupling reaction using a condensing agent such as DCC is carried out using a suitable solvent such as dichloromethane, chloroform, carbon tetrachloride, etc., using a condensing agent in an equimolar amount to about 4 times the molar amount of the starting compound. In addition, in a solvent such as halogenated hydrocarbons such as tetrachloroethane, dioxane, tetrahydrofuran, acetone, acetonitrile, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, etc., generally from about 0°C to 60°C,
Preferably, it can be carried out at about 0° C. to room temperature and for about several tens of minutes to about 120 hours. The elimination reaction of the protecting group after the above-mentioned coupling reaction can be carried out by a conventional hydrogenation method, reduction method, acidolysis using a strong acid, or the like.

Further, the polymerization reaction of each monomer can be carried out in the same manner as in the production of ordinary polystyrene. More specifically, repeating unit B (wherein R is a hydrogen atom or an acylamino group, and the amino group of the acylamino group may be protected with a protective group) alone or repeating unit A and repeating unit B A predetermined proportion of mixed monomers is dissolved in a suitable solvent, such as aromatic hydrocarbons such as benzene, chlorobenzene, xylene, toluene, and nitrobenzene, halogenated hydrocarbons such as carbon tetrachloride and methylene chloride, dioxane, and tetrahydrofuran. Polymerization initiator, e.g. 2.2'
- Add azobisisobutyronitrile (AIBN), benzoyl peroxide (BPO), etc. to 1% of the total amount of monomers.
/200 to 1/100 mol is added. The obtained reaction solution is frozen at about -10°C or lower, vacuum degassing and nitrogen substitution are repeated several times, and then the tube is sealed under normal pressure or reduced pressure, preferably at 30 to 90°C, more preferably at 50°C. ~2 hours at 70℃~
Allow to polymerize for several days. In addition, in the case of repeating unit B in which the acylamino group is protected with a protecting group, deprotection is performed as necessary after the polymerization reaction. The polymer is washed with a suitable solvent such as ether, methanol, hexane, etc., or the reaction solvent is removed by freeze-drying, etc., and then the polymer is washed with a suitable acid such as acetic acid, etc.
Dissolve in formic acid etc. and purify with ether.

In any of the above methods, the charging ratio of the monomer giving the repeating unit A and the monomer giving the repeating unit B is 0 and B≦1, preferably 0, assuming that A+B=1.
．． 03<B<0.5 or B=1, more preferably B is about 0. It is preferable to set it to a range that satisfies around 1 or B=1.

The proportion of the aminoalkylcarboxylic acid to be subjected to the modification coupling reaction with the aminomethylene groups in the above repeating unit B or the monomer providing the repeating unit B is such that at least 1/2 of all the aminomethylene groups are modified, preferably almost all of the aminomethylene groups are modified. It is appropriate that the amount is such that the methylene group is modified.

In this way, the styrenic polymer of the present invention can be produced. The identity of the styrenic polymer can be confirmed by instrumental analysis such as infrared absorption spectroscopy and thin layer chromatography. As a result of measurement by mutation chromatography, it is in the range of 10,000 to 80,000.

The styrenic polymer obtained above may be obtained in the form of an acid addition salt of the amino group present in its side chain, or it may be converted into the form of an acid addition salt using a normal acid according to a conventional method. You can also do it. Examples of acids that can form such acid addition salts include inorganic acids such as hydrochloric acid and perchloric acid, and organic acids such as acetic acid and formic acid.

The present invention also provides an adhesive cell culture carrier made of the above styrene polymer and/or its salt. The carrier can be obtained by shaping the above-mentioned material into a suitable shape, such as a membrane, fiber, or sphere. A particularly preferred form of the above-mentioned carrier is a film-like form, which is obtained by casting a solubilized solvent solution of the polymer of the present invention on a substrate such as a usual petri dish for cell culture, and then distilling off the solvent. It can be prepared by The solubilizing solvent solution for the polymer used above varies depending on the type of polymer, but for example, an acetic acid solution, a dioxane solution, a dioxane-acetic acid solution, an ethanol-acetic acid solution, a dimethylformamide solution, etc. are preferable. In this case, it is appropriate to neutralize with alkaline water and wash with deionized water after film formation.

