JPS5836385A - Obtaining method of cellular strain for producing interferon continuously - Google Patents

Abstract

PURPOSE:To prepare cells UNCL capable of producing human interferon of high units continuously without using an interferon inducer, by varying lymphocytes derived from human infants with Epstain Barr viruses. CONSTITUTION:Lymphocytes separated from a blood collected from human infants younger than two years after their birth by collecting the blood or other means are transformed by Epstaine Barr viruses to give a cellular strain UNCL capable of producing interferon of high units without using any interferon inducer.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to lymphocytes derived from the peripheral blood of human infants infected with Epstein-Barr virus (Epstein-Barr virus).
Virus, hereinafter referred to as EBV. ) by mutating it to enable successive multiplication and to continuously produce high units of human interferon (hereinafter referred to as IF) without the use of interferon-inducing agents such as viruses. It is.

IF has been recognized to exert preventive or therapeutic effects not only on various viral diseases but also on certain viral or non-viral tumors. However, in order to use IF for the prevention or treatment of these diseases, a large amount of IF is required, and on the other hand, because IF is highly species specific, IF must be produced from living human cells. The production of large quantities of IF from human cells has been a major obstacle to the therapeutic use of IF.

That is, conventional IF production methods include methods using leukocytes isolated from fresh adult human cells, methods using cultured human fetal-derived fibroblasts, and methods using lymphoid cell lines derived from Burkitt's lymphoma or leukemia patients. Alternatively, there are known methods for growing them within an animal body and using them. However, in order to obtain a large amount of IF using these methods, it is necessary to
A common problem is that F-inducing agents must be used. In other words, IF production only occurs within a certain period of time after a large amount of virus or other IF inducer is applied to these living cells, and the cells can only be used to produce IF once. This is extremely disadvantageous for mass production of IP, and the purification process may become complicated because the inducer, such as a virus, must be completely removed or rendered nontoxic during IP purification. On the other hand, although some cells that continuously produce IF are known, the number of mice that produce them is extremely small.

The present inventors first developed a cell line that can continuously produce high levels of IF without using any IF-inducing agent, by transforming lymphocytes derived from human fetuses or human neonates with EBV. We succeeded in obtaining a cell line UNCL that constantly produces high levels of IF without using IF, and obtained a patent.

As a result of further research, UNCL cells were obtained not only from the lymphocytes of fetuses and newborns, but also from infants, and those from these infants had a higher ability to produce IF than those obtained from fetuses and newborns. The present invention was completed based on the discovery that the IF is not only large but also grows normally even when IF accumulates in excess of IO'V/mlV, unlike that of fetuses and newborns.

UNCL cells in the present invention are obtained by transforming lymphocytes derived from human infants with EBV.

Lymphocytes derived from human infants refer to lymphocytes separated from blood collected from human infants less than two years old or by other means.

As a method for separating lymphocytes from blood, a method using gelatin or a specific gravity centrifugation method using Ficoll is most conveniently used, but other cell fractionation methods for fractionating lymphocytes may also be used.

The overview of the gelatin method is that 3 volumes of anticoagulated blood and P
Mix 1 volume of 3W/V% gelatin solution dissolved in B S (Phosphate Buffer Sa) in a test tube, leave it at 37°C for 30 minutes, and collect the transparent upper layer of the three separated layers. Then, gelatin is removed by centrifugation and washing to obtain a lymphocyte fraction.

Further, an example of the outline of the specific gravity centrifugation method using Ficoll is as follows. After mixing 1 volume of anticoagulated blood with the same amount of balanced salt solution, add 1.5 volumes of Ficoll-Einpaque solution (P
(manufactured by harmacia Fine Chemicals). After centrifuging at 400x9 for 30 minutes, the lymphocyte fraction collected in the middle layer was collected.
This is a method for obtaining lymphocytes by performing a washing operation of adding a balanced salt solution and centrifuging two to three times.

The EBV used in this test is, for example, monkey lymphoblastoid B-9578 cells persistently infected with this virus or Q+iR-w+t derived from human leukemia.

1,000-2.00 Or p m from cell culture solution
Virus obtained in the supernatant after removing cells by centrifugation for 5 to 15 minutes (J. H. Pope et al.
Int, J, Cancer4,8j? (1968), G.
, MNIer etal, , J, Viral, +18
．． 1071 (1976)), but EBV obtained from other cell types and by other methods may also be used.

