JPS60259187A - Method and cell line for obtaining plasminogen activating factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Plasminogen activator (PA) is a molecule of Argssa in putesminogen that produces 2-frequency plasmin molecules.
Va15e1 is a serine protease that exerts its action by hydrolyzing peptide bonds. Plasmin has general proteolytic activity and primarily attacks fibrin in the physiological environment of circulating blood. Plasminogen activators play an important role in the fibrinolytic system and contribute to the development of thromboembolic vascular diseases and by dissolving thrombus, clots or fibrillar deposits inside or outside the vessels. It has a strong influence on overcoming it.

Plasminogen activators have been isolated from body fluids, such as blood and urine, and 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 can produce the same oncogenic substance.
There can be large differences in the amount of plasminogen activator produced by cells of the same histology derived from a single type of cell independently transformed with genet).

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.

European Patent Application No. 113,319 A2 describes 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. Disclosed. European patent application 112
, 174 A2 describes a serum-incompatible medium capable of growing a wide range of suspensions and monolayers of cells, said medium containing fetuin, transferrin and substituted phosphatidyl-choline.

Journal of Biological Chemistry (J, Biol, Chem,.

256.7035-7041 (1981), that human cell lines of neoplastic origin, such as melanocytoma cells, are capable of producing substantial amounts of plasminogen activator. Are known.

Cells of malignant cell lines, such as cervical cancer, laryngeal cancer, epidermal cell carcinoma of the oral cavity, and colon and rectal cancer, have been shown to produce plasminogen activators (Mo
regular Ce1lular Biochemi
str ), v.

1.15. No. 2, pp. 149-153 (1977
)reference].

Two metastable prostate cancer cell lines, PC-3 and D
U-145, was reported to grow in long-term culture in a defined medium containing no serum, hormones or growth factors [Proc, Natl. Acad.
sci,, Vo, 78, No, 9.5673-5676
(1981) A gradual loss of 1° metastable potential tumorigenicity was observed after sequential ex vivo passages of human epidermal cell carcinoma. However, when it comes to plasminogen activators, high levels of production correlated well with retention and metastasis of tumorigenesis [Cancer Research
), 40.2310-2315 (1980)].

] Barnes Barnes and Sato, ``Growth of a Human Mammalian Tumor Cell Lines in Serum-Incompatible Medium.''
ry Tummour Ce1l Line in a
Serum-Free Medium)” (Nat
reported the growth of MCF-7 cells in serum-incompatible medium supplemented with insulin, transferrin, epidermal cell growth factor, prostaglandin F, and cold insoluble globulin (2007). The synthetic nutrient medium used in this study was Ham (
Ham's F-12 and Dulbecco
A 1:1 mixture with modified Eagle's medium supplemented with antibiotics, sodium bicarbonate, HEPES and sodium selenite.

None of these references estimate the yield of plasminogen activator from plasminogen activator-secreting cells as
There is no suggestion or teaching that such cells can be increased by growing them in a medium that is ligneously free of fetal calf serum.

The present invention provides methods for increasing the yield of plasminogen activator (FA) obtained from plasminogen activator-producing cell lines, and cells capable of producing relatively large amounts of plasminogen activator. Regarding lineage. This method involves growing plasminogen activator-producing cells growing in a suitable growth medium containing fetal calf serum (bovine serum) and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum), and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum), and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum), and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum), and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum), and growing plasminogen activator producing cells in a suitable growth medium containing fetal calf serum (bovine serum); The method includes growing the cells in the appropriate medium that does not contain fetal calf serum by passing the cells through one line.

Biologically pure tissue cultures capable of producing relatively large amounts of plasminogen activator are derived from adenocarcinoma of the human prostate [the American Type Culture Collection (ATCC)].
Cell Consignment Line (CRC)-1435, available from Culture Co 11 ection K). A biologically pure tissue culture capable of producing relatively large amounts of plasminogen activator when adapted according to the methods of the invention is ATCC CRC-1622.
and ATCC CRC-1579.

It is well known that the plasminogen activator described in Noboribetsu 1411 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
, including colon, ovarian, pancreatic, cervical, rectal, endometrial and fibroblast tumor cells. Preferred cell lines are mammalian tumor cells such as melanocytoma, prostate, breast, colon, ovary, pancreas and endometrium. Suitable cancer cell lines can be purchased from a depository, e.g. the American Typical Culture Collection (ATCC).
n Type Culture Co11ection
, 12301 Parklawn Drive, Roc.
kville.

MD 20852) or can be obtained from numerous researchers at universities, hospitals or research institutes. The identity of the cancer cell line is not critical; the methods of the present invention increase the yield of plasminogen activator from plasminogen activator-producing cells by about 5 to about 60 times. can be done.

