JPH06277038A - Cell culture medium - Google Patents

Abstract

(57) [Summary] [Object] To provide a cell culture substrate comprising a polyelectrolyte complex (PEC). [Structure] At least the surface is formed by PEC of chitosan as a cationic polyelectrolyte and a cellulose derivative as an anionic polyelectrolyte. [Effect] It is possible to easily provide a culture substrate having appropriate physical properties by appropriately selecting a combination and a blending ratio of chitosan and a cellulose derivative according to the property of cells to be cultured. Compared with the conventional method using biological components, there are no problems such as lack of uniformity among products, deterioration, and complexity of gel production, and it is inexpensive. Furthermore, functional culture can be performed without dedifferentiation of cultured cells and loss of function.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture substrate having at least a surface formed of a polyelectrolyte complex composed of a cationic polyelectrolyte chitosan and an anionic polyelectrolyte cellulose derivative. .

[0002]

2. Description of the Related Art In recent years, with the rapid development of biotechnology fields such as artificial organs and culturing animal cells and producing physiologically active substances such as vaccines and interferons from the cells, in vivo Studies on cell culture, such as studies for expressing the function of the cells in vitro in cells cultured in vitro, have been actively conducted. Although there are many types of conventional cell culture methods, a monolayer culture method in which cells are attached to the surface of a carrier made of glass or a synthetic polymer resin, for example, a petri dish, and a monolayer is formed with proliferation is mainly used. .

In order to grow adherent animal cells, the adhesion between the surface of the substrate and the cells is good, and the morphology and arrangement of the adhered cells are effective for spreading and proliferating the cells. Need to be present. Therefore, various improvements have been attempted by making the surface of the base material itself hydrophilic (JP-A-52-41291 and JP-A-57-22691). However, even if these base materials are used, it is insufficient to maintain the cell function originally possessed in the living body, and although the cells survive and proliferate, the cells are often dedifferentiated and lose their functions in most cases. Is. In order to prevent such loss of function, functional culture has been sought to try to express cell function by reconstructing tissue in vitro.
That is, an extracellular matrix is used as a base material for in vitro culture, and cells are intended to perform tissue construction. For example, culturing hepatocytes using a floating collagen gel as a substrate [Exp. Cell Res. , 9
4, 70 (1975)] or L-ascorbic acid 2-phosphate, which is a cofactor for collagen synthesis, is added to the culture system to activate collagen synthesis in cells and culture three-dimensionally [Biogenesis, 60 , 201 (1988)].

[0004]

However, since collagen itself is a biological component, it is expensive and has many problems such as lack of uniformity of products, deterioration, and complexity of gel production. Therefore, an object of the present invention is to provide a substrate which is made of a completely artificial synthetic material and is suitable for functional culture.

[0005]

According to the present invention, at least the surface is formed by a polyelectrolyte complex composed of chitosan as a cationic polyelectrolyte and a cellulose derivative as an anionic polyelectrolyte according to the present invention. Can be achieved by a cell culture substrate.

The invention will be described in detail below. The polymer electrolyte complex (polyelectrolyte) used in the present invention
yte complex: hereinafter also referred to as PEC),
It is a substance known per se. PEC can be instantaneously formed by mixing a solution of a cationic polymer which is a polyelectrolyte having a positive charge and a solution of an anion polymer which is a polyelectrolyte having a negative charge. The PEC thus obtained is soluble in a special ternary solvent (for example, water / acetone / low-molecular-weight salt having a specific composition), but is insoluble in general solvents. PEC can provide various polyelectrolyte complexes having various properties depending on the types of starting polymers (polyelectrolytes), their mixing ratio, preparation conditions, and the like.

In the present invention, chitosan represented by the following structural formula (1) is used as the cationic polymer. As is well known, chitosan is a product obtained by deacetylating chitin (β-poly-N-acetyl-D-glucosamine), and is β-poly-D-glucosamine. In formula (1), R ₁ is a hydrogen atom or an acetyl group, X ₁ ^- is a counterion, deacetylation degree from 50 to 100% (preferably 70 to 10
0%), q ₁ is 10 to 3000 (preferably 10 to 20)
00).

