GB2248628A - Cell culture in media comprising a calcium phosphate compound as carrier - Google Patents
Cell culture in media comprising a calcium phosphate compound as carrier Download PDFInfo
- Publication number
- GB2248628A GB2248628A GB9119709A GB9119709A GB2248628A GB 2248628 A GB2248628 A GB 2248628A GB 9119709 A GB9119709 A GB 9119709A GB 9119709 A GB9119709 A GB 9119709A GB 2248628 A GB2248628 A GB 2248628A
- Authority
- GB
- United Kingdom
- Prior art keywords
- cells
- serum
- carrier
- calcium phosphate
- free medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/10—Mineral substrates
- C12N2533/18—Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Adhering proliferative cells may be cultured in a serum-free medium on a carrier comprising a calcium phosphate compound such as hydroxyapatite. An apparatus for performing the method is described.
Description
1 TITLE
Cell Culturing in Serum-free Media. DESCRIPTION
The invention relates to a method of culturing adhering proliferative cells in a serum-free medium, to a carrier for use in the method and to an apparatus in which the method may be performed.
It is known in a cell culturing method to use a medium in which about 10% of an animal serum is added to a synthetic medium containing amino acids, vitamins, essential nutrients, inorganic salts and the like ("Tissue Culture Technology" Asakura Shoten, 1988). However, since the animal medium has variable qualities in different manufactured lots, it is necessary that a suitable lot be selected when cells are cultured. Furthermore, since supply of the animal medium is unstable, the method is not suitable for use on an industrial scale. Also, the use of a medium including animal serum has a disadvantage in that the separation of useful substances produced in the cells is difficult, since there are present serum proteins in the medium at a high concentration.
The invention provides a method of culturing adhering proliferative cells in a serum-free medium, the method comprising supporting the cells on a carrier material containing a calcium phosphate compound and contacting the supported cells with the serum-free medium.
The invention further provides a carrier for culturing adhering proliferative cells in a serum-free medium comprising a carrier material containing a calcium phosphate compound.
The invention also provides a columnar apparatus for continuously culturing cells, the apparatus being filled with a carrier according to the invention and adapted for the continuous passage of a serum-free medium through the carrier thereby continuously to proliferate and differentiate proliferative cells such that useful substances produced by the cells are contained within the eluate from the column.
Cells or adhering proliferative cells may be selected from strained osteoprogenitor cells MC3T3-E1, various fibroblasts, endothelial cells, muscle cells, nerve cells, tumour cells of the foregoing, the aforementioned cells into which an ecdemic or extraneous gene has been transduced, and mixtures thereof.
The calcium phosphate compound is preferably hydroxyapatite, tricalcium phosphate, tetracalcium phosphate, f luoroapatite, carbonate apatite or a mixture of two or more thereof. Most preferably, hydroxyapatite is used since hydroxyapatite has good adhering properties with the cells and allows the adhering proliferative cells to exhibit higher activity. The physical form of the calcium phosphate compound may be selected to suit the type of cell culture reactor in which it is to be used. For example, the calcium phosphate compound may be in the f orm of a porous body or granular particles. Preferably, the specific surface area of the calcium phosphate compound, as measured by the gas absorption method, is from 0.1 to 70 M21g.
The calcium phosphate compound may be prepared by known sintering processes. For example, the calcium phosphate compound may be controlled by the sintering temperature.
In the apparatus according to the invention, serum-free inedium is passed continuously through the carrier so that the aforementioned adhering proliferative cells are continously proliferated and differentiated. Accordingly, a useful substance produced in the cells is collected from an eluate obtained from the carrier. To f ill in the carrier into the column, f or example, the calcium phosphate compound may be firstly filled in into the column followed by adding a medium for adhering proliferative cells or a serum-free medium.
To prepare the serum-free medium, the prescription of an animal cell culture medium or Eagle's minimum essential medium may be used. A solution of inorganic salts, amino acids, vitamins, essential nutrients or mixtures thereof may be prepared. After the preparation, the solution may be sterilized. If needed, after sterilizing, cell growth factors, hormones, cell adhesion factors or mixtures thereof may be added to the solution.
