JPS606190A - Novel cell strain - Google Patents

Abstract

PURPOSE:To provide a novel cell strain which can be proliferated even in a serum-free medium as well as in a serum-containing medium, and can produce an interferon by the virus inducing process, by adapting a human lymphoblast cell which cannot be proliferated in a serum-free medium, using a specific method. CONSTITUTION:Human lymphoblast cell (preferably Namarva cell strain ATCC CRL-1432 originated from Burkitt lymphoma) which cannot be proliferated in a serum-free medium is cultured successively in the media having gradually decreasing serum concentration, and finally cultured in a serum-free medium. The lymphoblast cell is adapted to be proliferable in a serum-free medium by this process to obtain the objective cell strain. EFFECT:The interferon-productivity can be developed even by the vitamin A treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel human lymphoblastoid cells that have been adapted to grow in a serum-free medium by adapting human lymphoblastoid cells that do not proliferate in a medium that does not contain IIIL ffj, a method for preparing the same, and a method for preparing the cells. Concerning a method for producing interferon.

Animal cells normally proliferate in a medium supplemented with serum, and when the serum is removed from the medium, the proliferation stops and eventually dies. The development of serum-free media using various substances such as hormones and growth factors to replace serum is underway. However, when these cells grown in a serum-free medium are transferred to a serum-supplemented medium and cultured, they show a faster growth rate and a higher cell density than a blood-free medium.

A serum-free medium can also be advantageously used if there are cells that provide the same growth rate and cell density as in a serum-containing medium.

The present inventors have succeeded in culturing human lymphoblastoid cells, which are animal cells, and Zunawamo Burkitt's lymphoma-derived Namalva cell line, by sequentially transferring them to a medium with a low serum concentration, thereby obtaining a strain that can grow in a serum-free medium. I discovered that.

Even when these serum-free medium-acclimated cells are grown again in a serum-supplemented medium, there is no significant difference in growth from that in the serum-free medium. J: It was confirmed by observation using an electron microscope that the serum-free medium-acclimated octopus cells had different intracellular components from the parent cells. Furthermore, these serum-free medium-acclimated ♀11 cells retain the ability to produce interferon induced by virus, but the ability to produce interferon is lost by pretreatment with vitamin 8. Since the parent 11 cells exhibit production ability even after pretreatment with vitamins, it can be said that cells conditioned to serum-free medium are sensitive to vitamin A.

In the past, there have been examples of acclimating animal cells and growing them in low-serum or serum-free media. For example, Dokky.

Journal of Medical 5cienc
es Vel, 9. I) rl 97-105.198
In 2, a cell line from rat liver was acclimatized to obtain a cell line that can grow in a medium with serum concentration reduced to 1%. However, not all cells can adapt,
Also, like H a L; I cells, it took about 8 years.
Some cells were able to adapt to C.

There are no known cases of lymphoblastoid cells becoming able to proliferate and proliferate in a serum-free medium due to acclimation, and in particular, there are no known cells that exhibit the above properties.

The present invention will be described in detail below.

The present invention relates to novel human lymphoblastoid cells that have been adapted to grow in a serum-free medium by adapting human lymphoblastoid cells that do not increase in ML in a serum-containing medium.

1) Method of acclimatization As a parent strain for acclimatization, any human lymphoblastoid cell that cannot proliferate in a serum-free medium can be used. Suitable examples include Birkin 1 lymphoma-derived, lymphoblastoid Namalva strain, AT
CCCRL-1432 is mentioned.

The acclimatization method differs slightly depending on whether the culture is a monolayer or a suspension culture. In monolayer culture, the cells are attached to the culture vessel, so it is easy to change the medium, but when treating with trypsin, a serum-free medium is used to replace the trypsin inhibitor contained in serum. It is necessary to add an inhibitor pregnancy. The acclimatization period takes from one week to several months, depending on the rate of decrease in serum concentration and changes in species.

In suspension culture, "C" is 0.5 to s as the number of cells
It is preferable to maintain around 105 cells/ml. pH6.5
It is necessary to replace the medium to avoid the following. When replacing the medium, dilute it if the number of cells is large, and if the number of cells is small, centrifuge δ1] or "2/i?" The cell concentration is combined by ζ concentration by removal. The acclimatization period takes from one week to several months.

Taking suspension culture of Namalva cells as an example, the specific acclimation method is as follows.

The Namalva cell line is subcultured in a serum-containing basal medium supplemented with penicillin, streptomycin, glutamine, Hepes mildew, etc., and used as the parent cell line.