By utilizing the adhesive cell culture carrier of the present invention thus obtained, adherent cells can be efficiently cultured and maintained in a serum-free medium. The adhesive cells that can be cultured in a serum-free medium using the carrier of the present invention are not particularly limited as long as they are adhesive, and examples include human fetal foreskin fibroblasts, Chinese hamster lung fibroblasts, chimpanzee lung fibroblasts, Various fibroblasts such as chicken embryo fibroblasts and mouse metastatic fibroblasts, human uterine cancer cells, human fetal lung cells, human kidney cancer cells, primary monkey kidney cells, pituitary tumor cells,
Examples include epithelial cells such as primary human hepatocytes and cancerous cells such as these. In particular, hepatocytes are difficult to culture in vitro, as mentioned above, and by using the carrier of the present invention, serum-free culture of such hepatocytes becomes possible, which will lead to the development of hybrid artificial livers, bioreactors, etc. This will greatly contribute to the

In culturing the above-mentioned adherent cells using the carrier of the present invention, any of various known media can be used. The medium includes, for example, Minimum Essential (MEM) medium, Dulbecco's modified Eagle's medium, Williams E medium 1, L-
15 medium, RPMI-1640 medium, and the like. 2
It is preferable to add various commonly used growth factors to the equal medium, and it is of course possible to add serum, fibronectin, etc., although this is not particularly necessary.

The above-mentioned medium is also preferably adjusted to a pH of 7 to 8 using, for example, HEPES buffer, Dulbecco's phosphate buffer, etc., and the culture of cells in this medium is carried out under normal liquid culture conditions.
Specifically, carbon dioxide culture is preferably carried out at a temperature of 30 to 40°C, preferably around 37°C. The amount of cells seeded in the above medium is generally 1×104 to 104 per 111 of the medium.
Appropriately, the number is about 105.

Thus, by using the carrier of the present invention, adhesion, spreading, and proliferation of adherent cells can be achieved very well in a serum-free medium.

EXAMPLES Hereinafter, in order to explain the present invention in more detail, production examples of monomers, etc. for producing the polymer of the present invention will be given as reference examples, and then examples of production of the polymer of the present invention will be given as examples.

Furthermore, an example of cell culture using a carrier of the present invention prepared using the obtained polymer of the present invention will be given as a test example.

The substances obtained in each production example were identified by the following infrared absorption spectrum (IR) analysis and thin layer chromatography (
This was done by TLC') analysis.

1. IR analysis: Monomer samples were prepared as KBr tablets, and polymers that could not be crushed and KBr tablets could not be obtained were dissolved in an appropriate solvent to form a film, each manufactured by JASCO Corporation A302.
Measured using a model infrared spectrophotometer.

n, TLc: Using silica gel 60F254TLC plastic sheet manufactured by Metck as a thin layer carrier, the following 4
It was carried out using a seed developing solvent.

1) CMA; Chloroform: Methanol: Acetic acid (95:
5:5) 2) CMP; chloroform:methanol:pyridine (9
5:5:3) 3) Pa; n-butanol: acetic acid: water (4: 1: 5 upper layer) 4) Py; n-butanol: pyridine: acetic acid: water (15
:10:3:12) Furthermore, unless otherwise specified, the average molecular weight of the polymer is determined by gel permeation chromatography [Column: Superose 6 (manufactured by Pharmacia), developing solvent: Q, 05M
HCl, flow rate: 0.4xl/min], and the weight molecular weight ratio and asci in terms of polystyrene.

Reference Example 1 Purification of styrene monomer Commercially available styrene (10 to 20 ppm of P as a polymerization inhibitor)
- tert-butylcatechol added (manufactured by Wako Pure Chemical Industries, Ltd.) was washed with a 5% aqueous sodium thiosulfate solution and deionized water, further washed with a 10% aqueous sodium hydroxide solution, and then washed with water until neutral. , then dehydrated with barium oxide,
Styrene monomer was prepared by distillation under reduced pressure under a nitrogen stream.

