JP2757400B2 - Cell culture method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides a method for growing cells in a gel for immobilization.
Quantitatively increase the amount of voids in the gel required for its growth,
Further, the present invention relates to a culture method for allowing cells to reach a high cell density in a short time.

[Conventional technology]

Due to recent advances in biotechnology, production of high value-added substances such as pharmaceuticals has been performed in cultured cells. As the demand for production materials increases, it has become necessary to culture cells in large quantities. As a device for culturing a large amount of cells on an industrial scale, a jar fermenter or an air-lift fermenter similar to those used for culturing microorganisms is used.

Large-scale fermenter mass culture has various problems such as an increase in capital investment cost due to an increase in size, an increase in mechanical share, difficulty in separating the medium from the cells, and detachment and detachment of the cells.

However, the above problems are being solved in the direction of high-density cell culture and fixed culture. One method is to use a gel for cell immobilization such as alginic acid. That is, by immobilizing the cells in the gel, the mechanical shear and the difficulty in separating the cells from the medium, which were problems in the conventional large-scale culture, are solved, the exchange rate of the medium is increased, and the growth environment of the cells is increased. And high-density cultivation is possible.

[Problems to be solved by the invention]

By the way, in the immobilized cells, the cells can be easily separated from the culture solution, so that the exchange rate of the medium can be increased.
The cell density increases in proportion to the exchange rate. However, in order to culture cells in the gel for cell immobilization, there must be voids in the gel, and the amount of voids per gel has an influence on the reached cell density. It is considered that the formation of this void is caused by the non-uniformity of the cation concentration gradient originating from cells or bubbles. The amount of the void varies depending on the lot of the fixing agent, and also greatly depends on the conditions for gelation. As described above, there is a problem that the amount of the void cannot be controlled by the currently used method.

The present invention has been made as a result of diligent efforts to solve the above problems in the culture method by culturing cells in the voids in the cell fixing gel, and the purpose is to fix the cells. An object of the present invention is to provide a culturing method for quantitatively increasing the amount of voids in the gel required for the proliferation when the cells are propagated in the gel, and for allowing the cells to reach a high cell density in a short time.

[Means for solving the problem]

The present invention, which has achieved the above object, is to adhere cells to a soluble solid, fix the soluble solid to which the cells are attached to a cell fixing gel, and then dissolve the soluble solid. This is a cell culture method in which a void is formed in the gel and cells are cultured in the void.

The soluble solid used in the present invention has an affinity for cells and has substantially no or little adverse effect on cells, and is dissolved by means such as heat treatment and enzyme treatment. There is no particular limitation as long as it can be a non-solid substance, and examples thereof include beads made of a high molecular compound such as collagen beads, gelatin beads, agarose beads, and dextrose beads. The soluble solid is preferably 50-50
It is about 0 μm, more preferably about 150 to 250 μm.

The cells used in the present invention are not particularly limited as long as they are intended for growth, and may be any of animal-derived cells, plant-derived cells, and microorganism-derived cells, including genetically modified cells, transformed cells, and normal cells. And so on. The cells may be either adhesion-dependent cells or adhesion-independent cells. Preferred examples of the cells grown according to the present invention include, for example, microbial cells such as Escherichia coli and yeast, Chinese hamster ovary cells, adhesion-dependent animal cells such as mouse cells, hybridomas, blood cells and the like. Dependent animal cells, plant calli, insect cells and the like are exemplified.

Substances produced by the cells include, for example, erythropoietin, tissue plasminogen activator,
Examples include interferon, interleukin 1, interleukin 2, interleukin 3, monoclonal antibody, insulin, growth hormone and the like.

One feature of the present invention is that cells are adhered to a gel for cell immobilization after the cells are attached to the soluble solid. That is, when the cells are immobilized without adhering the cells to the soluble solid, the proliferation of the cells is extremely poor.

In the present invention, as a method for attaching cells to the soluble solid, for example, the cells and the soluble solid are mixed in a medium, and the cells are placed in a container at an optimum temperature for cell growth or intermittently stirred. Or a method of attaching cells to the soluble solid by continuous stirring (at this time, a substance having a high affinity for the cell on the surface of the soluble solid,
For example, the number of attached cells increases when an antibody or the like is applied).

The gel for cell immobilization used in the present invention is capable of immobilizing cells, has little hindrance to cells when performing a gelation reaction, and passes through peptides formed by cells or peptides produced by cells. There is no particular limitation as long as the protein flows out. As such a gel for cell immobilization, specifically, agarose,
Examples include natural polymers such as K-carrageenan, alginic acid, and alginates (eg, alkali metal salts such as sodium salts and potassium salts), and synthetic polymers such as polyvinyl alcohol, photocrosslinkable polyethylene glycol, and polypropylene glycol.

[Action]

In the present invention, first, a soluble solid to which cells are attached is prepared. The soluble solid can be easily prepared by the method described above.

Next, the soluble solid to which the cells are attached is immobilized on a cell immobilization gel. The term “immobilization” as used herein means to encapsulate a soluble solid substance having cells attached to a gel for cell immobilization.

