JPH0322973A - Constitutive production of colony stimulating factors (csfs) by human hepatoma cell line - Google Patents

Abstract

PURPOSE: To produce ganulocyte macrophage colony-stimulating factors in high yield by culturing HA22T/VGH of human hepatoma cell line in a conventional culture medium to produce colony formation-stimulating proteins and releasing them into the culture medium.

CONSTITUTION: HA22T/VGH of human hepatoma cell is cultured in a conventional medium (PRMI1640, IMOM, etc.) to produce ganulocyte macrophage colony formation-stimulating factors (GM-CSFs), and the products are released into the conventional culture medium. The cell line: HA22T/VGH can constitutively produce GM-CSFs in high yield. The taming medium which has been tamed by the cell line can produce a large quantity of GM-CSFs, has larger activity for stimulating human bone-marrow hemopoiesis in vitro than a conventional taming medium, and enables the detection of the activity at a high dilution (1:64).

COPYRIGHT: (C)1991,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the constitutive production of granulocyte-macrophage colony-forming factors (GM-CSFs) by human hepatoma cell lines.

As is well known, hematopoietic growth factors play an important role in stimulating stem cell proliferation and differentiation of one or more fully differentiated blood cell types I\. Among these factors, granulomonopoieLic
Granulocyte-macrophage colony forming factors (GM-CSFs) targeting progenitor cells show special effects on the production of granulocytes and monocytes/macrophages (
Eight. W. Burgess & D. Metcalf, B.
load 56:947, 1980; and N. S. Ni
cola & m. Vadas, Inuaunol.
Today 5:76.1984). rGM-CSFs
” refers to G-CSF, M-CSF and GM~
Unpurified GNCSF containing CSF f! -means.

Recently, there has been a widespread trend to use radiation and chemotherapy in the treatment of various cancers. However, these therapies include myelo-suppression.
This is accompanied by a side effect called s ion, which inevitably causes undesirable events such as a decrease in the number of white blood cells and a decline in immune function. Therefore, it is not uncommon for it to be extremely difficult to continue this type of therapy. CSFs (especially GM
- CSFs) in combination with the aforementioned treatments,
As expected, the cancer treatment was effective 1: {ru (M.Sh i
mamura et al., Illood 69:353, 198
7; and P. Mayer et al., Blood 70:
20B, 1987). CM about hematopoiesis and clinical use
- In order to facilitate research on CSFs and further promote genomic research, methods for producing GM-CSFs must be developed, such as large quantities of conditioned medium (CM) for purifying GM-CSFs. A cell line is required that will serve as the source of the stimulant.

From previous research, we have found that GM-CSFs can be produced6
It is known that there are four types of cells. That is, monocytes/macrophages (D.W. Gold et al., Launcet 2:
1397, 1972 and I. N. Rich et al., Exp
．． llemato1 14:738, 1986); fibroblasts (partial) (P.E.^ustin et al., J.Ce
ll Pbysio 402:334, 1980). Endothelial cells (GE Bagby et al., Blood62:0
63.1983 and G. M. Segal et al., J. I+
nmunol. 1381772, 1.987). and lymphocytes (R.G. Sbab et al., Illood5
0:81 1, 1977 and M. ．． LCli+i
Eike, Nature 248:7031974). However, large amounts of GM-
CSFs are difficult to obtain from normal tissue cells in vitro culture. GM-CSFs are derived from rGCTs isolated from certain tumor cell lines, such as fibrous histiocytomas that have metastasized to the lungs (U.S. Pat. No. 4,135,975;
(1979), r5637 J derived from human bladder cancer
(J. Fog Natl. Cancer Inst.
．． Monogr. 49+5.1987) and Hi1.
SK derived from lung cancer. -MES-1 (S.N.Zinz
ar et al., Exp. Hematl. 13:574,1
It was found that a higher yield could be obtained by culturing 985).

We surprisingly found that the human hepa1.-m cell line is GM.
- It has been discovered that GM-CSFs can be produced horizontally in high yields and has a significantly larger colony form or promoting ability compared to well-known GM-CSFs producing cells. This cell line was developed by the inventor C. Established by Cbang, Molecular & CellularB
io! ogy 3:1133-1137. Published in 1983. This system is D e p a r L +n to Me in Taipei City, Taiwan, Republic of China.
dical Research, Veterans
General llospiLal (and Gradu
ate Program of Microbiolo
gy and L+aunology, Nation
al YanH-+nin8+nedical col
available at eg.