Reference Example 2 Synthesis of aminomethylstyrene monomer (1) Commercially available chloromethylstyrene monomer (m-:p
-=7:3, manufactured by Tokyo Kasei Co., Ltd.) 61.0 g and potassium phthalimide (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 74.0 g, N, N'
-Dissolve in dimethylformamide (DMF) 2002A', react at 50°C for 4 hours, concentrate under reduced pressure, dissolve the resulting syrup in chloroform 240xl, and 0.2N
Washed four times with 200 zl of sodium hydroxide aqueous solution. More water 200z! After washing three times with
A solid was obtained by washing with hexane and drying.

This was recrystallized from methanol to obtain 76.0 g of vinylbenzyl phthalimide.

Melting point 76-77℃ Rf value CMA: 0.83 CMP: 0.5sPa
:0.92 Py :0.75(2) Above (1
) of vinylbenzyl phthalimide obtained in 20% of ethanol.
To this was added an ethanol solution of 25.0 g of 80% hydrazine hydrate (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) to
Boil at reflux for minutes. After concentration under reduced pressure, it was dissolved in 600 zl of potassium hydroxide aqueous solution (1:6 (w/v%)), extracted with ether, and diluted with 160 zl of 2% potassium carbonate aqueous solution.
After washing twice, the residue was dehydrated with anhydrous potassium carbonate, concentrated, and then distilled under reduced pressure (5.0 mmHg, 77-78°C) to obtain 23.8 g of aminomethylstyrene.

d2G=0.96 g/xi Rf value CMA: 0.05 CMP: 0.07Pa
:0.31 Py :0.67 Reference Example 3 Production of aminoacylaminomethylstyrene monomer (1) γ-Aminobutyric acid (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 4.1
4g of triethylamine (TEA) with 22zl of deionized water.
) 8.4zl and added 5-t-butyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine (Boc-8D P).

(manufactured by Peptide Institute) 10.6g dioxane 2211
The solution was added and allowed to react overnight. Add 70% deionized water to the reaction solution.
After washing with ethyl acetate 8011 twice to remove unreacted Roe-8DP, the mixture was cooled to 0°C and adjusted to pH 2 with 4N hydrochloric acid. The precipitated oil was dissolved in ethyl acetate 60
Extracted at z/(1 time) and 30y/(2 times), respectively.
Washed three times with 5011 5% hydrochloric acid (saturated with sodium chloride) at 0°C, and then three times with 50xl of saturated saline. After treatment with decolorizing charcoal, it was dehydrated over sodium sulfate, concentrated under reduced pressure, flushed with ether, and added hexane to the resulting clear syrup, frozen, and crystallized. Recrystallization from ethyl acetate-ether-hexane gave 7.2 g of N-Boc-aminobutyric acid.

Rf value CMA: 0.65 CMP: 0.46Pa
:0.84 Py :0.83 N obtained above
4.06 g of -Boc-aminobutyric acid and 2.66 g of the aminomethylstyrene monomer obtained in Reference Example 2 were added to 401 g of methylene chloride! After cooling to 0 to 5°C, 4.12 g of dicyclohexylcarbodiimide (DCC, manufactured by Kokusan Kagaku Co., Ltd.) was added, and a cobbling reaction was carried out at the same temperature overnight. The reaction solution was concentrated, 100 yi of ethyl acetate was added, the insoluble matter was removed in an oven, and the mother liquor was diluted with 10% aqueous citric acid solution.
00yJ (3 times), 100 zl of saturated saline (2 times),
The mixture was washed successively with 100if of a 5% aqueous sodium hydrogen carbonate solution (three times) and 100if of a saturated saline solution (twice), dehydrated with sodium sulfate, and concentrated under reduced pressure to obtain white crystals. This was recrystallized from ethyl acetate to obtain 4.0 g of NBOC-aminobutyrylaminomethylstyrene monomer.

Elemental analysis value (% as C18H26N203) Actual value: C67,64H8,25N8.96 Calculated value: C67,
73H8, 46N8.78Rf value CMA: 0.62
CMP: 0.56 (2) 5.25 g of 6-aminocaproic acid (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) with deionized water 2211 and TEA
8.4z/, and add Boc-8DP to this.
10. 6 g of dioxane 2211 solution was added and reacted overnight.

