FR2667324A1 - SUPPORT FOR CULTURE OF CELLS IN A SERUM FREE MEDIUM AND COLUMN TYPE DEVICE WITH SUCH SUPPORT. - Google Patents

Abstract

A support for culturing adherent proliferative cells in a serum-free medium, said support contains a support material of a calcium phosphate compound. A column type device for the continuous cultivation of cells, this device contains the aforementioned support lining said device for continuously passing serum-free medium through the support to ensure continuous proliferation and differentiation of cells proliferative adherent so that a useful substance produced in the cells is collected from an eluate from said carrier.

Description

Support for cell culture in a serum-free medium and

  column type device packed with said support.

  Technological background of the invention The present invention relates to a support for the culture of adherent cells or proliferative cells in a serum-free medium and to a device of the packed column type.

  of said support for the continuous culture of these cells.

  In a conventional manner, it is well known in a cell culture method to use a medium in which approximately 10% of an animal serum is added to a synthetic medium to which amino acids, vitamins, essential nutrients , inorganic salts and the like are

  added ("Tissue Culture Technology" Asakura Shoten, 1988).

  However, since the animal environment has variable qualities caused by manufactured batches, it is necessary that a

  appropriate batch is selected when cells are grown.

  In addition, since the supply of an animal environment is unstable, it was disadvantageous that the method could not be used on an industrial scale In addition, the use of

  medium in which animal serum is added presents the incon-

  comes that the separation of useful substances produced in cells is difficult due to the presence of

  serum proteins in the medium at a high concentration.

Summary of the invention

  An object of the present invention is to provide a support for the culture of cells or proliferative cells.

  adherent in a serum-free medium.

  Another object of the present invention is to provide a device of the column type provided with said support for the continuous culture of the cells, in which the cells proliferate continuously and differentiate and the useful substances produced in the cells are effectively collected by passage to through

the support in the column.

  The objects mentioned above and others of the

  present invention will be highlighted in the description which

  follows. In accordance with the present invention, there is provided a support for culturing adherent proliferative cells in a serum-free medium comprising a support material

  containing a calcium phosphate compound.

  Furthermore, in accordance with the present invention, there is provided a column type device for continuous cell culture, comprising the above-mentioned support lining said column, for continuously passing a serum-free medium through the support in order to to continuously proliferate and differentiate adherent proliferative cells so that a useful substance produced in the cells is collected from a

eluate from the support.

Brief description of the drawings

  Figure 1 is a graph showing the relationship between the concentration of lactic acid in the medium used for cell culture and the culture time according to

Example 1;

  Figure 2 is a graph showing the simi-

  milk in conjunction with Comparative Example 1;

  Figure 3 is a graph showing the similar relationship

  Laire in conjunction with Comparative Example 2;

  Figure 4 is a graph showing the simi-

  milk in conjunction with Comparative Example 3; and FIG. 5 is a diagram showing the results of the electrophoresis of the concentrated liquid obtained in accordance with

Example 2.

  Preferred Embodiments of the Invention Support for Cell Culture or Cells

  adherent proliferatives in a serum-free medium

  mement to the present invention is composed of a support material containing a calcium phosphate compound The adherent cells or proliferative cells can be selected from an osteoparent strain of MC 3 T 3-E 1 cells, of various

  fibroblasts, an endothetial cell, a muscle cell-

  the tumor, of a nerve cell, of the tumor cells of these cells, of the cells mentioned above in which an ecdemic or external gene has been incorporated by transduction and

mixtures of these.

  The calcium phosphate compound used as a support for the culture of adherent proliferative cells in a serum-free medium of the present invention can be preferably chosen from the following: hydroxyapatite, tricalcium phosphate, tetracalcium phosphate, fluoroapatite, apatite carbonate and mixtures of more preferably, hydroxyapatite can be used because hydroxyapatite has good adhesion properties with cells and allows adherent proliferative cells to exhibit superior activity. The form of the calcium phosphate compound can be selected varied according to the types of reactors for cell culture For example, the form may be a porous body or a granular particle In addition, the specific surface area of the calcium phosphate compound measured by a gas absorption method may preferably be in the interval of

0.1 to 70 m 2 / g.

