JPH0358270B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION Plasminogen activator (PA) is a activator in plasminogen that generates two-chain plasmin molecules.
It is a serine protease that exerts its action by hydrolyzing the Arg ₅₆₀ - Val ₅₆₁ peptide bond. Plasmin has general proteolytic activity and primarily attacks fibrin in the physiological environment of circulating blood. Plasminogen activator plays an important role in the fibrinolytic system,
By dissolving thrombi, clots or fibrinous deposits inside or outside the vessels, it strongly influences the occurrence and overcoming of thromboembolic vascular diseases. Plasminogen activators can be added to body fluids, e.g.
It has been isolated from blood and urine and from solid tissues of various histological sources. Mammalian cell cultures are also known to produce plasminogen activators. cell lines derived from different tissues of the same animal, resulting in the same histology derived from a single type of cell independently transformed with the same oncogenic agent. There can be large differences in the amount of plasminogen activator produced by different types of cells. There is an urgent need to obtain cells capable of producing increased amounts of plasminogen activator and to establish techniques to improve methods of using animal cells to produce plasminogen activator on an industrial scale. ing. Description of Related Art European Patent Application No. 113319A2 discloses human cell lines that can be grown in the absence of serum and other macromolecular growth factors and methods utilized to produce such cell lines. are doing. European Patent Application No. 112174A2 describes a serum-free medium capable of growing a wide range of suspension and monolayer cells, said medium containing fuetuin, transferrin and substituted phosphatidyl-cholines. Journal of Biological Chemistry (J.Biol.Chem., 256, 7035−
7041 (1981), it is known that human cell lines of neoplastic source, such as melanocytoma cells, are capable of producing substantial amounts of plasminogen activator. . Cells of malignant cell lines, such as cervical cancer, laryngeal cancer, epidermal cell carcinoma of the oral cavity, colon and rectal cancer, have been shown to produce plasminogen activators [Molecular Cellular Biochemistry
Biochemistry), vol. 15, No. 2149-153 (1977)]. Two metastable prostate cancer cell lines, PC-3
and DU-145, were reported to grow in long-term culture in defined media containing no serum, hormones or growth factors [Proc.
Natl.Acad.Sci., Vo.78, No.9, 5673−5676
(1981)]. A progressive loss of metastable potential tumorigenicity was observed after sequential in vitro passages of human epidermal cell carcinoma. However, for plasminogen activators, high levels of production correlated well with retention and metastasis of tumorigenesis [Cancer Research, 40, 2310-2315
(1980)]. Barnes and Sato
“Growth of a Human Mammary Tumor Cell Line in Serum-Free Medium”
Tummour Cell Line in a Serum-Free
MCF-7 in serum-free medium supplemented with insulin, transferrin, epidermal cell growth factor, prostaglandin F ₂ 〓 and cold insoluble globulin in “Medium Medium)” (Nature, October 1979).
Reports on cell growth. The synthetic nutrient media used in this study were Ham's F-12 and Dulbecco's modified Eagle.
culture medium supplemented with antibiotics, sodium bicarbonate, HEPES and sodium selenite. Both of these references demonstrate that the yield of plasminogen activator from plasminogen activator-secreting cells can be increased by growing such cells in medium essentially free of fetal calf serum. Not suggested or taught. SUMMARY OF THE INVENTION The present invention provides methods for increasing the yield of plasminogen activator (PA) obtained from plasminogen activator-producing cell lines, and cell lines capable of producing relatively large amounts of plasminogen activator. Regarding. This method involves expanding plasminogen activator-producing cells grown in a suitable growth medium containing fetal calf serum (bovine serum) and passing through a series of growth media in which the amount of fetal calf serum is sequentially decreased. Subculturing the cells includes adapting the cells to a medium essentially free of fetal calf serum. Biologically pure tissue cultures capable of producing relatively large amounts of plasminogen activator have been used to develop adenocarcinomas of the human prostate [American type].
Culture Collection (ATCC) (the
Cell Consignment Line (CRC) -1435 available from American Type Culture Collection K]. Biologically pure tissue cultures capable of producing relatively large amounts of plasminogen activator when adapted according to the methods of the invention are ATCC