As a method for transforming lymphocytes with EBV, for example, human infant-derived lymphocytes obtained by the above method are added to 5×+05 ml of animal cell culture column 1 supplemented with 5 to 15 V/V% fetal bovine serum. 5X10' pieces/m
e, and set the EBV to 10.
' ~I 0611) 507 ml and incubated at 36-37°C in the presence of 3-6V/V% COg.
After standing for 5 hours, the cells were collected by centrifugation, washed 2 to 3 times by centrifugation to remove excess EBV,
Washed cells were added to a cell culture medium containing 5 to 20 V/V96 fetal bovine serum at 58I05 to 5X10' cells/ml.
3 to 6 V/V% CO,,3
A method of obtaining transformed lymphoblastoid cells UNCL by subculturing them at 6 to 37°C for 1 to 2 months is preferably used, but a similar method using EBV may also be used. good.

In addition, TD ao refers to the transformation of lymphocytes by inoculating serial dilutions of EBV into lymphocytes.
The activity in the diluted solution where there is a 50% probability that n will occur is 1
This is the unit of viral activity.

This is how it was obtained! The F sustained production cell line UNCL may be continued to be cultured under the above conditions and used for IF production, but if necessary, it may be separated from the medium by centrifugation or the like.
5-15V/V96 orchid fetal or reconstituted serum and 10-1
5V/V% DMSO (Dimethyl 5ulfo
xide) or in a cell culture medium supplemented with glycerin at a concentration of 106 to 108 cells/ml, -8
It can also be stored at low temperatures below 0°C.

The IF sustained producing cell line UNCL of the present invention, similar to those obtained from fetuses and neonates, produces IF without an IF inducer.
and the production is 1.000 units or more per 106 cells after culturing for 2 to 4 days under the above culture conditions,
The cell doubling time in the growth phase under the above culture conditions is 2
4-48 hours, the cells are EBNA (EBV
However, EBV was not detected in these 9 cells or in the culture medium by electron microscopy or EBV infection experiments, and Ig was detected among the immunoglobulins present on the cell surface.
The distribution of M or IgD is 20% or more, the immunoglobulin present in the culture medium in which these cells are cultured contains 5096 or more IgM, and the number of chromosomes is 44 to 48. It is believed to be the same as the IF persistent producing cell line IJNCL obtained from a newborn baby.

1F is manufactured as follows. Namely, 1. UtlCL cells were added to the nutrient medium for culturing animal cells.
Add to about 10' pieces/ml, 3-6V/V
When the pH of the medium is 6.0-7.5, air containing 96% CO1 is added.
Fill or aerate the container so that the temperature is 35 to 38°C, and culture by leaving it for 3 to 6 days or stirring gently. After culturing, the culture solution is centrifuged, and the separated cells are added to a new medium at a concentration of about 105 to 106 cells/ml to continue culturing, while 1. IF produced is collected from the centrifuged supernatant. .

The nutrient medium is not particularly limited as long as it is for animal cell culture, but for example, fetal bovine serum or serum 1 to 1
A nutrient medium containing 5% V/V is suitable for the present invention. Examples of such media include EagleS MEM medium, RPMI (640 medium,
Examples include Dulbecco's Modified MEM medium.

During culture, metabolic inhibitors such as actinomycin D1' cloheximide, 5-bromdeoxyuridine, 5-iododeoxyuridine, hydrocortiscin, prostaglandin, butyric acid, dibutyl-31sl-
The axillary IF produced by adding a substance that changes metabolism such as cyclic AMP, increasing or decreasing the concentration of serum or serum albumin as appropriate, or changing the pH of the medium, redox potential, or culture temperature. It is possible to further increase the

0 The IF produced in the culture solution is purified and concentrated by known purification methods such as salting out, ultrafield filtration, dialysis, centrifugation, freeze drying, etc., and then purified by ion exchange, gel filtration, abity chromatography, etc. Highly purified IF can be obtained by further combining methods such as focusing fractionation and electrophoresis.

The IF obtained in the present invention is heated at 56°C for 1 hour or at pH
2 is stable even if left at 4°C for 24 hours.

Further, even if centrifuged for 1 hour using too or oooxy, no precipitation occurs, and antiviral activity is lost when treated with trypsin or anti-human leukocyte IF antiserum.

Since these physicochemical properties are the same as those of human leukocyte interferon, the IF of the present invention is considered to be of the same type as human leukocyte interferon.

The IF sustained production cell line tJNcL of the present invention has a proliferation rate comparable to that of general human-derived cells, and this cell line
By subculturing every 6 days, IF production ability and proliferation ability can be maintained for a long period of 12 months or more.

If the UNCL cells of the present invention are used, for example, while culturing in a suspension cell culture container, the culture medium is taken out from one outlet, and the increased amount of new culture medium is injected from the other inlet, thereby continuously increasing the IF in high units. Moreover, it can be generated by logarithmic increase. In other words, whereas in the conventional method using an IF inducer, cells could only be used once, the present invention makes it possible to continuously mass-produce IF, making it possible to put IF into practical use. This provides a major means of achieving this goal.