[0008]

[Chemical 1]

In the present invention, a cellulose derivative represented by the following structural formula (2) is used as the anionic polymer. Formula (2)
R ₂ is a hydrogen atom, a carboxymethyl group, a sulfuric acid group or a phosphoric acid group, and q ₂ is 10 to 15000 (preferably 20 to 5000).

[0010]

[Chemical 2]

When the above chitosan as the cationic polymer and the cellulose derivative as the anionic polymer are reacted by a usual method, --N ⁺ H ₂ R in the above formula (1) is obtained.
₁ N ⁺ atom, COOH group in the above formula (2), SO ₃
PEC can be easily prepared by reacting with at least a part of the H group and / or the PO ₃ H group. That is, the concentration ratio of each of the above aqueous solutions of chitosan and cellulose derivative (10 ⁻⁵ mol / liter to 10 ⁻² mol / liter) to the cation seat of the cation polymer and the anion seat of the anion polymer (cation seat / anion seat). Is 0.25-4.
The reaction may be carried out by mixing in an aqueous solution within a range of 0, preferably within a range of 0.4 to 2.5. When the concentration ratio between the cation seat and the anion seat is outside the range of 0.25 to 4.0,
It is not preferable because PEC becomes difficult to be formed. As a solvent for dissolving each polymer, purified water, various buffer solutions (eg, phosphate buffer solution, etc.), or a mixed solution thereof with a water-miscible organic solvent (eg, methanol, ethanol, acetone, etc.) can be used. . Since this reaction is relatively active, the pH, ionic strength, temperature, etc. of the solution can be in a relatively wide range, but generally pH 3 to
9, carried out at an ionic strength of 0 to 1.0 and 20 to 60 ° C. The polyelectrolyte complex thus obtained can be used as a cell culture substrate by directly forming it as a cell culture substrate or coating it with a conventional appropriate material.

In the present invention, the charge balance of PEC to be produced can be easily changed and adjusted by changing the combination of chitosan and the cellulose derivative and the compounding ratio. That is, PE having various charge balances
By using C, the surface charge of the cell culture substrate is adjusted,
It is possible to appropriately select the surface characteristics according to the usage conditions. The charge balance of PEC used in the present invention is −
It can be selected in the range of 6 to +6. Here, the charge balance expresses the charge state of PEC by the concentration ratio of each cation seat and anion seat of the cation polymer and the anion polymer which are the starting materials. For example,
When the concentration ratios of the cation seat of the cationic polymer and the anion seat of the anionic polymer used are the same, the charge balance of the produced PEC is ± 0. If the concentration ratio is higher than this (that is, if the concentration at the cation seat is higher)
The charge balance is positive, and smaller (i.e., higher at the anion seats) and negative. When the concentration ratio is 1.5, the charge balance is +2, and when the concentration ratio is 0.5, the charge balance is -3.3. The charge balance can be easily adjusted by changing the mixing amount of the cationic polymer solution and the anionic polymer solution having the same concentration.

By changing the pH of the solution, the salt concentration, and the content of the water-miscible organic solvent during the preparation of PEC, it is possible to freely adjust the physical properties (eg hardness and elasticity) of the produced PEC. The cell culture substrate of the present invention can be formed from PEC itself because it can be easily molded into particles, plates or films. Also PE
Since C can be easily and easily coated on various materials, it is also possible to form a cell culture substrate of the present invention by coating PEC on a suitable carrier or a conventional substrate. Further, it may be formed of a portion consisting of PEC itself and a portion of PEC coated on a suitable carrier or conventional substrate.

As a carrier capable of coating PEC or a conventional cell culture substrate, a suitable material (glass,
Synthetic polymer resins such as cellulose, polyethylene, polyvinyl chloride, polyacrylic acid, polystyrene, polyester, polyisoprene, polypropylene and polyamide,
Examples include natural polymers such as cotton and paper, regenerated resins such as cuprophane and rayon, metals, ceramics and the like), plates, culture vessels, culture bags, films, fibers, microcarriers, beads and the like. Since the conventional cell culture substrate has already been controlled in shape, size, and pore diameter of the porous material, simply coating PEC
The cell culture substrate according to the present invention in which various standards are controlled can be easily produced.

