JPS6225975A - Human cultivated and established cell - Google Patents

Abstract

PURPOSE:A human cultivated and established cell 'OC-1', obtained from primary culture of a human ovarian tissue and capable of producing a plasminogen activator in a culture medium with a high productivity and permanent cultivation. CONSTITUTION:A human cultivated and established cell obtained by subjecting a cultivated cell group derived from a human ovarian tissue to subculture isolating a single cell group having the high ability to produce a plasminogen activator (hereinafter abbreviated to PA). The cell is in the oval form with 20-80mum size, but sometimes assumes an amorphous form (the major axis/minor axis ratio is increased when the cell density is low, but close to spherical form in the case of single layer formation). The cell is capable of multiplying in Eagle MEM culture medium, and preservable at -196 deg.C for a long period. The ability to produce the PA is stable for a long period, and PA having about 50,000 daltons molecular weight and single enzyme activity ban d by enzymography can be produced in a culture medium with high productivity. The permanent cultivation can be carried out.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel human cultured cell line.

In detail, Sara has a plasminogen activator (hereinafter also referred to as PA) that has a single enzymatic activity band with a molecular weight of approximately 50,000 daltons in enzymography, and is highly produced in the medium and can be cultured permanently. This invention relates to a cultured cell line derived from human ovarian tissue.

[Conventional technology]

Many cultured cell lines have been known that produce plasminogen activators in the culture medium or within the cells. For example, cultured human cell lines (Bows cells) and MCl-7 cells derived from human breast cancer cells are famous for producing tissue plasminogen activator (hereinafter also referred to as tPA) with a molecular weight of approximately 70,000. be. On the other hand, urokinase (hereinafter referred to as IJ
Although IJK-type PA is famous, it is already known that IJK-type PA is produced from human kidney cells and the like.

[Problem that the invention seeks to solve]

In recent years, PA has been increasingly sought to originate from human cultured cells, and there is an industrial need for cell lines that can produce it at higher concentrations.

The purpose of the present invention is to capture cell lines that produce PA in higher concentrations.

[Means for solving problems]

The present inventor searched for PA in cultured cells derived from various tissues in the human body, and as a result of various searches, found that cultured cells derived from human ovarian tissue have remarkable PAA bioactivity, and further developed this cultured cell group. Through passage, a single cell group with high PA productivity was isolated, and this was established as a cultured cell line, leading to the completion of the present invention.

That is, the present invention relates to a human cultured cell line [0C-1] obtained from the primary culture of human ovarian MI tissue and characterized in that it produces plasminogen activator.

The cell line of the present invention uses PA as follows, i.e., the molecular weight is about 5.
10,000, it is IJK type, but it appears only in the molecular weight of about 50,000 LJ with enzyme activity, and low molecular weight type like IJK (molecular weight of about 3
On the other hand, PA, which has extremely high affinity for fibrin, exhibits type A, and also has the properties of a PAA precursor, as its enzyme activity is enhanced by plasmin treatment etc., is cultivated. It is produced in fluid,
This is a novel human cultured cell line that has never existed before.

The present inventor named this human cultured cell line "0C-1".

The cell line of the present invention has the following properties in addition to the above-mentioned PAA bioactivity.

(1) Morphological characteristics It exhibits an elliptical shape with a width of about 20 to 80 μm (more specifically, a short axis of about 20 to 40 units and a long axis of about 50 to 80 units), but it may sometimes have an amorphous shape. Particularly when the cell density is low, the short axis becomes even smaller and the long axis becomes even larger.

When formed as a single layer, it becomes nearly spherical. The nucleus occupies about 1/3 to 1/4 of the cell, and is concentrated in the protoplasm.
Many intracellular granules are observed.

(2) Proliferative (Proliferative in Eagle MEM medium) Synthetic penicillin (Lilacillin, manufactured by Narita Pharmaceutical Co., Ltd.) 100
~300ppm, 0.2M glutamine 1% (V/V)
, fetal bovine serum (hereinafter also referred to as FC5, (, IBCO
(manufactured by Russ 4) in Eagle MEM medium containing 10%
(hereinafter also referred to as complete medium).