Deionized water 6011 was added to the reaction solution, and ethyl acetate 8011 was added.
After washing twice with 1 to remove unreacted Boc-8D P,
The mixture was cooled to 0°C and adjusted to pH 2 with 4N hydrochloric acid.

The precipitated oil was dissolved in ethyl acetate 60z/(once) and 30%
yA' (2 times) and 5% hydrochloric acid at 0°C.
11 three times, then twice with 40 zl of saturated saline,
After decolorizing charcoal treatment, dehydrate with sodium sulfate, concentrate under reduced pressure,
The resulting clear syrup was crystallized by treatment with ether-petroleum ether. Recrystallization from ether-petroleum ether gave 7.5 g of N-Boc-aminocaproic acid.

Rf value CMA: 0.47 CMP: 0.53Pa
:0.79 Py :0.77 N obtained above
4.64 g of -Boc-aminocaproic acid and 2.66 g of the aminomethylstyrene monomer obtained in Reference Example 2 were dissolved in 40xl of methylene chloride, and after cooling to 0 to 5°C, 4.12 g of DCC was added, and the mixture was kept at the same temperature overnight. A cutting reaction was performed. The reaction solution was concentrated, 100 z/ml of ethyl acetate was added to remove insoluble matter, and the mother liquor was diluted with 1 ml of 10% citric acid aqueous solution.
00zl (3 times), saturated saline 100zA' (2 times),
5% sodium bicarbonate aqueous solution 1001! (3 times) and 100 zA' saturated saline solution (2 times), dried over sodium sulfate, and concentrated under reduced pressure to obtain white crystals. This was recrystallized from ethyl acetate-ether-hexane and N
5.6 g of -Rot-aminocaproylaminomethylstyrene monomer was obtained.

Elemental analysis value (% as 02GH3ON203) Actual value: C67, 86H9, 19N8.83 Calculated value: C69,
18H8,93N8.07Rf value CMA: 0.62
CMP: 0.59 (3) 6.38 g of 8-aminocaprylic acid (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was mixed with 221 g of deionized water! and T.E.A.
8.4zI and Boc-8DP to this.
10. 6g of dioxane 22yl solution was added and reacted overnight. Add 601 g of deionized water to the reaction solution, and add 80 g of ethyl acetate! unreacted Boe-8D after washing twice with
After removing P, the mixture was cooled to 0°C and adjusted to pH 2 with 4N hydrochloric acid.

The precipitated oil was treated with ethyl acetate 60zf (once) and 30zf.
zJ (2 times) and 5% hydrochloric acid 501 at 0°C.
! 3 times and then twice with 40xl of saturated brine, treated with decolorizing charcoal, dried over sodium sulfate, concentrated under reduced pressure, crystallized by flashing with ether, and recrystallized from ether-hexane to obtain N-Boc. -Aminocaprylic acid 7.8
I got g.

Rf value CMA: 0. so CMP: 0.57Pa
:0.89 Py :0.87 Above N-Hoe
- 5.20 g of aminocaprylic acid and 2.66 g of the aminomethylstyrene monomer obtained in Reference Example 2 were mixed into 401 g of methylene chloride! After cooling to 0-5℃, DCC4
, 12g was added, and the cobblestone reaction was carried out overnight at the same temperature.
The reaction solution was concentrated, 100z# of ethyl acetate was added to remove insoluble matter, and the mother liquor was dissolved in 100yj of 10% citric acid aqueous solution (3
times), saturated saline 100'11 (2 times), 5% sodium bicarbonate aqueous solution 100yf (3 times), saturated saline 10
The organic layer was washed successively with Oyj (twice), dehydrated with sodium sulfate, concentrated under reduced pressure, and flushed with ether to obtain white crystals. Recrystallized from ethyl acetate-ether-hexane to give N
4.2 g of -Boc-aminocaprylylaminomethylstyrene monomer was obtained.