In general, the immobilization is carried out by mixing a target cell or a soluble solid substance to which the cell is attached with an aqueous solution for cell immobilization.
This is carried out by adding a valent cation, increasing the temperature, or decreasing the temperature to cause gelation and confine cells. For example, when alginic acid is used as a cell-immobilizing gel, an alginic acid solution forms a gel and solidifies in the presence of divalent cations, and this property is used to form a calcium alginate gel. This allows immobilization of the soluble solids with the cells attached. That is, the soluble solids on which cells have grown (for example,
A cell suspension and collagen suspension) are mixed with an alginate aqueous solution, and the mixture is dropped into a calcium chloride solution. As a result, calcium alginate gel beads are formed, and a gel of soluble solid calcium alginate, Immobilization on a gel for fixing cell-adhered cells is performed.

At that time, the concentration of the alginate in the alginate aqueous solution is usually 0.5 to 5%, preferably about 0.75 to 3%. In addition, calcium chloride solution
Suitably it is 200 mM, preferably 10-100 mM.
The mixing ratio of the medium suspension of the soluble solid to the alginate aqueous solution is about 0.5 to 2 parts by weight with respect to 1 part by weight of the former.

Thereafter, the soluble solid substance immobilized on the cell immobilization gel is dissolved. The dissolving means varies depending on the type of the soluble solid substance and the like, and usually includes heat treatment, decomposition with an enzyme, and the like. The heat treatment is usually performed by heating to about 30 to 42 ° C. In particular, gelatin beads at 37 ° C
It can be dissolved by heating to a certain degree.
In addition, as the enzyme used in the decomposition by the enzyme, for example, collagenase is used for collagen beads, agarase is used for azerose beads, and dextranase is used for dextrose beads. In the present invention, since there is little damage to cells,
Enzymatic lysis is preferred. For example, when the soluble solid is collagen beads, this is immersed in a collagenase solution (preferably, a collagenase solution having a concentration of about 100 units / ml) to dissolve the collagen beads. Thus, the cells attached to the collagen beads proliferate in the voids after the collagen beads have been dissolved.

〔Example〕

Next, the present invention will be described in more detail with reference to Examples,
The examples described below do not limit the invention.

Example 1 Using a 1.5 spinner flask as a culture apparatus, microcarrier culture was performed. In addition, human erythropoietin-producing cells were cultured to produce human erythropoietin. As the medium, DME medium + 10% F
Using CS.

Collagen beads to which cells were attached by spinner flask culture and a sterile 2% sodium alginate solution were prepared. The collagen bead suspension and a 2% sodium alginate solution were mixed at a ratio of 1: 1 (weight ratio). This is 5
It was dropped into 0 mM calcium chloride physiological saline to prepare calcium alginate gel beads. About 20 in this state
After standing for a minute, the solution was replaced with a collagenase solution. As it is
After incubating at 37 ° C. for several hours, the cells were washed with a medium and culture was started. The medium was replaced once every 3 to 4 days, and the concentration of human erythropoietin was measured.

According to the cell culture method of the present invention, it can be seen that the space occupied by the collagen beads in the calcium alginate gel is now a space for cell growth (see FIG. 1). When this is observed over time, cells increase only in the voids as seen on days 6, 15 and 18 (see FIGS. 2, 3 and 4, respectively). To be observed.

Comparative Example 1 When only cells were immobilized on a calcium alginate gel without attaching cells to a soluble solid substance and treated in the same manner as in Example 1, cell proliferation was not observed despite the sixth day of culture ( (See FIG. 5).

Comparative Example 2 When treated in accordance with the procedure of Example 1 except that collagenase treatment was not performed, cell proliferation was observed only on the surface of the collagen beads, and there was no tendency for cells to extend into the gel and increase ( (Fig. 6).

Experimental Example 1 As a result of culturing in Example 1, Comparative Example 1 and Comparative Example 2, the time-dependent change in the concentration of human erythropoietin secreted into the culture solution was examined. The result is shown in FIG. In FIG.
○ indicates Example 1, 比較 indicates Comparative Example 1, and △ indicates Comparative Example 2.

As is evident from the results shown in FIG. 7, the voids formed by collagenase treatment and dissolution of collagen beads work effectively, and the human erythropoietin productivity remains extremely high on day 21 of culture. Was observed.

〔The invention's effect〕

As described above, according to the present invention, it is possible to quantitatively prepare the cell-proliferable space in the cell-immobilizing gel, thereby increasing the attainable cell density. Thus, there is an effect that it is possible to improve the production amount of a protein produced by cells and useful as a pharmaceutical such as erythropoietin.

[Brief description of the drawings]

1 to 6 are photographs showing the morphology of an organism showing cells during culture. FIG. 1 is a photograph on day 6 of culture in Example 1 (×
80). FIG. 2 is a photograph on day 6 of culture in Example 1 (×
30). FIG. 3 is a photograph on the 15th day of culture in Example 1 (×
30). FIG. 4 is a photograph on day 18 of culture in Example 1 (×
30). FIG. 5 is a photograph on day 6 of culture in Comparative Example 1 (×
80). FIG. 6 is a photograph on day 18 of culture in Comparative Example 2 (×
30). FIG. 7 is a graph showing the change over time in human erythropoietin production in Example 1, Comparative Example 1 and Comparative Example 2.

Continuation of the front page (72) Inventor Shinji Miki 2-1-1 Katata, Otsu City, Shiga Prefecture Toyo Boseki Co., Ltd. (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 5/00 -5/28 C12N 11/00-11/18

Claims (1)

(57) [Claims] After the cells are attached to the soluble solid, the soluble solid to which the cells are attached is immobilized on a gel for cell immobilization, and then the soluble solid is dissolved to form voids in the gel. And culturing the cells in the voids.