The PEC can be coated on a carrier or a conventional substrate by a method such as coating, spraying or dipping. The PEC solution may simply be contacted with the carrier. For example, a chitosan solution and a cellulose derivative solution are mixed, and the solution is mixed with a carrier or a conventional base material at 0.5.
After being kept in contact for about 48 hours, the carrier thus treated or a conventional substrate is washed with physiological saline or purified water, and then air-dried at room temperature or heated to about 50-100 ° C. to be dried. At this time, for example, a salt such as NaCl is added in an amount of 0.01 to 5
M, by generating PEC in an atmosphere range of 0 to 100 ° C. and contacting or immersing it with a carrier or a conventional substrate, the coating treatment time of PEC is shortened (for example, by increasing the temperature), and the temperature is mild. It is also possible to coat PEC under conditions.

The cells that can be cultivated using the cell culture substrate of the present invention comprising the chitosan-cellulose polyelectrolyte complex thus prepared are not particularly different from those cultivated on the conventional cell culture substrate. However, they are all so-called adherent animal cells such as epithelial cells and fibroblasts. Further, the culturing method using the cell culture substrate of the present invention is not particularly different from the conventional culturing method, and a conventionally known culturing method can be applied depending on the culturing scale and the purpose of culturing.

[0017]

A cell culture substrate using PEC similar to that of the present invention is disclosed in JP-A-63-79587.
However, the PEC described in this publication uses a quaternized polyethyleneimine as a cationic polymer, whereas the PEC of the cell culture substrate according to the present invention uses a polysaccharide having a bulky ring structure in the main chain as a cationic polymer. Therefore, the PEC of the present invention has a much higher degree of restraint on internal rotation and less freedom in steric arrangement of dissociative groups, as compared with the PEC described in the above publication, and thus has a regular ladder shape.
er) PEC having a structure is easily formed, and the constitutional components and the overall structure are considerably different, so that the physical properties as a culture substrate are also different. That is, the adhesion between the cell and the base material interacts via an electrostatic binding force in addition to the effect of the adhesion factor contained in serum. Therefore, it is considered that the hydrophilicity / hydrophobicity of the substrate surface, the surface energy, the microdomain structure, etc. are deeply involved.

In the case of a polysaccharide PEC, it is easily conceivable that it has properties other than the properties of the other PECs described in the above publications and the like. For example, depending on the type, density and position of the dissociating group, even if the PEC as a whole is theoretically neutral, a free dissociating group that does not participate in the ionic bond is generated, which may affect cell adhesion or proliferation. Or
It is considered that PEC itself expresses a function that could not be predicted in the past, such as the possibility that PEC itself has a factor function for cell growth. Furthermore, since PEC also functions as a hydrogel, it is considered that physical surface mobility, excluded volume effect, etc. are also involved. It is considered that these several factors are combined to exert a function as an extremely suitable cell culture substrate, which has never existed before.

[0019]

EXAMPLES The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention. The average molecular weight described in the following examples is the number average molecular weight measured by the vapor pressure drop method. The polymers used in the following examples and their abbreviations are shown below. (1) Cationic polymer (chitosan) CS: chitosan (deacetylation degree 100%; average molecular weight about 5000) (2) anionic polymer (cellulose derivative) CCEL: carboxymethyl cellulose (functional per pyrano-acid group Group introduction rate 0.9; average molecular weight about 18,000
0) PCEL: Phosphorylated cellulose (functional group introduction rate per pyranose acid group 0.5; average molecular weight about 160,000) SCEL: Sulfated cellulose (functional group introduction per pyranos acid group) Ratio 0.8; average molecular weight about 120,000)

