JPS633786A - Collagen-containing capsule - Google Patents

Abstract

PURPOSE:Collagen-containing capsules multiplying cells in high density, obtained by covering a specific gelatinous composition with a film formed by bringing a fluid such as polyanion polysaccharide, etc., into contact with a solution of an edible chitin derivative, etc. CONSTITUTION:A medium for animal cell culture such as UEI mouse medium, etc., is optionally blended with an antibiotic to give a culture solution (A). Then NaOH and NaHCO3 are dissolved in water to give a solution (B). Then 5-20ml of 0.1-1wt% aqueous solution of I type collagen at pH 3 is blended with 0.5-3ml solution A, 0.5-3ml solution B and 0.5-3ml cell dispersion obtained by peeling off an animal cell (e.g. adhesion-dependent normal cell derived from human) with trypsin/PBS(-)(Dulbecco's) solution to give a gelatinous composition (C). Then, the component C is incorporated with 1-5wt% aqueous solution of polyanion polysaccharide (salt), cooled with ice and the prepared mixed solution is dripped through a needle having 100mum-1.5mm inner diameter to 0.5-5wt% aqueous solution of soluble chitin derivative (salt) of pH 6-7 to form the liquid drops into capsules.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to cell culture. More specifically, the present invention relates to a capsule that allows cells to grow at high density in a unit culture medium.

(Prior art) Capsules for cell culture are made using a two-step encapsulation method (first, a temporary capsule is made from calcium alginate gel, a permanent membrane is installed, and then the gel is mixed with a liquid again). Also, a method of encapsulating a cell suspension containing a cell growth medium in one step using a soluble chitin derivative and its salt (Nikkei Sangyo Shimbun).

January 22, 1985, Japanese Unexamined Patent Publication No. 6O-175539) is known.

(Problems to be Solved by the Invention) Among conventional capsules for cell culture, in the case of the two-step encapsulation method (Japanese Patent Publication No. 6O-38111), the operation during capsule manufacturing is complicated and time-consuming. Great damage to 9 cells.

In addition, when the collagen gel a degree is 0.1 weight or less,
Insufficient gelation 7t-af>K gel strength.

Some cells cannot serve as a fixed matrix material (three-dimensional growth matrix).

Therefore, it is not possible to grow cells at a high density per unit culture medium. Therefore, in order to obtain sufficient gel strength, collagen gel with a high concentration of 0.1% by weight or more, 1% -
If an attempt is made to encapsulate the collagen, the collagen will gel before the encapsulation process is completed, making it difficult to reliquefy the calcium alginate gel. If calcium alginate is not rehydrated, cell proliferation will be adversely affected.
give.

' Mayu, a method for encapsulating a cell suspension containing a culture medium in one step using a soluble chitin derivative or its salt (
Nikkei Sangyo Shimbun, January 22, 1986.

In the case of JP-A-6O-175539), since the core is a fluid, it is not suitable for culturing adhesion-dependent cells.

The present inventors focused on the fact that adhesion-dependent cells grow three-dimensionally in collagen glue that has sufficient gel strength. However, in this case, the collagen gel contracts as it proliferates.

Cells die. The present inventors solved this problem by culturing in the presence of a support (Special JlI Introduction 6).
O-162875).

As a result of further investigation, we embedded cells into collagen glue,
A polyanionic polysaccharide or a fluid based on its salt or a mixture thereof.

When the chitin was formed by contact with a solution of a soluble chitin conductor or its salt and was surrounded by a nine-layer membrane and cultured as a capsule of 100 μm to 51 μm, cell proliferation was observed inside the capsule. As the culture continues, the cells proliferate three-dimensionally, and the capsule coating acts as a support, causing the gel to shrink even though the cells are growing at a high density. This led to the discovery that this was not possible and led to the present invention.

(Means for solving problems) The present invention relates to animal cells and a medium for culturing the animal cells.

A gel-like composite material composed of collagen is formed by contacting a fluid based on a polyanionic polysaccharide salt alone or a mixture thereof with a solution of a soluble chitin fat conductor or a salt thereof. It concerns a capsule enclosed in a membrane.

In the present invention, b5 obstruction cells include insect cells, fish cells, amphibian cells, reptile cells, avian cells, or mammalian cells, and may be floating cells or adhesion-dependent cells. The attachment ratio may be normal cells or cancer cells.