Elemental analysis value (% as C2□H34N203) Actual value: C70,54H9,16N7.71 Calculated value: C70,
42H9,33N7.47Rf value CMA: 0.71
CMP: 0.72 (4) 11-aminoundecanoic acid (Aldrich Chemica
6.38 g (manufactured by Co., Ltd.) was suspended in a mixture of deionized water 2211 and TEA 8.4zA', and Boc
A solution of 0.6 g of -8DPI in dioxane 2211 was added and reacted overnight. Add deionized water 6011 to the reaction solution,
Unreacted Boc-8 was washed twice with ethyl acetate 8011.
After removing DP, the mixture was cooled to 0°C and adjusted to pH 2 with 4N hydrochloric acid. The precipitated oil was extracted with 60yJ of ethyl acetate (once) and 30zA' (twice), and then extracted with 501! of 5% hydrochloric acid at 0°C. 3 times, then saturated saline solution 401! After washing with water twice and treating with decolorizing charcoal, it was dehydrated with sodium sulfate, concentrated under reduced pressure, and the resulting crystallization was recrystallized from ether-hexane to obtain 8.0 g of N-Boc-aminoundecanoic acid.

Rf value CMA: O, as CMP: O, asPa
:0.78 Py :0.84 Above N-Boc
- 6.04 g of aminoundecanoic acid and 2.66 g of the aminomethylstyrene monomer obtained in Reference Example 2 were dissolved in methylene chloride 4011, and after cooling to 0 to 5°C, DCC
Add 4.12g of citric acid and let it cumulate overnight at the same temperature, concentrate the reaction solution, add ethyl acetate Loot/ to remove insoluble matter, and add the mother liquor to 10% citric acid aqueous solution 100y/(
3 times), saturated saline 100z7 (2 times), 5% sodium bicarbonate aqueous solution 100z/(3 times), saturated saline 10
0z! (twice), dehydrated with sodium sulfate, concentrated under reduced pressure, and recrystallized the obtained white crystals from ethyl acetate-ether-hexane. .8g was obtained.

Elemental analysis value (% as C25H4oN203) Actual value: C71,70) 19.62 N7.08 Calculated value: C
71,96H9,83N6.72Rf value CMA:0.
72 CMP: 0.77 Reference Example 4 Production of polyaminomethylstyrene (AM-100) Aminomethylstyrene monomer (m-
: p-=7 : 3) 8. 8g and polymerization initiator (2
, 2'-azobisisobutyronitrile.

AIBN (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 55 mg and benzene 201
! The solution was dissolved in water, degassed under reduced pressure while cooling to -20°C, and replaced with nitrogen. After repeating this process several times, the tube was finally sealed under reduced pressure and polymerized at 60°C. After 3 days, the upper layer of the phase-separated liquid was decanted, and the lower layer was diluted with 10 methylene chloride.
The precipitated polymer was taken out in a furnace and dried to obtain the desired polymer as a white powder.

Elemental analysis value (+C9H1□N+., %): CHN Actual value near 7,07 9. 14 8. 59 Calculated value: 81.21 8.26 10.53 Average molecular weight: 2.
0X104 Reference Example 5 Production of copolyaminomethylstyrene-styrene (1) Styrene:aminomethylstyrene=9:1 copolymer (A
Production of M-10) 65.6 g of the styrene monomer purified in Reference Example 1 and 9.4 g of the aminomethylstyrene monomer prepared in Reference Example 2
Dissolve in benzene 200zAF and add AIBN to this.
After adding 0.57 g and degassing under reduced pressure at -20°C and repeating nitrogen substitution several times, the tube was sealed under reduced pressure and polymerized at 60°C. Two days later, the reaction product was poured into methanol, washed several times with methanol, and dried to obtain the desired copolymer as a white powder.

Elemental analysis value ((-CtiHs, 3N+-8,%)2C)lN Actual value: 89. 83 7.75 1. 44 calculated value:
90.93 7.76 1.31 Intrinsic viscosity number: [η)
=0.20~0.23 (25°C, in benzene) Viscosity average molecular weight: 2.9~3.5X104 (K=L, L
3xlO' α=0.73) (2) Styrene:aminomethylstyrene=7:3 copolymer (AM-30),
5:5 copolymer (AM-50), 3 near copolymer (AM-70) and 1:9 copolymer (AM-9)
Production of acetate salt of 0) The styrene monomer purified in Reference Example 1 and the aminomethylstyrene monomer prepared in Reference Example 2 were each taken at a predetermined charging ratio, with a total of 50 m and +++ ol of the monomers, dissolved in dioxane 1511, and charged into a polymerization tube. is. A to this
After adding 04 g of IBN O and freezing in a refrigerator, deaeration and nitrogen substitution were repeated, polymerization was carried out at 60° C. for 2 days under reduced pressure. The reaction product was lyophilized to remove dioxane, dissolved in acetic acid, and purified by pouring into ether. In addition, AM-70 and AM-90 had some insoluble parts,
The soluble portion was used for the following tests.