Example 1: PEC coated dish
Preparation of CS, which is a cationic polymer, was prepared using a 1% acetic acid solution (pH 6.0) so as to have a concentration of 1.62 mg / ml (10 ⁻² M as an ion seat; hereinafter also referred to as 10 ⁻² UM), CS
It was a solution. 2.14 mg of CCEL, an anionic polymer
/ Ml of a concentration (10 ^-2 UM) and so as distilled water (pH 7.
0) to prepare a CCEL solution. Equal amounts of these solutions were mixed to form PEC and immediately the PE
1 ml of the C solution was poured into a cell culture dish (NUNCLON DELTA), allowed to stand at room temperature overnight, the supernatant was removed, and then dried at 65 ° C. overnight. After drying, pour 1 ml of distilled water to wash the dish and dry again at 65 ° C to remove PEC.
A coating dish was obtained. Perform the same operation using PCEL and SCEL instead of CCEL above, and
S-PCEL and CS-SCEL coating dishes were prepared respectively. The following test was conducted using a dish not coated with PEC as a control.

Example 2: Evaluation of cell adhesiveness As cells, periodontal ligament-derived periodontal ligament cells (hereinafter referred to as HPLF)
), And the culture solution is Dulbecco's Modified Eagle's
A culture medium containing 10% fetal bovine serum (hereinafter, referred to as FBS) in Medium (hereinafter, referred to as DMEM) was used. Using this culture medium, HPLF cell suspension (8 × 10 ⁴ cell
s / ml) was prepared. 1.5 ml of this suspension was poured into each dish prepared in Example 1 (12 × 10 ⁴ cells).
s / dish), cultured at 5% CO ₂ and 37 ° C. for 24 hours.
After that, the supernatant is collected, and further 20 mM phosphate buffer (pH
7.4) By rinsing the dish with 1 ml,
Non-adherent cells were completely recovered. The non-adherent cells were counted under a phase contrast microscope using a hemocytometer (Birker Turk type) to determine the adhesion rate. The results are shown in Table 1.

Example 3 Evaluation of Cell Proliferation Using the culture medium used in Example 2, an HPLF cell suspension (2.5 × 10 ⁴ cells / ml) was prepared. This suspension 2
ml was poured into each dish prepared in Example 1 (5 × 10 ⁴ cells / dish), 5% CO ₂ and 4 ° C. at 37 ° C.
Cultured for a day. Then, the floating cells were removed together with the supernatant, and the dish was rinsed with 1 ml of 20 mM phosphate buffer (pH 7.4). 1 ml of 20 mM phosphate buffer (pH 7.4) containing 0.02 W / V% EDTA and 0.25 W / V% trypsin
Was injected and incubated at 37 ° C. for 10 minutes, and then pipetting was performed to detach and collect the adhered cells. In addition, add 20 mM phosphate buffer (pH 7.4) to the dish.
The cells were completely recovered by rinsing with 1 ml. The collected cells were counted using a hemocytometer as in Example 2. The results are shown in Table 1.

[0023]

[Table 1] Dish Adhesive Proliferative cell morphology CS-CCEL ○ ○ R, A CS-PCEL ○ ○ S CS-SCEL ○ ○ S Control ○ △ S

In Table 1, ◯ indicates good, Δ indicates slightly poor, R remains round, A indicates aggregated form, and S indicates extension. Table 1
As is clear from the above, the cell culture substrate of the present invention has the same degree of adhesion as the control, but is superior to the control in terms of proliferation. Therefore, the PEC coated dish is superior to the control in terms of growth rate and long-term culture. In addition, judging from the cell morphology, especially CS-CC
It was confirmed that the cells were three-dimensionally grown by using the EL coated dish.

[0025]

EFFECTS OF THE INVENTION Since PEC consisting of chitosan and a cellulose derivative is used for the cell culture substrate of the present invention, a combination and a blending ratio of chitosan and a cellulose derivative are appropriately selected depending on the properties of cells to be cultured. Thus, a culture substrate having appropriate physical properties can be easily provided. Further, as compared with the case of using a biological component, there are no problems such as lack of uniformity among products, deterioration, and complexity of gel production, and the cost is low. Furthermore, functional culture can be performed without dedifferentiation of cultured cells and loss of function.

Claims (1)

[Claims] 1. A cell culture substrate, at least the surface of which is formed of a polyelectrolyte complex composed of chitosan as a cationic polyelectrolyte and a cellulose derivative as an anionic polyelectrolyte.