Among mammalian cells, mouse, rat, Chinese hamster, human, horse, monkey, and bovine cells are often used. Among adhesion-dependent cells, fibroblast-like cells are used, and among them, the effect on normal diploid cells is remarkable. say. in particular.

MEM, Medium j 9
9. Ham's medium.

DPMI-1640 medium, Weymouth medium, etc., and mixtures thereof are preferred. In some cases.

To this, supplementary fluids such as animal serum and vitamins, Gross 7 Actor, etc. may be added.

As the collagen in the present invention, first base collagen is mainly used. Among In collagen, neutral salt soluble collagen, acid soluble collagen, enzyme soluble collagen,
Atelocollagen is used.

The af of lff1 collagen is generally in the range of 0.1 to 0.5% by weight, and if it deviates from this range, it will affect the careful growth. Within this range, it is possible to encapsulate the gel with a film at an optimal collagen gel concentration depending on the type of animal cell. For human normal diploid fibroblasts, 0.2 fold th1% is optimal.

What is the polyanionic polysaccharide or salt thereof of the present invention?

Polysaccharides or salts thereof that form polyanionic polymers in aqueous solutions include low methoxy pectin, carrageenan, carboxymethyl cellulose, and alginic acid.

Fundroitin sulfate, etc., or its salts are preferred.

Among these, carboxymethylcellulose sodium is common.

The soluble chitin derivative of the present invention can be obtained by chemically treating chitin to increase its reaction activity, and chitosan obtained by deacetylating chitin or a salt thereof is preferable. In some cases, chitosan is further chemically treated to improve the methyl ratio,
May be succinylated. Soluble chitin derivative 7
An aqueous solution of the salt of t exhibits viscoelasticity characteristic of polyionic polysaccharides, and upon capsule formation, the shape of the capsule is guided to a spherical or long spheroidal shape.

A 7 ton capsule of the present invention is manufactured as follows.

Distilled water 1tK Commercially available powdered medium 50-200? Then, if necessary, add antibiotics (Ambiciri, gentamicin, tylosin, etc. to a certain extent) to dissolve and sterilize by filtration to obtain Solution A. 0.05 N sodium hydroxide aqueous solution a1
00ml, then carbonated water; add 1-4f of l-thorium and add the following or 0.08N sodium hydroxide aqueous solution 100sdiC1N-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid 2-5f is dissolved and filtered to obtain liquid B.

Add 0.5 to 3 d of the above solutions A and B to an aqueous solution of pH 3.0, 5 to 20, containing 0, t to 1 t of collagen, under water cooling.
Solution 0.5 to 3- were added sequentially, and the monolayer cultured animal cells were further treated with trypsin-ethylenediaminetetraacetic acid disodium/PBS (→ (Dulbetzko) solution or trypsin/PBS←) (Dulbetzco). In the solution, add 0.5 to 3 - 0.5 - 3 - peeled skin cells @114 solution and stir.

The collagen aqueous solution is commercially available Cellmatrix.
(manufactured by Nitta Gelatin Co., Ltd.) or one extracted from rat tail, calf skin, etc.

After uniformly mixing a 1 to 5 weight aqueous solution of a polyanionic polysaccharide or a salt thereof into the mixed solution, inject into a solution of a soluble chitin derivative or a salt thereof in a well-ice-cooled state,
Form a capsule. The soluble chitin derivative or its salt is preferably contained in the solution in an amount of 0.5 to 5xii%. The solution of the salt of soluble chitin derivative '1fc has a pH of 6.0.
Adjust the osmotic pressure to ~7.0 and adjust the osmotic pressure to 280-590 m with salt.
Adjust osm K. When dropping, the inner diameter is 100 μm.
~1.5! Using a No. II needle, a droplet is formed and then dropped into a solution of the soluble chitin derivative or its salt. When dropped, the polyanionic polysaccharide or its salt on the fLS surface forms an ionic bond with the soluble chitin derivative or its salt.

Forms a gel coating and becomes a capsule. When a film is formed on the surface, the soluble chitin derivative or its salt.

No longer penetrating into the capsule interior, this ionic bonding reaction stops at the capsule surface. In this manner, optimal conditions for collagen gelation are maintained inside the capsule.

9. In some cases, a laminar air flow may be sent to this. In some cases, the minute ratio of liquid tablets is measured. Usually, a diameter of about 100 μm to 5 μm is suitable for agitation culture. The capsule formed is 1
It has a spherical or long spherical shape, and the collagen inside gels in about 30 minutes. however.

Since collagens other than type I usually do not cause gel flaw, a mixture with type I is used in some cases.