One of the objects of the present invention is to produce GM-CSFs using a hepatium cell line. This phasic system HA22T/VCII can constitutively produce GM-CSFs in high yield and is also a valuable source of pure GM-CSF.

Another object of the present invention is to provide a conditioned substrate adapted by the H822T/VCI1 cell line that is capable of stimulating in vitro explantation of bone marrow cells. This conditioned medium has a molecular weight of 12.4 to 29 Kd and a molecular weight of 22.53 Kd0 (;M
- G-CSF identified as CSF. Contains M-CSF and GM-CSF (; Contains M-CSFS.

Yet another object of the present invention is to develop bone marrow cells in vitro.
The purpose is to provide a way to stimulate the preceptor. This: f
i't large c1, culture medium IIA22T/VGHJ{
H Ha system t''Am (e L) consists of inoculating this conditioned medium with bone marrow abocytes to induce granulocyte colonies, R3 cell colonies, granulocyte and monocyte colonies, and eosinophil colonies.

Hereinafter, the present invention will be explained in more detail based on the attached drawings.

In the present invention, in order to develop a simple and effective production method for GM-CSFq, we used the hebatome cell line H^22T/VG for the horizontal production of GM-CSFs. ! Use.

The H822T/VGIII cell system used in the present invention is 56
It was established from four different specimens of hepatocellular carcinoma obtained from a 2000-year-old Chinese man. This cell line and its virulence are known as Mole
cufar & Cellular Biology
3:1133-11371983. This cell line 41 is capable of constitutively producing M-CSFs in high yield. Conditioned medium conditioned with this cell line retains large amounts of GM-CSFs and its ability to stimulate human bone marrow hematopoiesis in vitro is superior to that of conventional conditioned medium, i.e. 'r-)GCT-CM, 5637CM. , placenta 1C
M, lung 1 CM and hilus 18-L-CM, and activity could be detected at considerably higher dilutions (1:64). The type of colony formed by inoculating human bone'ti KfA cells into B. M
cndelou+Ike's method (8m.J.Hemato1
5:151, 1978) and G. R. John
nson & D. Metcalf's method (Exp.H
cmatol 8:549, 1980). After fixation and staining of the cultures, colony types were determined by microscopic morphological examination. The results showed that the majority (75%) of the colonies formed were of the granulocytic type, but other evening and eve colonies (monocytes, eosinophils, and granulomonocytes) were also included. did. These crystal fruits contain CS containing H822T-CML.
F protein or GM-CSFs or Kanoichi CSF
The activity of GM-CSFs was investigated using a CFU-GM assay as known (S.Y. Wang et al., [Ilo
od 65:1181, 1985). Comparative experiment Teha, 1
1 A 2 2 T/V G }I cells'ζ conditioned medium is larger than conventional lobe in vitro
Ohi1・Bone marrow growth spur? lj. demonstrated ability. Therefore, the medium represents a valuable source for producing pure GM-CSF for use in laboratory and clinical research.

In order that the invention may be better understood, 41 limited examples are given below. Note that "1 part" and "%" are "parts by weight" and "heavy weight ≦" unless otherwise indicated.

Table 1 Live cell line 11^22T/VGI1 and its characteristics ゜'Mc
General & Cellular Biology
3:1133-1137.1983". For routine culture, these cells were cultured in PRMI 1640 or I containing fetal calf serum (FCS, Flow Laboratories) at a concentration of 2 x 1057 m
It was suspended in NDH medium (GIBCO laboratories). This cell suspension i({1/5+Ill Tt
The cultures were then incubated at 37 °C in an atmosphere containing 5% CO2 in the air. Cells were grown to confluency (
After 3 days of incubation), subculture was performed.

A conditioned medium was formed with I1^22T/VGI+4{B cells necessary for the XAmu2+jj^22T-CM to lsummer GM-CS^ assay and colony type analysis. l{822 containing large amounts of GM-CSFs
1'-CM is also used to purify GM-CSF.

5x 10'/ml {i^22T/V(:Ill cells) were incubated in RI'MI 1640 medium with 5% FCS at 37°C and the supernatant was collected after 5 days of incubation. Conditioned medium conditioned by 22T/VGHM cells (herein 11^22T-CM
) was subjected to tests such as colony formation promoting activity.