As shown here, a preferred high yielding producer of plasminogen activator is human prostate adenocarcinoma, ATC.
CCRL-1435. Biologically pure tissue cultures capable of producing relatively large amounts of plasminogen activator when adapted according to the methods of the invention are AT
CCCRC-1622 and ATCCCRC-1579
It is.

As used herein, the term "suitable growth medium" refers to
Waymouth (7)M
B 752/1, Dulbecco's Minimum Essential Medium (Dul
becco Minimal Es5ential M
(MEN) and Ham's F-12 medium from about 1:1:l to about 3:1:1, respectively.
It means a medium as described in detail hereinafter, containing a ratio (by weight) of 2:2.

This method is 1. A parental cancer cell line that produces plasminogen activator is selected and this cell line is first grown to confluency in an appropriate growth medium containing about 5 to about 20% fetal calf serum. ). After the cell line reaches full growth, the cells are removed, usually by triscination, and subcultured in an appropriate growth medium of the I series containing decreasing amounts of fetal calf serum. The rate of withdrawal of 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 an appropriate 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, successive reductions in factors of 2 have been found to be the 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 (fet
ufn), bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone, and dexameth hasone, the point during the sequential reduction of fetal calf serum at which these growth factors must be added is known to those skilled in the art. can be easily determined and will vary depending on factors such as the type of cell line being cultured and the specific conditions under which the culture is grown.

It is sufficient, however, that 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; 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 herein is Waymouth's MB752/l
Medium, DulbecCo MEN and Ham's F-12 medium (GIBCOLabo)
rations, Grand l5land, New
(commercially available from York). The ratio (weight) is 1 for each
:1=1 to 3:1:2.

Preferred media, hereinafter referred to as "SYC" media, are Waymouth's MB752/l, Dulbecco MEN and Ham's F-12 media, 1:l each: l
(by weight) Consists of a mixture.

It has been observed that cell growth is optimal when the appropriate medium used is SYc medium.

Waymouth MB752/
In the complete absence of 1, cell growth is greatly retarded. These media have the compositions shown in Table I.

As previously mentioned, the appropriate growth medium was adjusted to 50 to 150 mL as the concentration of fetal calf serum decreased with each successive inoculation.
mg/l fetuin; 50-150 mg/l bovine serum albumin; 5-10 mg/l insulin; 5-40 mg/l transferrin; 5-200 J
Lg/l of 5α-dihydrotestosterone; and 50
Supplementation with ~200 μg/l dexamethasone will be required. Insulin and transferrin are important growth factors; in their absence, all other growth factors were added to supplement the appropriate growth medium.
It was observed that after one passage the cells would die.

The following examples further illustrate the invention.

Implementation 1” After receiving from the trust, human prostate adenocarcinoma, ATCC
CRL-1435, Earl's Minimum Essential Medium (Earl
e's Minimal Es5e,nt ial M
Ejium) (MEM) (commercially available from GIBCO) was stabilized by growing to full growth. This procedure was performed as follows. 25 ml of MEM and 10% fetal calf serum were placed in a 175 ml culture flask, maintained at 37° C. in a 5% CO□ atmosphere, and adenocarcinoma cells on 5×10S were added. Parent cells were removed from the flask by addition of 5 ml portions of 0.25% trypsin.

The parental cells from the above stabilization procedure were then added to a pre-prepared volume of SYC medium containing 10% fetal calf serum 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 procedure was repeated in S2 containing 2.5% and 1.25% fetal calf serum, respectively.
Two replicates were performed using YC medium. Fetuin (75 mg/ml) at a fetal calf serum concentration of 1°25%;
Bovine serum albumin (75mg/ml); insulin (8mg/l); transferrin (25mg/l)
); 5α ~ dihydrotestosterone (100 μg/l
) and dexamethasone (100 ILg/l). Cells reached full growth in approximately 10 days.

Cells were then passaged into SYC medium containing the same growth factors as in the 1.25% fetal calf serum passage, except that fetal calf serum was absent. Cells reached full growth in approximately one week.

The cell line was then stabilized by passages of human cells through SYC medium without calf serum (containing the same growth factors as above) for 10 times. The decision to stabilize is
This was achieved by observing the intermittent growth pattern of each passage and the amount of plasminogen activator (FA) produced. After 10 passages, biologically pure modified whole-grown cells (designated PC-3f and deposited with the ATCC under accession number CRL-8539) were
Plasminogen activator production was evaluated by the following procedure [Advances in Chemical Fibrinolysis and Thrombolysis].
s and engineering] orombol 5is), Vo, 3
, pp. 315-322].

A 100 pl portion of the culture fluid (obtained after full-scale growth) was added to 1
00μm human plasminogen (0゜33mg/ml
) for 15 minutes at 37°C, followed by 250 IL of Tris buffer c.
o, i mol Tris; 0, 2 mol cy) NaCl;
pH 7,4) and 250 Kawa-1 (7) tripeptide substrate (Valeu-1ys-p-nitro-anili
de;Kabi.