When observed under a microscope, a membrane is formed and animal cells are observed three-dimensionally fixed within the membrane by collagen gel. All of the above operations are performed aseptically. This method can be directly scaled up. Friend, this can be done on a small scale.

The next turnips produced by the above procedure are placed in a stirred culture vessel.
It is used by suspending it in the animal cell culture medium. The 7'C cannon embedded in the capsule is protected from the shear force caused by V and stirring, and is kept in a physically mild state.

After culturing for 1 day, viable cells can be observed under a microscope. If the culture is continued, the cells will proliferate smoothly and can be used for producing substances for ';fi. Three-dimensional proliferation of cells was observed inside the capsule on the 7th day of culture, demonstrating the effect of the collagen glue9.

The produced useful substances can be accumulated inside the capsule or can be released outside the capsule depending on the upper limit of molecular permeability of the capsule coat. The upper molecular permeability limit of the capsule coating can be controlled by the concentration of the polyanionic polysaccharide salt alone or in a mixture thereof, and by the a degree of the salt of the soluble chitin derivative.

(Effect of the invention) When animal cells are sealed in a capsule together with the animal cell culture medium and collagen gel and suspended and stirred in the animal cell culture medium, z, oxtocells/TRt
Implant with gel, maximum 5. OX 10 cells/a
Proliferate with 1tgel'j.

(Example) Example 1 Eagle's MEM powder (flow) 96 t/l, ampicillin 5001 L9/l
Dissolved in distilled water at a concentration of , and filter sterilized. This solution is A
Liquid binding ratio 0.05 N sodium hydroxide aqueous solution 100% to sodium bicarbonate 2.29%. N-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid 4.77ft-dissolved,
Filter sterilization friend. This solution was designated as Solution B.

Cel 1matrix I A (Nitta Gelachi 7,
3,01L9/td.

pH 5,0) 14,1 dVcs Under ice-cooling, 2 d of solution A and 2 d of solution B were added sequentially, and monolayer cultured HEL cells (Dainippon Hiroyaku Co., Ltd.) were filter-sterilized using disodium trypsin-ethylenediaminetetraacetate. / PBS(-) (Dulbetzko) solution and peel off'fi- cell suspension (2,OX
10' cells/d) Add 2 td f and stir L
lt.

Add 2 dft of this solution [5 wt% carboxymethylcellulose sodium salt aqueous solution and mix uniformly without forming bubbles, and use this solution as solution 6c.
Prepare a weight-related aqueous solution and add alkali to adjust the pH to 6.0.
Then, adjust the osmotic pressure f 290 mosm K with NaC4. To this solution 50-, the above solution Cl was slowly added dropwise through a needle with an inner diameter of 0.51 II while stirring, and the average particle size was 2.
m-rich capsules were formed. Eagle's capsules were added to 10% fetal bovine serum.
Culture in MEM medium 40°C with stirring in the presence of air containing 5% carbon dioxide A at 1' (37C).The container is a spinner flask (Sakude Kagaku) with a capacity of 100 d, and the container is 1 rotation speed SO.
Run at rpm.

4. After 10 days of culture. OX 10 cells/11I
Obtain the cell density of tgel. Instead of HEL cells, M
Lg cells (ddY mouse lung cells two major Japanese M4 drugs), CG
BQ cells (goose embryo sternum-derived cells, two major Japanese #i)'i
A similar effect was obtained using i-.

Example 2 When forming the droplets of Example 1, a laminar air flow was used to form capsules with an average particle size of 600 μm.

The obtained capsules were cultured with agitation in a growth medium, and on the 10th day of culture, 2. OX 10' cells/
The cell density of dgel was obtained.

Example 3 HEL cell membrane of Example 2) I/C7'' (Fl
ow) 7000 (Human fetal foreskin-derived cells, two major Japanese pharmaceuticals)
Capsules were prepared using sodium carboxymethylcellulose glue and sodium alginate in 9.

When the obtained capsules were cultured with stirring, 5.5% was obtained on the 14th day of culture. A cell density of OX 10 cells/m gel was reached.

Example 4 The HEL cells of Example 2 were replaced with Eagle's +7 embryonic chondrocytes, and capsules were made using Ham'r F'-12 in Eagle's MEM. Created friend.

The resulting capsules were mixed with ham (H) containing 10 grams of fetal bovine serum.
Am's) When agitation culture was performed in F-12 medium, 1. OX 10' cells
/wtget cell density was obtained.