CRC-1622 and ATCC CRC-1579. DETAILED DESCRIPTION OF THE INVENTION It is well known that plasminogen activators can be produced by tumor cells in culture. Examples of tumor cells from mammals that can be used in the present invention include melanocytoma, prostate, breast, colon, ovarian, pancreatic, cervical,
Includes rectal, endometrial and fibroblastic tumor cells. Preferred cell lines include mammalian tumor cells, melanocytoma, prostate, breast, colon, ovarian,
These are the pancreas and endometrium. Suitable cancer cell lines can be obtained from repositories such as the American Type Culture Collection (ATCC).
Type Culture Collection, 12301 Parklawn
Drive, Rockville, MD 20852) or can be obtained from numerous researchers at universities, hospitals, or research institutes. The origin of the cancer cell line is not very important; the method of the invention can increase the amount of plasminogen activator obtained from plasminogen activator-producing cells by as much as about 5-60 times. . As shown here, a preferred high yield producer of plasminogen activator is human prostate adenocarcinoma, ATCC CRL-1435. Other biologically pure tissue cultures that can produce relatively large amounts of plasminogen activator when adapted according to the methods of the invention are ATCC CRC-1622 and ATCC CRC-1579. As used herein, the term "appropriate growth medium" refers to Waymouth's
MB752/1, Dulbecco Minimal Essential Medium
(MEN) and Ham's F-12 medium in a ratio of about 1:1:1 to about 3:1:2, respectively (by weight)
means a medium as described in detail hereinafter. This method involves selecting a parental cancer cell line that produces plasminogen activator and growing this cell line first in a suitable growth medium containing about 5 to about 20% fetal calf serum ( confluency). After the cell line reaches full growth, the cells are removed, usually by trypsinization, and subcultured in a series of appropriate growth media containing decreasing amounts of fetal calf serum. The rate of reduction in fetal calf serum can be easily determined by sequentially reducing the calf serum and testing the viability of cell growth. For example, parental cells can be grown to full growth in a suitable growth medium containing approximately 10% fetal calf serum. The cells are then removed and subcultured for at least one passage in an appropriate growth medium containing approximately 5% fetal calf serum. After the cells reach full growth, they are removed again and subcultured for at least one passage in a suitable growth medium containing approximately 2.5% fetal calf serum. This procedure is repeated until the appropriate growth medium used is essentially free of fetal calf serum. In the present invention, it has been found that a sequential reduction by a factor of 2 is a preferred method. It has been discovered that various combinations of commercially available growth media are suitable for use in the present invention. However, as the concentration of fetal calf serum decreases, the appropriate growth medium used in the method of the invention must be correspondingly supplemented with the following growth factors: fetuin, bovine serum albumin, insulin. , transferrin,
5α-dihydrotestosterone and dexamethasone. The time during which these growth factors must be added during the sequential reduction of fetal calf serum can be readily determined by one skilled in the art and depends on the type of cell line being cultured and the culture being performed. This will vary depending on factors such as the specific conditions you are experiencing. It is sufficient that the growth factors must be added at a time when the viability of the cells would otherwise be threatened due to a decrease in the concentration of fetal calf serum; however, if a suitable medium is The addition of the growth factors described above is unnecessary and difficult when subsequently growing the cells in a suitable growth medium. lead A suitable growth medium for use as described here is Waymouth's
MB752/1 medium, Dulbecco
MEN and Ham's F-12 medium (GIBCO Laboratories, Grand Island, New
commercially available from Yowk). The ratio (by weight) can range from 1:1:1 to 3:1:2, respectively. Preferred media, hereinafter referred to as "SYC" media, are Waymouth's MB752/1, Dulbecco's MEN and Ham's
F-12 medium, respectively, in a 1:1:1 (by weight) mixture. Cell growth was observed to be optimal when the appropriate medium used was SYC medium. In the complete absence of Waymouth MB752/1, cell growth is greatly retarded. These media have the compositions shown in the table.