  To prepare the calcium phosphate compound used

  as support for cell culture or proliferative cells

  adherent feratives in a serum-free medium of the present invention, the well-known sintering method can be used For example, the calcium phosphate compound can be

  controlled by the sintering temperature.

  In addition, a column type device for continuous cell culture in accordance with the present invention is

  composed of the support mentioned above lining said device.

  The serum-free medium is continuously sent through the support so that the adherent proliferative cells

  mentioned above proliferate continuously and differentiate.

  Consequently, a useful substance produced in the cells is collected from an eluate obtained from the support. To fill the column with the support, for example, the calcium phosphate compound can be first loaded in the column and then a medium is added for adhesion of proliferative cells or

  well a serum-free medium is added.

  To prepare the serum-free medium, the prescription of an animal cell culture medium or the minimum essential medium of Eagle can be used. A solution of inorganic salts, amino acids, vitamins, essential nutrients or mixtures thereof can be prepared. After preparation, the solution can be sterilized. If necessary, after sterilization, cell growth factors, hormones, cell adhesion factors or their mixtures can be added to the solution.

  In the support for the proliferation of Icellules proliferated

  adherent ratives in a serum free medium in accordance with the present invention a serum free medium can be used and

  thus it is advantageous that a medium having a predefined composition

  completed can be fed, while the cultivation operation is easy without requiring the selection of an appropriate batch for

a cell.

  In addition, the column-type device for the continuous culture of cells, loaded with the support is extremely useful as a bioreactor because the cells proliferate continuously and differentiate and the useful substances produced in the cells adhering to the support are efficiently separated and

  purified in the absence of superfluous proteins.

  Examples of the Invention The present invention will be explained with reference to the illustrative examples. It is understood that these examples should not

  in no case limit the scope of the invention.

Example 1

  A tubular column having a size of 2.0 cm in diameter and 3.2 cm in length is filled with granular hydroxyapatite particles having a specific surface of m 2 / g and a particle size of 0.3 to 0 , 5 mm Then, 1 x 106 osteoparent MC 3 T 3-E 1 cells previously purified in

  strain are seeded into the hydroxyapatite particles.

  For 3 days after seeding, the osteoparent cells are cultured in o-MEM medium including 10% fetal bovine serum. Next, the cells are cultured in serum-free "-MEM medium and the medium is exchanged with a new medium. x-MEM serum free once every two days The concentration of lactic acid in the medium which has been cultured and exchanged is measured by the NAD reduction method using L-lactate dehydrogenase The measured values are shown

in figure 1.

  Comparative examples 1 to 3 The same processes are carried out according to example 1 respectively except that the charge of the column consists of granular particles of alumina in comparative example 1, granular particles of zirconia in the example comparison 2 and granular particles of titanium oxide in the example

  Comparative 3, each particle size being from 0.3 to 0.5 mm.

  Each lactic acid concentration in each of the media used is measured similarly to that of Example 1 respectively. The result of Comparative Example 1 is shown in Figure 2, that of Comparative Example 2 is shown in Figure 3 and that of Comparative Example 3 is shown in Figure 4 respectively.

Example 2

  A tubular column with a volume of 50 ml and 3.0 cm in diameter and 7.5 cm in length is charged with granular particles of hydroxyapatite with a particle size of 0.3 to 0.5 mm to constitute a column type device for the continuous culture of the cells After inoculation of 8.2 × 105 MC 3 T 3-EL cells on the hydroxyapatite particles, the mi Lieu serum-free is sent continuously at a rate of elution of ml / d to obtain 2.4 L of a mi Culture place Then, the medium obtained is concentrated to 50 ml and a concentrated liquid thus obtained is subjected to electrophoresis on polyacrylamide SDS gel As a result, proteins produced in cells are detected The result of electrophoresis is shown in the

figure 5.

Claims (2)

  1 A support for the culture of proliferative cells   adherent ratives in a serum-free medium characterized in that it comprises a support material containing a compound calcium phosphate.   2 A device of the column type for the continuous culture of cells, characterized in that it comprises the support according to claim 1, filling said device for continuously passing a serum-free medium through said support to cause continuous proliferation and differentiating adherent proliferative cells so that a useful substance produced in said cells is collected from an eluate from said support.