With the carrier according to the invention, a serum-free medium may be employed and thus it has an advantage that a medium having a predetermined composition can be supplied, while the culturing operation is easy without the need to select a suitable lot for a cell.
Further, the apparatus according to the invention is extremely useful as a bioreactor since the cells are continously proliferated and differentiated and the useful substances produced in the cells adhering to the carrier are efficiently separated and purified in the absence of superfluous proteins.
The following Examples illustrate the invention, in conjuction with the accompanying drawings, of which:
Figures 1 to 4 are graphs showing the relationship between lactic acid concentration in the cell culture medium used in Example 1 and in Comparative Examples 1, 2 and 3 respectively and the culture time period; and Figure 5 is a chart showing the results of electrophoresis of the concentrated liquid obtained in accordance with Example 2.
Example 1
A tubular column having a diameter of 2.0 cm and a length of 3.2 cm was filled with granular particles of hydroxyapatite having a specific surface area of 10 M2/g and a particle size of from 0.3 to 0.5 mn. Then, 1 X 106 cells of of from 0.3 to 0.5 mm. Then, 1 X 106 cells of osteoprogenitor cells MC3T3- El having preliminarily been strained were seeded in the hydroxyapatite particles. For three days after seeding, the osteoprogenitor cells were cultured in an a-MEM including 10% fetal bovine serum. Following that, the cells were cultured in a serum-free a-MEM and the medium was exchanged for a new serum-free a-MEM once two days. A concentration of lactic acid in the medium which had been cultured and exchanged, was measured by a NAD-reduction method using L-lactate dehydrogenase. The measured values are shown in Figure 1.
Comparative Examples I to 3 Example 1 was repeated except that the column was filled with alumina granular particles, zirconia granular particles and titanium oxide granular particles, each having a particle size of from 0.3 to 0.5 mm, in Comparitive Examples 2, 3 and 4 respectively. The lactic acid concentration in each of the used media was measured as described in Example 1.
The results of Comparative Examples 1, 2 and 3 are shown in Figures 2, 3 and 4 respectively.
ExapRIe 2 A tubular column having a volume of 50 ml, a diameter of 3.0 cm and a length of 7.5 cm was filled with hydroxyapatite granular particles having a particle size of from 0.3 to 0.5 mm to make a column type device for continuously culturing cells. After 8.2 X 105 cells of MC3T3-El cells were seeded onto the hydroxyapatite particles, the serum-free medium was continuously caused to flow at an eluting rate of 70 M1/day to obtain 2.4 1 of a cultured medium. The medium obtained was concentrated to 50 ml and the concentrated liquid thus obtained was subjected to SDS polyacrylamide gel electrophoresis. As a result, the proteins produced by the cells were detected. The results of the electrophoresis is shown in Figure 5.
4, k
Claims (6)
1. A carrier for culturing adhering proliferative cells in a serum-free medium comprising a carrier material containing a calcium phosphate compound.
2. A columnar apparatus for continuously culturing cells, the apparatus being filled with a carrier according to claim 1 and adapted for the continuous passage of a serum-free medium through the carrier thereby continuously to proliferate and differentiate adhering proliferative cells such that useful substances produced by the cells are contained within the eluate from the column.
3. A method of culturing adhering proliferative cells in a serum-free medium, the method comprising supporting the cells on a carrier material containing a calcium phosphate compound and contacting the supported cells with the serum-free medium.
4. A method according to claim 3 in which the carrier material is contained within a column and the serum-free medium is passed through the column, such that useful substances produced by the cells are contained within the eluate from the column.
5. A method of culturing adhering proliferative cells in a serum-free medium, the method being substantially as described herein with reference to either of the Examples.