Elemental analysis value (%); AM-30 acetate (C11,9H1o, INO, 300
, 6) 'CHN Actual value near 8. oo 7.65 3. 10 Calculated value: 81. 73 7. 72 3. 21AM-50
Acetate (C9,5H11,5NO,501,0)'HN Actual value near 2. 35 7. 93 4. 25 Calculated Value Near 6.79 7,74 4.71 AM-70 Acetate (
010, IH12, 9NO, 701, 4) 'CHN Actual value: 68. os s, 07 5. 41 calculated value near 2. 90 7. 75 5. 89AM-9
0 acetate (CIo, 7H14,3NO,901,8)
'HN Actual value=62. 05 7. 79 5. 99 Calculated value: 69.77 7.76 6.85 Example 1 Production of polyaminoacylaminomethylstyrene hydrochloride 5 rAmol of each of the four types of Roe-aminoacylaminomethylstyrene monomers produced in Reference Example 3 were dissolved in dioxane 3011. and AIBN3 as an initiator.
．． After adding 8 mg of the mixture, freezing it in a refrigerator, repeating deaeration and nitrogen substitution, and polymerizing it under reduced pressure at 60° C. for 7 days, a polymer suspended in dioxane was obtained. After adding methylene chloride to this and dissolving the polymer, 4N
-HC#/dioxane was added and the mixture was stirred at room temperature for 40 minutes for deprotection, concentrated, flushed with methanol, added with ether, filtered, and dried.

The above polyaminobutyrylaminomethylstyrene (
AM-100-m), polyaminocaproylaminomethylstyrene (AM-100-V), polyaminocaproylaminomethylstyrene (AM-100-■) and polyaminoundecanoylaminomethylstyrene (AM-
The respective hydrochloride salts of 100-X) were obtained as superhygroscopic yellow gums.

Average molecular weight: AM-100-III hydrochloride: 2.3X104AM-1
00-V Hydrochloride + 2.2X104AM-100-■ Hydrochloride: 2.8X104AM-100-X Hydrochloride: l, 3X
104 The IR analysis diagrams of each polymer are as follows.

AM-100-III hydrochloride: Figure 1 AM-100-■ Hydrochloride: Figure 2 AM-100-X hydrochloride: Figure 3 Example 2 Manufacture of copolyaminoacylaminomethylstyrene-styrene DCC copolymer AM-10 obtained in 1)
Coupling reactions were carried out with four types of spacer reagents using The procedure is as follows.

That is, AM-102.0g (amino content 1.03nm
ol) was dissolved in methylene chloride 200z7, and each of the three aminoacylic acids obtained in the first half of each of (1) to (4) of Reference Example 3 with the N-terminus protected by a Boc group was dissolved in methylene chloride 200z7.
After adding equivalent amount (6,20 mmol), D at 0-5 °C
28 g of CCl was added and a coupling reaction was carried out for 2 days.

After the above reaction was completed, DCUtea produced as a by-product was removed, and the reaction product was poured into a large amount of methanol, and saturated brine was added to precipitate a white precipitate. The precipitate was decanted, washed with methanol, deionized water, evaporated and dried. Which N
Even when -Boc-aminoacylic acid was used, the yield of the target product was in the range of about 95-98%.

Next, 1.0 g of the copolymer produced above was mixed with 151 g of dioxane.
1! 4N-HCA'/dioxane was added thereto, and the mixture was allowed to stand at room temperature for 1 hour, and then concentrated under reduced pressure and flushed with methanol. Appropriate amount of methylene chloride (approximately 2
A solution obtained by adding and dissolving triethylamine 37A' in 00zA' was added dropwise to the above methanol solution, and saturated saline was added to the vaginal fluid to form a precipitate, which was decanted and mixed with methanol and then with methanol. Washing with deionized water, evaporation, and drying yielded the desired aminoacyl group-modified copolymer in the form of a white powder.