The IF activity in the following examples is based on FL derived from human amniotic membrane.
Cells and V SV (Vesicular Stoma)
titisVirus) using the cytopathic suppression method (Iizuka Noise et al., Kinakaku Igaku, 29,660 (+974)).

Cell Production Example 1 1 ml of peripheral blood from an infant was aseptically collected with saline and immediately fractionated into lymphocytes by specific gravity centrifugation using Ficoll Isopanc. This lymphocyte compartment was treated with 3 times the amount of Eagle MEM (Minimum Essent).
After repeating washing three times by adding ialMedium medium (manufactured by Hinaga Pharmaceutical Co., Ltd.), centrifuging, and discarding the supernatant,
RPMI-1640 with 10V/V% fetal bovine serum
The washed lymphocytes were added to a medium (manufactured by Nippon Pharmaceutical) and suspended at a nucleated cell density of 3XIO' cells/ml. B-95-
EBV grown in 8 cells was treated with 5X IO'TD,
After culturing at 37° C. for 2 hours, the cells were collected by centrifugation, followed by washing three times by adding Eagle MEM medium, centrifuging, and discarding the supernatant. Washed cells were washed 1. The cells were implanted in RPMI-1640 medium containing OV/V% fetal bovine serum at 3XIO' cells/ml, and incubated at 36-37°C with 5V/V96 Co.
Culture was performed under the following conditions. Cultures continued for 1.5 months, during which time RPM containing +ov/v% fetal bovine serum was added every 5 days.
Either an equal volume of I-1640 medium was added or a half volume of the medium was replaced with this medium.

Four cases of infant peripheral blood and seven cases of adult peripheral blood were subjected to such treatment, and the spontaneous production of IF was measured after each cell was transformed. The results are shown in the table below. This production amount is calculated by adding 5 x 10' cells/cell to RPMI-1640 medium containing IOV/V96 fetal bovine serum.
Implanted to be me, SV/V% CO□, 37
The results were obtained by measuring the activity of the culture supernatant after culturing for 5 days at ℃.

1F activity
/l -12610,+//-13155,ISA<o,+KN
O,2 adult peripheral s
u'<o・1 blood lymphocyte M
l<O・lUE
, 0.3KU
'<0. IFU
<0.1 1F Production Example 1 Pour RPM-1640 medium containing IOV/V% fetal bovine serum into a glass culture bottle, and add cell line tJNcL to it.
-11 and the disordered strain UNCL-8 obtained from cord blood were implanted at a cell density of 5 x 10 cells/ml.
After blowing in 54CO, seal it tightly and 36. 5 at ~37℃
Cultured for 1 day. This culture solution was centrifuged at 1.00 rpm for 10 minutes to collect the supernatant, and the remaining cells were added with the above medium to a cell density of 5×105 cells/ml and cultured in the same way as above. , the above operation was repeated 5 times. The results of measuring the IF activity of each culture supernatant are shown in the table below.

1F Cell circulation (ml) x+o'U/)++j' (
XIO' U) 1 10 1.5
(1,5) 2 45 1.0 (4
,5) UNCL-II 3 +80 0
．． 98 (+7) 4 810 0
．． 97 (79)5 3.000 0.
95 (285)e 1101□, 5 (1,5) 2 15 ,t,a (1,9
5) UNCL-8318' 1.0 (1,8
)4 20 1.0 (2,0
)5, 20 0.98 (1,9
6) 1F Production Example 2 Dulbeccoa Modified M containing 5 V/V% fetal bovine serum in a glass culture tank with a volume of 31
EM (manufactured by Nissui Seiyaku ■) medium 1. Add OOQ+nl and add cell line UNC'L-10 to it at a cell density of 5XI.
The cells were implanted at 5 O/me and kept at 37°C.

Add 5 V/V% C02 to this at a flow rate of 300 me/m.
While blowing the mixture into the culture tank, stirring was continued at a rotation speed of 60 to 150 rpm using a suspended stirrer in the culture tank, and the culture was continued for 5 days.

After stopping stirring and aeration and allowing the cells to settle for 4 hours, 400 ml of the supernatant was taken out by suction and immediately centrifuged at 1.20 rpm for 15 minutes to remove the contaminated cells. . Next, the same medium as above was added at 1,000 mA' from the other port, and after culturing under the same conditions for 4 days, the culture solution was removed from the culture tank and heated at 1.20 rpm.
The culture supernatant was obtained by centrifugation for 5 minutes.

When the IF activity of each culture supernatant was measured, it was found that IF activity of each culture supernatant was measured once and 9. I x 106 units, the second -E clearing solution contained 6.8 x 10' IF.

Patent applicant: Ajinomoto Co., Inc.

Claims (1)

[Claims] A method for producing human interferon persistent producing cells UNCL, which comprises transforming lymphocytes derived from human infants with Ibstai/Barr virus.