(3) Culture conditions and culture format Proliferates well at pH around 7 and around 37°C. Also, 5%
Cultivation in an incubator with CO2 and 95% air is preferred. The culture is carried out in a monolayer on the bottom of a glass or plastic flask (manufactured by CoRNING) or a petri dish. Usually 5 x 10'c
If seeded at a cell concentration of ells/ni+ medium, 7~
A monolayer is formed in 10 days. They also adhere well to carriers such as microbeads and proliferate well.

(4) Subculture is possible without limit. That is, after monolayer formation, cells were detached with trypsin 0.2%, and 5X 104c
Subculture is possible without limit by inoculating the cells on a new culture medium to obtain cells/ml medium and repeating subculture. We have been cultivating subcultures for five years already.

(5) Cryopreservation - Can be easily stored for long periods at 196°C. That is, 1
1.0 to 0% dimethyl sulfoxide + 90% complete medium
Cells were cultured in liquid nitrogen (−1
It can be easily stored for a long time at 96°C.

+61 PAA production capacity The above PAA production capacity is stable over a long period of time, with no decline observed.

(7) Enzymatic properties of PA produced ro(, - A more detailed explanation of PA produced by IJ cells is as follows.

"Immediately after monolayer formation of OC-IJ cells, the culture medium was changed to serum-free medium and cultured for 1 day. Regarding PA produced in the culture medium,
5DS-polyacrylamide electrophoresis method [Fuichichi + -
(Nature), 2g-7,680-685 (+
970) ) and enzymography [Anal Biochemistry = (Anal, formerly oche+s
, .

■? Translated by 164-172 (19B2))
The molecular weight and enzymatically active portion of A were measured. Immunological properties were examined from the degree of attenuation of the activity of the enzyme by reacting with rabbit anti-JK antibody and rabbit anti-tPA antibody. Also,
The affinity of concanavalin-A to fibrin was examined by concanavalin-A Sepharose column chromatography and fibrin-Sepharose column chromatography.

The above results are as follows.

■Published in 50,000~53,000■Enzyme activity: Only has a molecular weight of 50,000~53,000, and low molecular weight types (30,000>> do not exist).

- Affinity for fibrin: Yes - Affinity for concanavalin-A: Yes - Immunological properties iUK type The 10C-IJ of the present invention is expected to be a cell line capable of stably supplying PA due to the characteristics described above.

〔Example〕

1"O (>IJ), examples are given below regarding the method for producing this cell line, the culture method for this cell line, its cytological properties, etc.

Example 1 i [) Preparation of C-IJ Human ovarian tissue, freshly removed by surgery, weighing approximately 0.0 g was thoroughly washed with Eagle MEM medium 3001 containing 1000 ppa+ of liracilin, and then washed with ophthalmological scissors or the like. The tissue was then shredded into tissue pieces of 11-3 or smaller. The tissue pieces were collected and resuspended in Eagle's MEM medium 8 (1+1) containing 300 ppa+ of wallacillin, and trypsin 0.5
% (V/V), 10ml, collagenase (200U/+
++1) was added, and cells were dispersed by enzymatic digestion in a 2001 capacity beaker. At this time, the stirring speed was set to IOOrpm using a stirrer, the temperature was set to 37°C, and the digestion time was set to within 60 minutes. After completion of cell dispersion, stirring was stopped, undigested tissue and other residues were allowed to settle naturally, and the supernatant 501 was taken, and an equal volume of complete medium was added thereto to stop the enzyme reaction.

100 ml of this cell suspension was dispensed into Plusdec centrifuge tubes using an appropriate device, centrifuged at 500 rpm for 11.5 minutes, and the cells were collected. The collected cells were resuspended in complete medium and incubated at 1.0-2. Seed into plastic flasks (75 cJ) at a cell concentration of OX 106 cells/ml,
The cells were incubated at 37° C., 5% CO2, and 95% air, and after 1 to 2 days, non-adherent cells and microscopic tissue fragments were removed by replacing the medium. Under the above conditions, the medium was replaced every week, and at the same time microscopic observation was performed to confirm the growth of adhered cells.