Stockho'1m, Sweden) was added.

This mixture was incubated for a further 3 min, after which the reaction was stopped by the addition of 1 OoJLl of 50% acetic acid. The absorbance of the reaction mixture was read at 405 nm and then the commercially available urokinase (Calbino
chem, LaJolla, Calif.) was compared to 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.

The test results are listed in Table II. CTA is the amount of plasminogen activator produced by cells per ml of culture fluid.
Expressed in units [The Urn Committee Meeting on the Trombolic Agents of the Q National Bar 1
Institute (the Comm1ttee on
Thr.

Mbolytic Agents of the Nat
ional He&rt In<t, i++1te). Thromb, Death, Haem
or, p. 21.259 (1969)], and as a control the parental cell line (ATCC-1435
) (1) The activity of fy7° minogen activator was also determined.

Human J (Activity of FA, CTA units/ml) Cell line 48 hours 72 hours 96 hours ATCCCR L-143511,518,5 death PC-3f (A TCCCRL -8539) 28.5 45.0 70.4 Above test As the results show, passage of the parental cell line through serum-incompatible medium produced certain cell lines. This cell line has the plasminogen activity produced by the parent cell line.
#±18 FsmTΔmb ratio il, te, 70.4CT
A unit of plasminogen activator was produced, representing an almost 4-fold increase. Advantageously, the modified cell line was able to survive for an additional 24 hours.

Tsutsuta 11 Human pancreatic cancer cell line, ATCC-1, as the parent cell line
420, the procedure described in Example I was repeated. The biologically pure modified cell line obtained by the procedure described herein is designated PaCa-2f and has been deposited at the ATCC under accession number ATCCCRL-8725. The test results obtained are summarized in the table ■ below:
Person 1 (FA activity, CTA units/ml) Cell line 4
8 hours 72 hours 96 hours ATCC 14 20, 2,57, 5 dead PaCa-2f (ATCCC RL-8725, 415, 025, 2 5) As the above test results indicate, passage of the parental cell line through serum incompatible medium The surrogate inoculation 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.

B-1 Mouse melanocytoma cell line as the parent cell line
6 [Jacks.

The procedure described in the Examples was repeated using the following methods: The biologically pure modified cell line obtained by the procedure described herein was designated B-16. The test results obtained are summarized in the table ■ below: Person 1 (PA(7) activity, CTA units/m'l) Cell line 2
4 hours 48 hours 96 hours B-16 0.70 1.
20 Dead cell line 96 hours B-16--- B-163,2 Biologically pure modified cell line exhibits a 3-fold increase in plasminogen activator and 48 hours at 24 hours
showed a 2-fold increase in plasminogen activator over time. Furthermore, the modified cell line was able to survive for up to 96 hours.

EXAMPLE 111 The procedure described in the Example 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 an uninvented method is designated KLE and designated AT with accession number ATCCCRL-8726.
Entrusted with CC. Plasminogen activator activity was measured 48 hours after the cells reached full growth,
The test results obtained are shown in Table V below.

Person N (Activity of PA, CTA units/m Lateral lineage 48 hours ATCC CRL-162 20,05 KLE (ATCCCR3,o3 L-8726) Modified cell line, KLE as shown in Table V
showed a 60-fold increase in plasminogen activator production over the parental cell line at 48 hours.

Taketsuta Ken N. ATC with accession number ATCC-1579 as the parent cell line.
The procedure described in the Example was repeated using a melanin-deficient melanocytoma cell line available from C.C. The biologically pure modified cell line obtained by the method of the invention is designated as ML and has accession number ATCCCRL-
8724 and was deposited with the ATCC. The activity of plasminogen activator was measured 48 hours after the cells reached full growth, and the test results obtained are shown in Table 1 below.

Table j (FA(7) activity, CTA units/ml) Cell line 4a time ATCCCRL-157 90a KL (ATCCCCRL o, o7-8724) a The level of activity was below the detection limit of the assay.

Patent applicant: Miles Laboratories, Inc.

Claims (1)