``Tbe Ilemopoietic stimula''
ting factors” (M. Netealr, ^
n+sterdam: Elsevier, 1984), pages 101-102, or as described in S. Y. W
[1load 65:1181.198
The activity of GM-CSFs was measured using the CFUGM assay as described in 5.

i.e., McCo supplemented with 10% heat-inactivated FCS.
5 x 10' low-density, non-adherent human bone marrow cells were implanted in medium h1, which was formed by adding 0.3% agar (Dirco) to 5^ medium of y. H^2 for this assay system
2T-CM or other cellular CM at a final concentration of 10% (vol.
/vol) added 7 pieces. Incubate the cultures at 37°C in a 30°C tube and count the number of colonies and clusters after 7 days of incubation. After this counting procedure, the clusters can be fixed and stained and also used for colony type analysis.

After counting colonies, the agar plate was fixed on the spot with 59≦glutaraldehyde dehydrated with methanol, and then fixed with Luxol-Fast-Blue and 11
arris hematoxylin
n) (B. Meddelow et al., 8m. J. lIema
to1 5:151, 1978 and G. R. Jobs
on & D. Metcal ( Exp.IIemat
o1 8:549, 1980). 100-2
Colony types were determined by morphologically examining 100 colonies under a 00x microscope. As a result, most of the colonies formed by stimulation with II^22T-CM (75%) were granulocytes, but
Other types of colonies (monocytes, eosinophils and granulomonocytes) were also included. These results indicate that the CSF protein contained in H822T-CM is GM-CSF or G
This suggests that it is a mixture of M-CSF and G-CSF.

As a result of LIXU kinetic observation of GM-CSFs production, the level of GM-CS^producers became remarkable in II^22T cultures cultured for 5 days, and peaked on the 7th day of culture. (See Figure 1). Also,
The activity could be detected at a fairly high dilution (1:64) (see Figure 2). Real JjdfLL: ゛(1
) Stability against heat treatment The activity of GM-CSFs contained in I1^22T-CM remains stable even when heated at 56°C for 30 minutes (see Table 1).
.

Large influence of ripening treatment on GM-CSFs activity of Wheat-1L]1^22T-CM % relative to control (untreated) group.

(2) Stability on pll value The optimum ρ11 for obtaining the effect of promoting colony formation was 7. If this value is greater than 9 or less than 5, the activity is significantly reduced (see Table 2).

Table 2 r on GM-CSFs activity of I1^22T-CM★
Influence of o4 ★ II A 2 2-CM is seed/Z p
Adjust to the 11 value and heat at 4°C for the first time in the cFu assay.
It was stored for 7 days.

★★This activity is expressed as a percentage of the activity in the normal state (pll7).

(3) Determination of molecular weight Physical purification and molecular fi (1.w.) determination using known methods (K. Weltz et al., J. Exp. Mcd. 156
:454, 1982; and v. p. R. Ghio et al.
Blood 69:1340, 1987>.

Molecular weight measurements were carried out as described in f.+1A22T-CM4: (NI1.)2SO. 3Q
% saturation. The precipitate was centrifuged (
11H7.2 PBs) and dialyzed against the same buffer. Concentrate obtained by dialysis (0.
1 ml) of Superose T12 (Pharma
cia) gel p percolumn (1.0 x 100c+Q
) was filled. a Adjust B to 0.4 mt/min, II
I. W. was calibrated with the following markers: blue dextran (2,OOOK), alcoholdehyde I.17 genease (150K), IlS8 (67.3K).
). Carponic anhydrase (29K) and Sil.
Chromium C (12.4K). CFt
CSFs were assessed by l-ON assay. CSF
m. of active eluted protein. tu. 4. ! 12.4
~29Kd (see Table 3). Based on the calibration curve <
+n of CSF proteins. lol. is approximately 22.53K
It was d.

This is llA22T-CM4:
;M-CSF” (7).

Reduces gallbladder and Toe CSF production by H A 2 2 T / V G R R
Comparisons were made between dB follicles and five well-known GM-CSF producing cells: GCT, 5637, Pll^ stimulated lymphocytes, placenta and fetal lung. A conditioned medium was prepared according to the method described in Example 2, and the CSF activity of the proteins produced by the cells was measured by the assay method described in II 3. Analyzed using the method described.