The parent cell lines were collected by centrifugation and cultured for about 1 to 2 months in a medium of each blood/naFf degree while gradually decreasing the serum concentration, changing the medium by half every 2 to 40 minutes. Culture in the same manner for about 1 to 2 months in a serum-free medium that does not contain serum.

As the basal medium, RPMI 1.640 (Nissui Pharmaceutical Co., Ltd.), MEM (manufactured by Hinaga FM Pharmaceutical Co., Ltd.), Darhe 2 Co. MEM (manufactured by Hinaga Pharmaceutical Co., Ltd.), Ham F 10, Nomu) F
12 (manufactured by Flow Lab), etc. are used.

Penicillin, streptomycin, glutamine.

The amount of Hepes relaxation solution added is 5 to 100 U/m1, respectively.
．． 5-100μg/ml, IO+1-400nw/ρ
and 1-30M.

As a blood-free 16 medium, base 11) with penicillin.

A medium containing streptomycin, glutamine, and Heves hydroxide was added, as well as insulin, transferrin, gasterone (EGF), fibroblast growth factor, and serum substitutes. lll growth promoting factor, platelet-derived growth factor, etc. are added. In addition, it may be effective to add unsaturated fatty acids, ethanolamine, etc.

In this example, as the protein component C, a minimum amount of transferrin and incibulin was added. Also selenite.

Pyruvate was used for purposes such as detoxifying harmful heavy metals and replenishing nutrient glands. Transferrin is 0.1-100 p
g/m+, 67 or O, I −] Ou g/
ml, insulin is 0.3-100 it g/m
+, preferably 1 to 10. czg/m+, selenite is 0.11-1O0x10-7. & Completed 0.5~
10XIOM, pyruvate is used in a concentration range of 0.1-100mM, preferably 0.5-20mM.

The obtained serum-free medium-conditioned cells of the present invention can induce interferon by being induced with an inducing agent. ↓−Starts to give birth. Accordingly, the present invention also provides a method for producing interferon using the cells.

Hereinafter, I will describe the method for producing terferon, and then culture it, dilute the obtained cell solution, or separate the cells and treat them with an fIAi' inducer in a fresh medium at a concentration suitable for interferon production. do. Prior to treatment with an inducing agent, the cells may be treated with a treatment agent, isolated and collected, and then resuspended and cultured in a fresh medium, which will be even more effective.

In this way, interferon is produced in the culture solution, and after a certain period of time, interferon production reaches a certain value, so
Collect this.

Regarding the treatment method using a treatment agent, when lymphoblastoid cells are used, treatment with a known inducing agent is performed for 24 hours to 4 hours.
Before 8 hours, soybean butylate, dimethyl sulfoxide (abbreviated as I) MSO), vitamin A, 5-
More preferable results can be obtained by treating with a treating agent such as bromodeoxyuridine alone or in combination with each other. After the cells were treated with the above-mentioned treatment agent, they were collected aseptically and resuspended in fresh medium.
-t o o o ttΔU/ml). The cell concentration may range from 1 to 20×10 cells/ml, but preferably 4 to 7×10 cells/ml.
is desirable.

After these induction treatments, the cells were cultured for 18 to 48 hours at 28 to 37°C to produce interferon, and the interferon titer reached its maximum value around time J (15 to 2 hours after induction).
Cells 1. Ship ti'l and collect interferon.

Interferon titer measurement was performed using human amniotic membrane 111 [vesicle W
IS II cells and VSV
This is done by the dye uptake method using Stomatitis virus).

Examples of the interferon production medium used in the present invention include R 11M I-1640 medium supplemented with penicillin, streptomycin glutamine, Hepes relaxation solution, etc., and Eagle M IF. M medium is suitable, and various serums are used.
% are attending. Furthermore, a serum-free medium is also effective in facilitating the purification and separation of interferon.

Use a serum-free medium and use insulin instead of serum in the bipedal medium, preferably 1-10 Mg/ml, trans7y. Phosphorus 0. 1
~10, O p g/ml, jiT or 1 ~10, Ll g/ml. ! lli selenic acid 0.1-100XIO M, preferably 0.5-l
OXI O-7M. Pyruvate triuno, 0.1
~l O (1mM, ~T or (1.5 - 2
Add 0 mM, optionally +lu tnsilbumin 0, 1-2 0 0 11 g/ml, &T
Lt. 1-50 Mg/ml is suitable. Furthermore, it is also good to add various kinds of rumons, vitamins, etc.