Example 5 The HEL cells of Example 1 were replaced with mammary gland epithelial cells, and Eagle's MEM glue [Medium 1 containing 10% fetal bovine serum] was used.
A friend who made a 99K capsule. Medium 1 containing the obtained T capsule with fetal bovine serum took
99 for 7 days with stirring to reach a final density of 4. Ox 1
0' cells/a (get the gel.

Example 6 The HEL cells in Example 1 were replaced with cultured vascular smooth muscle cells, and capsules were prepared. The obtained tomo capsules were grown in growth medium for 7 days.
Culture for 1 day and reach a final density of 2.0×1 cell.
ls/sd gel f 1hi.

Example 7 Bovine skin was treated with pepsin and extracted, and a total of atelocollagen was extracted. 3. Solution 14 with pH 3.0 containing OIR9
-, add solution A 2- of Example 1, and adjust the pH to 7 with NaOH.
, OicF, and further cultured in monolayer of CV-1 cells (Japanese M chestnut derived from African green monkey kidney).

Trypsin-ethylenediaminetetraacetic acid disodium treated cell SI fluid (2,OX 10' cells/d
) Add 2-, and then add 5-di aqueous solution 2- of sodium carboxymethyl cellulose to prepare capsules. The resulting capsules were cultured in Eagle's MEM medium containing 10% fetal bovine serum for 10 days to reach a final density of 4. OX 10” cells/
I got sdget as a friend.

Example 8 Capsules were prepared using atelocollagen glue 5 obtained by treating and extracting the bovine skin of Example 7 with pepsin and acid-soluble collagen obtained by extracting rat v with acetic acid. The resulting capsules were cultured in growth medium for 12 days to reach a final concentration of 2. OX 10'cells 7m gel
f Tokuji.

Example 9 Medium 199 powder (Gipco)
100 p, gentamicin 5009 was dissolved in 1 tK of distilled water, filtered and sterilized. This solution was designated as D.

Capsules were prepared by using KDi from Example 2 and AYa (Human Amnion Derived Epithelial Cells) for HEL cells. The obtained capsules were cultured with stirring in a growth medium, and 3, OX 10 cells were grown in 7 days.
Obtain a cell density of s/-gel.

Example 10 4.77 t of N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid was dissolved in 100% of a 0.08 N aqueous sodium hydroxide solution, and sterilized by filtration. This melting overnight was designated as E.

Use Eak instead of liquid B in Example 1 to make capsules. The obtained capsules were cultured with stirring in a growth medium.
Q days 1. OX 10' cells/1ntgel
Obtain a cell density of .

Comparative Example Cellmatrix IA (Mr. Gelatin Co., Ltd.)
, 3. Om9/l, pH 3,0) Example 1 to 21-
Solution A 3-1 and Solution B 3- were added sequentially under ice-cooling, cultured in a monolayer on a colander, and precipitated HEL cells (Dainippon Pharmaceutical Co., Ltd.) were sterilized by filtration using trypsin-ethylenediaminetetraacetic acid (I).
Peel off fixed cells with J Lamb/PB S (-) (Dulbetzko) solution and remove with SR solution (4.9X 10' cells).
s/d) 3ttk, mix uniformly, and drop the resulting suspension into a 96-well multi-plate in 300 μt portions. After that, I left it at 37C for 10 minutes and did a gel session.

Remove the collagen gel beads from the multi-plate,
Eta k (Eagle's) MEM 100 - A companion for performing agitation culture in MEM 100-. Collagen beads shrink over time, and the cell density after 6 days was 6. OX 10 Cells/s
dgel, and almost no cell proliferation was observed.

Claims (6)

[Claims] (1) A gel composition consisting of animal cells, the animal cell culture medium, and collagen is combined with a fluid based on a polyanionic polysaccharide or a salt thereof or a mixture thereof, and a soluble chitin derivative or a salt thereof. A capsule surrounded by a film formed by contact with a solution. (2) The capsule according to claim 1, wherein the animal cell is an adhesion-dependent cell. (3) The capsule according to claim 1, wherein the animal cells are adhesion-dependent normal cells. (4) The capsule according to claim 1, wherein the animal cells are human-derived adhesion-dependent normal cells. (5) The capsule according to any one of claims 1 to 4, wherein the soluble chitin derivative is chitosan. (6) Collagen concentration is greater than 0.1% and 0.5%
A capsule according to any of claims 1 to 5 which is smaller.