6. A calcium phosphate compound f or use as a carrier material in the culture of adhering proliferative cells in a serum-free medium.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2260522A JPH04141084A (en) | 1990-10-01 | 1990-10-01 | Carrier for serum-free cell culture |
Publications (3)
Publication Number | Publication Date |
---|---|
GB9119709D0 GB9119709D0 (en) | 1991-10-30 |
GB2248628A true GB2248628A (en) | 1992-04-15 |
GB2248628B GB2248628B (en) | 1994-09-07 |
Family
ID=17349140
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9119709A Expired - Fee Related GB2248628B (en) | 1990-10-01 | 1991-09-16 | Cell culturing in serum-free media |
Country Status (4)
Country | Link |
---|---|
JP (1) | JPH04141084A (en) |
DE (1) | DE4131375C2 (en) |
FR (1) | FR2667324B1 (en) |
GB (1) | GB2248628B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2254085A (en) * | 1991-03-02 | 1992-09-30 | Mitsubishi Materials Corp | Carriers for culturing adhering cells |
US10689615B2 (en) | 2014-01-29 | 2020-06-23 | Daikin Industries, Ltd. | Temperature-responsive base material, method for producing same, and method for evaluating same |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0175286A2 (en) * | 1984-09-14 | 1986-03-26 | MCW Research Foundation, Inc. | In vitro cell culture system and method |
JPS63198970A (en) * | 1987-02-16 | 1988-08-17 | Kirin Brewery Co Ltd | Hollow sphere of calcium phosphate |
JPH0367584A (en) * | 1989-08-04 | 1991-03-22 | Mitsubishi Materials Corp | Carrier for cell culture |
JPH1042311A (en) * | 1996-07-23 | 1998-02-13 | Matsushita Electric Ind Co Ltd | Image signal processor |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61282072A (en) * | 1985-06-07 | 1986-12-12 | Asahi Optical Co Ltd | Substrate for cell culture medium, device for cell culture and method therefore |
WO1988000967A1 (en) * | 1986-08-04 | 1988-02-11 | The University Of New South Wales | Serum free tissue culture medium containing polymeric cell-protective agent |
JPH02180707A (en) * | 1988-12-31 | 1990-07-13 | Tonen Corp | Production of phosphorus compound grain assemblage |
-
1990
- 1990-10-01 JP JP2260522A patent/JPH04141084A/en active Pending
-
1991
- 1991-09-16 GB GB9119709A patent/GB2248628B/en not_active Expired - Fee Related
- 1991-09-20 DE DE19914131375 patent/DE4131375C2/en not_active Expired - Fee Related
- 1991-10-01 FR FR9112069A patent/FR2667324B1/en not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0175286A2 (en) * | 1984-09-14 | 1986-03-26 | MCW Research Foundation, Inc. | In vitro cell culture system and method |
JPS63198970A (en) * | 1987-02-16 | 1988-08-17 | Kirin Brewery Co Ltd | Hollow sphere of calcium phosphate |
JPH0367584A (en) * | 1989-08-04 | 1991-03-22 | Mitsubishi Materials Corp | Carrier for cell culture |
JPH1042311A (en) * | 1996-07-23 | 1998-02-13 | Matsushita Electric Ind Co Ltd | Image signal processor |
Non-Patent Citations (2)
Title |
---|
In Vitro 1990, 26(3) Pt.2, 56A, Abstract 164. * |
J. Ferment.Bioeng. 1990, 70(3), 164-168. * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2254085A (en) * | 1991-03-02 | 1992-09-30 | Mitsubishi Materials Corp | Carriers for culturing adhering cells |
GB2254085B (en) * | 1991-03-02 | 1995-05-31 | Mitsubishi Materials Corp | Cell culturing in serum-free media |
US10689615B2 (en) | 2014-01-29 | 2020-06-23 | Daikin Industries, Ltd. | Temperature-responsive base material, method for producing same, and method for evaluating same |
Also Published As
Publication number | Publication date |
---|---|
DE4131375C2 (en) | 1994-01-20 |
GB9119709D0 (en) | 1991-10-30 |
FR2667324A1 (en) | 1992-04-03 |
DE4131375A1 (en) | 1992-04-02 |
FR2667324B1 (en) | 1995-06-16 |
GB2248628B (en) | 1994-09-07 |
JPH04141084A (en) | 1992-05-14 |
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Legal Events
Date | Code | Title | Description |
---|---|---|---|
PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19970916 |