【table】

[Table] As mentioned above, as the concentration of fetal calf serum sequentially decreases with each passage, the appropriate growth medium is mixed with 50-150 mg/fetuin; 50-150 mg/bovine serum albumin; 10mg/insulin; 5-40mg/transferrin; 5-40mg/transferrin;
It will be necessary to supplement with 200 μg/5α-dihydrotestosterone; and 50-200 μg/dexamethasone. Insulin and trasferin are important growth factors, and in their absence the cells will die after one passage in which all other growth factors are added and the appropriate growth medium is supplemented. was observed. The following examples further illustrate the invention. Example: Adenocarcinoma of the human prostate after receipt from the depository;
ATCC CRL−1435, Earle's Minimal Essential Medium (MEM)
(commercially available from GIBCO) was stabilized by growing to full growth. This procedure was performed as follows. 25ml MEM and 10%
of fetal calf serum into a 175 ml culture flask,
It was maintained in a 5% CO ₂ atmosphere at 37° C. and over 5×10 ⁵ adenocarcinoma cells were added. Parent cells were removed from the flask by addition of 5 ml aliquots of 0.25% trypsin. The parental cells from the above stabilization step were then added to the previously prepared volume containing 10% fetal calf serum.
Added to SYC medium and maintained at 37°C. Cells reached full growth in approximately one week, after which they were removed with trypsin as described above and added to a volume of SYC medium containing 5% fetal calf serum. Cells were again maintained at 37°C until full growth was reached. This step each 2.5%
and repeated twice using SYC medium containing 1.25% fetal calf serum. At a fetal calf serum concentration of 1.25%, fuetuin (75 mg/ml); bovine serum albumin (75 mg/ml); insulin (8 mg/ml);
mg/); transferrin (25 mg/); 5α-
Supplement with dihydrotestosterone (100 μg/) and dexamethasone (100 μg/). Cells reached full growth in approximately 10 days. Then 1.25 except that fetal calf serum is absent.
Cells were sub-inoculated into SYC medium containing the same growth factors as in the sub-inoculation of % fetal calf serum. Cells reached full growth in approximately one week. Then, contain the cells above without containing calf serum
Cell lines were stabilized by 10 passages through SYC medium (containing the same growth factors as above). Determination of stabilization was achieved by observing identical growth patterns and amounts of plasminogen activator (PA) produced during each passage.
After 10 passages, biologically pure modified fully grown cells (designated PC-3f and accession no.
CRL-8538 to the ATCC) was evaluated for plasminogen activator production by the following procedure [Progress in Fibrinolysis and Thrombolysis].
Thorombolysis), Vol. 3, pages 315-322]. 100μ portion of culture fluid (obtained after full growth)
100μ human plasminogen (0.33mg/ml)
Incubation for 15 minutes at 37°C with 250μ Tris buffer (0.1
mol Tris; 0.2 mol NaCl; PH 7.4) and
250 μ of tripeptide substrate (Valeu-lys-p-
nitro-anilide; Kabi, Stockholm, Sweden)
was added. The mixture was incubated for a further 3 minutes, after which the reaction was stopped by the addition of 100μ of 50% acetic acid. absorption of the reaction mixture
Read at 405 nm and then use commercially available urokinase (Calbinochem., La Jolla,
CA) and a standard curve of urokinase generated by the same procedure. This assay technique allows quantification of plasminogen activator produced by cells by measuring the amount of plasmin produced by activation of plasminogen from plasminogen activator present in the culture fluid. Record the test results in the table. The amount of plasminogen activator produced by a cell is expressed in units of CTA per ml of culture fluid [The Committee on Thrombolytic Agents of
The National Heart Institute (the Committee on Thrombolytic Agents of
The National Heart Institute). Thromb.Diath.Haemor., 21, p. 259 (1969)]. As a control, the activity of plasminogen activator of the parental cell line (ATCC-1435) was also determined.

Table: As the above test results show, passage of the parental cell line through serum-free medium produced certain cell lines. This cell line produced up to 70.4 CTA units of plasminogen activator compared to the maximum 18.5 CTA units of plasminogen activator produced by the parental cell line, representing an almost 4-fold increase. Advantageously, the modified cell line was able to survive for an additional 24 hours. Examples Human pancreatic cancer cell lines as parental cell lines,
The procedure described in the example was repeated using ATCC-1420. Biologically pure modified cell lines obtained by the procedure described here
PaCa−2f and accession number ATCC CRL
Deposited at ATCC under -8725. The test results obtained are summarized in the table below:

Table: As the above test results show, passage of the parental cell line through serum-free medium produced certain cell lines. This cell line produced up to 25.2 CTA units of plasminogen activator, an almost 3-fold increase, as well as an increase in 24 hour viability. Examples Mouse melanocytoma cell line B-16 [Jackson Lab.,
Bar Harbor, Maine) was used to repeat the procedure described in the Examples. The biologically pure modified cell line obtained by the procedure described herein was designated B- _16f . The test results obtained are summarized in the table below:

Table: Biologically pure modified cell lines showed a 3-fold increase in plasminogen activator at 24 hours and a 2-fold increase in plasminogen activator at 48 hours. Additionally, the modified cell line was able to survive for up to 96 hours. EXAMPLES The procedure described in the Examples was repeated using ATCC-1622, an endometrial adenocarcinoma cell line (available from ATCC) as the parental cell line. The biologically pure modified cell line obtained by the method of the invention is designated KLE _f and has accession number ATCC
Deposited at ATCC under CRL-8726. The activity of plasminogen activator was measured 48 hours after the cells reached full growth, and the test results obtained are shown in the table below.