Claims: 1. Growing plasminogen activator-producing cells in a suitable growth medium containing fetal calf serum, and growing said suitable growth medium containing decreasing amounts of fetal calf serum. sub-inoculating such cells through one lineage, whereby said cells are able to reach full growth in said medium suitable growth medium which is lignically free of said fetal calf serum, and said cell is capable of producing plasminogen activator at a level greater than that of an initial plasminogen activator-producing cell; How to increase. 2. Plasminogen activator-producing cells are found in melanocytoma, prostate, breast, colon, ovary, pancreas, mouth, rectum,
Claim 1 which is a mammalian tumor cell selected from the group consisting of endometrial and fibroblast tumor cells.
The method described in section. 3. The method of claim 2, wherein the mammalian tumor cells are human cancer cells 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 ATCCCRL-1435. 6. The method according to claim 1, wherein the suitable growth medium is SYC medium. 7. The method according to claim 6, wherein the SYC medium contains fetuin, bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone, and dexamethasone. 8. Fetuin is present at a concentration of 50-150 mg/l (7); bovine serum albumin is present at a concentration of 50-150 mg/l (7)
, / ]; insulin is present at a concentration of 5-10 m
present at a concentration of g/l; transferrin is present at a concentration of 5 to
5α-dihydrotestosterone is present in a concentration of 5 to 200 g/l; and dexamethasone is present in a concentration of 50 to 200 g/l. Method. 9. Fetuin is present at a concentration of 75 mg/l; bovine serum albumin is present at a concentration of 75 mg/l; insulin is present at a concentration of 8 mg/l and nidtransferrin is present at a concentration of 25 mg/l. 8. A method according to claim 7, wherein: 5α-dihydrotestosterone is present at a concentration of 100 mg/l; and dexamethasone is present at a concentration of 100 kg/l. 10. The method of claim 1, wherein the concentration of fetal calf serum is sequentially reduced by a factor of 2 through each of said passages. 11. Human prostate adenocarcinoma cell line, ATCCCRL-
1435, in a suitable growth medium containing fetal calf serum, and sub-inoculating such cells through a series of said suitable growth medium containing decreasing amounts of fetal calf serum. , whereby said cell line is capable of reaching full growth in said suitable growth medium essentially free of said fetal calf serum, and said cell line is comprised of early plasminogen activator producing cells. A method of increasing the yield of plasminogen activator obtained from a human prostate adenocarcinoma cell line, ATCCCRL-1435, characterized in that it is capable of producing plasminogen activator at levels greater than that of the line. 12. The method according to claim 11, wherein the suitable growth medium is SYC medium. 13. The method according to claim 12, wherein the SYC medium contains fetuin, bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone, and dexamethasone. 14.7x twin is present at a concentration of 50-150 mg/l: bovine serum albumin is 50-150 mg/1
insulin is present at a concentration of 5 to Long/l; transferrin is present at a concentration of 5 to 40 mg/l; 5α-dihydrotestosterone is present at a concentration of 5 to 20 mg/l;
present at a concentration of 0 gg/l; and dexamethasone at a concentration of 5
14. A method as claimed in claim 13, wherein the compound is present in a concentration of 0 to 200 gg/1. 15. Fetuin is present at a concentration of 75 mg/l; bovine serum albumin is present at a concentration of 75 mg/l; insulin is present at a concentration of 8 mg/l; nidtransferrin is present at a concentration of 25 mg/l; 5α 15. A method according to claim 14, wherein - dihydrotestosterone is present at a concentration of 110 Ox/l; and dexamethasone is present at a concentration of 1100 p/l. 16. The method of claim 11, wherein the concentration of fetal calf serum is sequentially reduced by a factor of 2 through each of said passages. 17. A chemically defined growth medium designated as SYC medium. 18. The chemically defined growth medium of claim 17 containing 18, fetuin, bovine serum albumin, insulin, transferrin, 5α-dihydrotestosterone and dexamethasone. 19. Contains fetuin at a concentration of 50 to 150 mg/1t7) 6 degrees; Contains bovine serum albumin at a concentration of 50 to 150 mg/l; Contains insulin at a concentration of 5 to 10 mg/l; and Nidoltransferrin at a concentration of 5 to 40 mg. Contains 5α-dihydrotestosterone at a concentration of 50-20
0. and dexamethasone at a concentration of 5 g/l;
19. A chemically defined growth medium according to claim 18, present in a concentration of 0 to 200 gg/l. 20. Fetuin at a concentration of 75 mg/l; bovine serum albumin at a concentration of 75 mg/l; insulin at a concentration of 8 mg/l; transferrin at a concentration of 25 mg/l: 5α-dihydrotestosterone. 1OOJ1. and dexamethasone at a concentration of 100.g/l; A chemically defined growth medium according to claim 20, present in a concentration of g/l. 21, PC with ATCC accession number CRL-8539
A biologically pure culture of a plasminogen activator-secreting cell line designated -3f. 22, Pa with ATCC accession number CRL-8725
Biologically pure culture of a plasminogen activator secreting cell line designated Ca-2f. 23, KL with ATCC accession number CRL-8726
Biologically pure culture of a plasminogen activator-secreting cell line designated E. 24, ML with ATCC accession number CRL-8724
and biologically pure cultures of plasminogen activator-prone cell lines. 2. A plasminogen activator secreted by the cell line according to claim 21. 2. A plasminogen activator secreted by the cell line according to claim 22. 2. A plasminogen activator secreted by the cell line according to claim 23. 2. A plasminogen activator secreted by the cell line according to claim 24.