This primary culture was carried out for about 1 month, and then the cells were detached with 0.2% trypsin to yield 5 to 10 x 104 cells.
, /+1 cell concentration in a new culture flask (25 cd
) and then start subculturing. When passage was repeated every 10 to 14 days, by the 3rd to 5th generation, cells with strong adhesiveness and proliferative ability were selected, and a single cell type was obtained. Thereafter, passage was repeated every week at a seeding rate of 5 x 104 calls/ml, and in this way, the cell line [0C-IJ of the present invention] was obtained.

Example 2 OC-IJ cells, which have been subcultured for iro(>IJ proliferative and PA production), were treated with trypsin 0.2
% out of contention, 1. OX 105 cells were seeded in a 30ml 1-diameter plastic jar (manufactured by CoRNING) containing 2ml of the above complete medium, and incubated at 37°C and 5% CO2.
Cultured under partial pressure. Then, the number of cells was measured every day, and PA activity within the cells and in the culture supernatant was measured. That is, the cells were peeled off from the petri dish using a rubber policemen, a portion was counted using a hemocytometer, and the rest was placed in Triton-X.
Solubilize with 0.1% of -100 and cell lysate (Ce
PA activity was measured using the fibrin plate method together with the culture supernatant. In addition, P.A.
UK (manufactured by Mochida Pharmaceutical Co., Ltd.) was used as the standard substance, and the activity was expressed in UK terms (Ill/ml). The results are shown in Figure 1, the cell growth curve, and Figure 2, extracellular PA (・),
The activity of intracellular PA(0) is shown in each figure. from this result,
"We found that OC-IJ stores almost no PA inside the cells, but secretes a significant amount of PA outside the cells.

Example 3: Bead culture of roC-1'J "OC-1
j cell 1. OX 107cells, dry weight W20
Microbeads equivalent to 0 tag (cytodesox 1,
(manufactured by Pharmacia) in a complete medium in an 801/100 ml spinner bottle and allowed to stand for about 4 hours.

At this point, it was confirmed by microscopic observation that almost 100% of the cells had adhered to the beads. Culture was performed at 37° C. in 100% air with a stirring speed of 60 rpm. On the third day of culture, 50% of the medium was replaced. The bead suspension was sampled every day, and the adhered cells were solubilized together with the beads with IN NaOH, etc., and the protein was immobilized [Lawry
[method], and the PA activity of the superior synovial fluid was measured by the fibrin plate method described above. The results are shown in Figure 3. In FIG. 3, * indicates the amount of cellular protein, and O indicates the PA activity value in the medium. Regarding l)A production ability of “OC-]J cells,
Table 1 shows the production amounts by various culture formats.

Table 1 [Action/Effect] The established cell line of the present invention has the ability to produce PA having the enzymatic properties described above, and is useful for producing the PA.

[Brief explanation of the drawing]

Figure 1 shows the growth curve of OC-IJ, and Figure 2 shows the growth curve of OC-IJ.
Figure 3 shows the intracellular and extracellular PA production amounts of IJ.
The growth curve of OC-IJ by bead culture and the extracellular PA production mechanism are shown, respectively. Fig. 1: 8 soft (B) Fig. 2: PA; @-L (IU/ml) Fig. 3: (m9/ml > PA Shiran・Lukushi 1 (10/ml) 1.5 to 6 0 To 0. 5 / ・～・ 14 o/°/ 3 ・to・' 0.5 2 @-・O Chaos Daily income (days) l l f)□＾ 61 enterprise 345678 4 meals Number of days (days)

Claims (2)

[Claims] (1) Human cultured cell line "OC-1" obtained from primary culture of human ovarian tissue and characterized by producing plasminogen activator. (2) Claim No. 1 having the following cytological properties:
) The human cultured cell line "OC-1" described in section 2. [1] Cell morphology: exhibits an oval shape of 20 to 80 μm, but may sometimes exhibit an amorphous shape. [2] Cell growth temperature: Proliferates well at around 37°C. [3] Cell growth pH: Proliferates well at around pH 7. [4] Subculture: Subculture is possible without limit. [5] Cryopreservation: Can be easily stored at -196°C for a long period of time. [6] Plasminogen activator production ability: Yes. [7] Growth in Eagle MEM medium: Proliferates. [Synthetic penicillin (lilacillin, manufactured by Takeda Pharmaceutical Company) 1
00-300ppm, 0.2M glutamine 1% (v/v
), Eagle's medium containing 10% (v/v) fetal bovine serum)
]