[Table] As shown in the table, the modified cell lines,
KLE _f showed a 60-fold increase in plasminogen activator production over the parental cell line at 48 hours. Example: With accession number ATCC-1579 as the parent cell line.
The procedure described in the Examples was repeated using a melanin-deficient melanocytoma cell line available from ATCC. The biologically pure modified cell line obtained by the method of the invention is designated ML _f and has been deposited at ATCC under accession number ATCC CRL-8724. The activity of plasminogen activator was measured 48 hours after the cells reached full growth, and the test results obtained are shown in the table below.

[Table] Atsuta.

Claims (1)

[Scope of Claims] 1. Plasminogen activator-producing cells are grown in a suitable growth medium containing fetal calf serum, and the content of said suitable growth medium is sequentially reduced in fetal calf serum. the step of subculturing such cells through one lineage, thereby enabling said cells to reach full proliferation in said suitable growth medium essentially free of said fetal calf serum, and said increasing the yield of plasminogen activator obtained from plasminogen activator-producing cells, characterized in that the cells are capable of producing plasminogen activator at a level higher than the level of the initial plasminogen activator-producing cells. Method. 2. The plasminogen activator producing cell is a mammalian tumor cell selected from the group consisting of melanocytoma, prostate, breast, colon, ovarian, pancreatic, cervical, rectal, endometrial and fibroblast tumor cells. The method according to claim 1. 3. The method of claim 2, wherein the mammalian tumor cell is a human cancer cell selected from the group consisting of melanocytoma, prostate, breast, colon, ovary, pancreas, and endometrium. 4. The method according to claim 3, wherein the tumor cell line is human prostate adenocarcinoma. 5. The method according to claim 4, wherein the human prostate adenocarcinoma is ATCC CRL-1435. 6 A suitable growth medium is Waymouse's MB752/
1 medium, Ham's F-12 medium and Dulbecco's
MEM medium 1:1:1 to 3:1:2 (weight ratio)
2. The method according to claim 1, wherein the SYC medium consists of a mixture of. 7. The method of claim 6, wherein the SYC medium also contains feutuin, bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone and dexamethasone. 8 Fetuin is present at a concentration of 50-150 mg/; bovine serum albumin is present at a concentration of 50-150 mg/; insulin is present at a concentration of 5-10 mg/; transferrin is present at a concentration of 5-40 mg/. 5α-dihydrotestosterone is 5~
8. The method of claim 7, wherein the dexamethasone is present at a concentration of 200 μg/; and dexamethasone is present at a concentration of 50 to 200 μg/. 9 Fetuin is present at a concentration of 75 mg/; bovine serum albumin is present at a concentration of 75 mg/; insulin is present at a concentration of 8 mg/; transferrin is present at a concentration of 25 mg/; 5α-dihydrotestosterone is present at a concentration of 100 μg/ and dexamethasone is present at a concentration of 100 μg/. 10. The method of claim 1, wherein the concentration of fetal calf serum is sequentially reduced by a factor of two through each passage of said subculture. 11 Human prostate adenocarcinoma cell line ATCC CRL
-1435, are grown in a suitable growth medium containing fetal calf serum, and such cells are grown through a series of said suitable growth media sequentially containing decreasing amounts of fetal calf serum. , whereby said cell line is able to reach full proliferation in said suitable growth medium essentially free of said fetal calf serum, and said cell line is free of initial plasminogen. ATCC, a human prostate adenocarcinoma cell line characterized by being able to produce plasminogen activator at levels higher than those of activator-producing cell lines;
A method according to claim 1 for increasing the yield of plasminogen activator obtained from CRL-1435. 12. The method of claim 11, wherein the suitable growth medium is SYC medium. 13 The SYC medium contains fuetuin, bovine serum albumin, insulin, transferrin,
13. The method of claim 12, which also contains 5α-dihydrotestosterone and dexamethasone. 14 Fetuin is present at a concentration of 50-150 mg/; bovine serum albumin is present at a concentration of 50-150 mg/; insulin is present at a concentration of 5-10 mg/; transferrin is present at a concentration of 5-40 mg/. 5α-dihydrotestosterone is 5~
14. The method of claim 13, wherein the dexamethasone is present at a concentration of 200 μg/; and dexamethasone is present at a concentration of 50 to 200 μg/. 15 Fetuin is present at a concentration of 75 mg/; bovine serum albumin is present at a concentration of 75 mg/; insulin is present at a concentration of 8 mg/; transferrin is present at a concentration of 25 mg/; 5α-dihydrotestosterone is present at a concentration of 100 μg/ and dexamethasone is present at a concentration of 100 μg/. 16. The method of claim 11, wherein the concentration of fetal calf serum is sequentially reduced by a factor of two through each passage of said subculture.