Aminobutyryl modified copolymer rAM-10-■'',
Aminocaproyl modified copolymer rAM-10-VJ
, aminobutyryl modified copolymer rAM-10-■
” and aminoundecanoyl modified copolymer as rAM-
Let it be 10-XJ.

The IR analysis diagram of each polymer is as follows.

AM-10-m: Figure 4 AM-10
-V: Figure 5 AM-10-■:
Figure 6 AM-10-X:
FIG. 7 Example 3 Preparation of carrier for adhesive cell culture A carrier (membrane-like material) for adhesive cell culture was prepared using the polymer of the present invention obtained in the above example. The procedure is as follows.

Polymer of the present invention obtained in Example 2: AM-10-I
I was made into a 5% dioxane solution, cast on a polystyrene culture dish (film thickness: approximately 30 μg/cm 2 ), dried, and sterilized by ultraviolet irradiation (irradiation time: 3 to 5 minutes) to prepare a culture carrier.

Test Example 1 Hepatocyte Culture Test 5% FC3 was added to the culture carrier prepared in Example 3 (hereinafter referred to as the "invention group"), a commercially available polystyrene culture dish (hereinafter referred to as the "control group"), and the above control group. The hepatocyte compatibility of the culture medium (hereinafter referred to as "comparative group") was tested by primary culture of canine hepatic parenchymal cells as follows.

That is, hepatocytes isolated and purified from adult dogs were seeded in the culture dishes of each group at a cell density of 5 x 104 to 105 cells/(ra 2).The seeding medium was 10'M dexamethasone and 10-8M insulin. Williams E medium (Flow Labo
(manufactured by Ratories, Idle, Inc.) was used.

After culturing the above-mentioned hepatocytes in a carbon dioxide culture device for 3 to 4 hours, the culture medium was replaced with a culture medium. This culture medium is 10'M
Using Shi-15 medium (manufactured by Flora Laboratories) containing dexamethasone, 10-"M insulin, 10'M glucagon, Long/zl hEGF (manufactured by Earth Pharmaceuticals), 30mg/A?proline, and 50,000 U/aprotinin. there was.

After 1 and 3 days, the medium was replaced, and after 4 days, hepatocyte D
The amount of NA and urea synthesis ability were measured by the following method.

<DNA amount measurement> Literature (Anal, Biochem, 92. 49
7-500 (1979)).

<Urea synthesis ability measurement> Literature (Clin, Chem, 11. 113 (19
The urea synthesis ability was measured according to 65)).

The results obtained for the present invention group and the comparison group are shown in Figures 8 and 9 in relative values (%) to the control group.

FIG. 8 is a bar graph showing the amount of DNA synthesis, and FIG. 9 is a bar graph showing urea synthesis ability.

From the above figures, the carrier of the present invention is Fe2 (fetal bovine serum)
It is clear that hepatic parenchymal cells can adhere and proliferate without the addition of serum, and thus can express their functions at a high level.

[Brief explanation of the drawing]

FIG. 1 to FIG. 7 are diagrams showing the results of infrared absorption spectrum analysis of the polymer of the present invention. FIGS. 8 and 9 are graphs showing the results of culturing hepatocytes using the adhesive cell culture carrier of the present invention prepared from the polymer of the present invention. (Above) Figure Hama map

Claims (2)

[Claims] (1) Repeating unit A: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ and Repeating unit B: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [R is a hydrogen atom or group ▲There are mathematical formulas, chemical formulas, tables, etc.▼ ( n is an integer of 1 to 10). ] in a ratio that satisfies A+B=1 and 0<B≦1, the weight average molecular weight is in the range of 10,000 to 80,000, and at least 1/2 of all repeating units B are other than hydrogen atoms. A styrenic polymer and its salt, which is a unit having. (2) An adhesive cell culture carrier made of the styrenic polymer and/or its salt according to claim 1.