74! The results are shown in Table 3, and the subtypes of induced CM cony are shown in Table 4. Phase 1 of promoting colony formation
Ability {j11^22T-CM>[;CT-CM>Placenta-CM>Fetal Ruto CM 5637-CM> PH8-L
-CM no IlIn -r Af. : , lIA22
The number of colonies induced by T-CHI was 94±3 per bone marrow cyst 5×10.

11^22T/VC:I1 (:M-CSFs production and other GM-CSA production fl{t! cells) (14-C
Comparison with SFs production 1: The culture medium was incubated with various cells for 5 days.

2: CFU-(;M assay system CMwofi4
? If added at 10% (vol/vol), GM-C
The results for the S8 level are shown as the number of GM colonies per 5 x 10' bone marrow cells.

3: Compare the data with IIA22T/VGH-CM
Ta.

4: Contains GM-CSFs (:CT-CM(ma(:I
Obtained from BO Laboratories.

Globular and monocyte types, Eo is eosinophil type.

5:5837 is a well-known cell line that constitutively produces GM-CSFs.

Twenty-one subtypes★ GM colonies grown in agar gel were fixed in situ and treated with Luxol FasL Blue and Harri.
Stained with hematoxylin. Subtype analysis is 1
This was done by counting 00 colonies under a 200x microscope. ★★C is granulocyte type, N is monocyte type, GM is granulocyte type

[Brief explanation of the drawing]

Figure 1 shows 1{^2 2 T/V depending on the culture time.
This is a graph showing the results of kinetic observation of GM-CSFs production by G II cells, showing that the level of GM-CS^ production became significant in I1^22T/VG}l cultures cultured for 5 days, and after 7 days of culture. It shows that it reaches its peak in about a day. Figure 2 is a graph showing the titration results of GM-CSFs in conditioned medium conditioned with 11^22T/VGH cells, showing that activity could be detected at a fairly high dilution (1:64). . Figure 3 is a graph showing the measurement results of the molecules of the GM-CSF protein produced by IIA22T/VGH,l cells, and the molecule Hk (m.u+.) of the eluted protein with CSF activity is It shows that it is 12.4 to 29Kd. Engraving of drawings (no changes in content) No. of CFLJ-GM Colony's Sudan Bangin 1'2
l(e) j@%4r,n 1;n゜7ta HA22T axis Jt)at
: GM-CSFS ojLL Figure I Procedural Amendment 8. Details of the amendment (1) In the first line of page 4 of Mei II, the text ``rCSF'' is amended to ``l CSA''. ■ Amend page 18 of Mei III as shown in the attached sheet. 1. Indication of the case: 1999 Patent Application No. 10954 @ 2. Name of the invention Co2 formation promoting factor (C) by human hebatome cell line
SFS) Constitutive Land 3. Relationship with the case of people who straighten their sleeves

Claims (11)

[Claims] (1) A method for producing a conditioned medium conditioned with colony formation promoting factor (CSFs) cells, the method comprising:
A22T/VGH was cultured in normal medium, thereby
A method comprising producing and releasing SF protein into said normal medium. (2) The method according to claim 1, wherein the culturing step for producing a conditioned medium is carried out by incubation in a humid atmosphere containing 5% CO_2 in the air. (3) The CSF protein contained in the medium according to claim 1, having a molecular weight of 12.4 to 29 Kd. (4) The CSF protein according to claim 3, which has an apparent molecular weight of 22.53 Kd. (5) GM-CSF including G-CSF and GM-CSF
5. The CSF protein according to claim 3 or 4, consisting of s. (6) The protein according to claim 5, consisting of GM-CSF. (7) The CSF protein of claim 3, which is capable of stimulating human bone marrow progenitor cells to form four types of colonies, including granulocytes, monocytes, granulomonocytes, and eosinophils. (8) A method for stimulating the in vitro growth of human bone marrow cells, the medium comprising human hepatoma cell line HA22T.
/VGH, inoculating this conditioned medium with human bone marrow cells, and incubating the resulting culture. (9) The method according to claim 8, wherein the medium contains GM-CSFs. (10) A CSF-containing medium for growing bone marrow cells in vitro, obtained by culturing the human hepatoma cell line HA22T/VGH. (11) The medium according to claim 10, which contains a CSF protein having a molecular weight of 12.4 to 29 Kd.