The interferon 4 system using the cells of the present invention can be used in blood cells.
1 when cultured with IIi-supplemented medium and virus induced.
When the concentration of 111 supernatant is low, there is an advantage that the potency is high when using a serum-free medium.

Examples of the present invention are shown below.

Example 1.

l) Serum-free medium/acclimation of I2 P M l- containing 0% calf fat blood li
1 640 medium (manufactured by J-Sui Seiyaku Co., Ltd.) was used as the basal medium, penicillin 25 U/m + Strep 1゛7, Isin 25 μ [
/n+1, glutamine 400+ng/l! ．． Burkinton's lymphoma-derived lymphoblastoid cell line Namaruha (ΔT C
CCRL-1432) cells at 1
0% (Vtl/V) IY beef blood soaked RPM t-164
0 medium as the basal medium, penicillin 251 μg/ml, streso1-mynon 25 μg/ml, glutamine 4 [1
0mg/II and IM Heves 1 fluid (pl+7.
0) I (]mM/ml (hereinafter referred to as N B
S 1.0) was inoculated into IO ml and cultured at 37°C for 84+ passages for 450 days. Passage 8x10 every 2 days
A total of 1111 cells were counted to give 5♀Ill II capsules/ml.

Except for reducing the serum concentration of iυ cells to 5%.
The cells were cultured for 4S in the same medium as described above (hereinafter referred to as FN13S5).Subculture was also carried out in the same manner.The serum concentration was adjusted to 3.
% (N13S 3), 15% (N13S], 5) and cultured in the same manner as described above and acclimatized for 1114 generations.

111 cells that had been acclimated to 1.5% serum-containing medium and had stable growth for 1J were acclimated to blood-free ti'l medium.

Serum-free medium contains 25 U/ml of penicillin and 2 ml of strebtomycin in RI) M1-1640q+ medium.
5μg/ml. Glutamine 400mg/I! , IM Heves buffer (p II 7. [1) I OmM/m
1. Imprin 3 μg/ml l.Lanceferrin 5 μg/ml 1. Serenite], serum-free medium containing 25XlOmM/ml pyruvate 5M/ml (hereinafter referred to as ``1'r
``I)S'' was used.

For conditioning of serum-free medium, 7 x 105 cells/ml of cells conditioned with medium containing 1.5% blood were inoculated into serum-free medium and incubated for 3
This was carried out by culturing the t+4th generation at 7°C for 450 minutes.

Passaging was carried out for 2 days at 7×] (+5 tm cells/…
I counted the cells in a bubble as shown in step 1.

Growth was stable and cell morphology was stable like the parent strain ♀1■
The cell was i5. One strain of these adapted cells was named IkuT-1 strain.

The regression of habituation is shown in Table 1 J0 Table 1 rcs 10 22-24- Ni3Si(l I(l
NB S 5 5 23-2545 NI333: (7,9xlP 24-2845NBS
1.51.5 --' 25-28451 TPS O
'6.2
1% (V/V) DM suspended in 10 ml of TPS medium
Add SO and l m M sodium butyrate, 37
The cells were cultured at ℃ for 24 hours. Centrifuge the cells (1,50 Or
p.m.

5 minutes) was collected and suspended in 10 ml of S medium, Mundai virus 500 IIΔLJ/ml was added, and after culturing at 37°C for 8 hours, the temperature was transferred to 28°C, and the interferon activity was measured after 14 hours. Examined. Interfaith 1
:I activity was determined using the method described above. l 1. (lOO
Interferon activity of LJ/ml was observed.

So Jhl! +1 row 3゜Interf-i +: Vitamin Δ sensitive NB53 conditioned cells for I production 4.56 × I (1” l'llll
Cells 7'ml, I<1'- cells 4xl [l'' cells/ml
,') J Example 2? , Nico.

1% I) Instead of treatment with M SOJ and sodium phthalate
1) M SO and 5 X I (] M 1.:l”y Min△i,
: J: Go to line 7 as in Example 2 except for performing Z+ processing.
,Ta. The results are shown in Table 2.1゜ff12Table Interferon Activity LJ/+nl DMS Former Sodium Butyle Il 50.000 11.
000D MSO+Vitamin/＼ ] 2. OO
(10 As a result, the K1'-1 strain was treated with vitamin Δ,
Example 4 It is clear that the interferon-producing ability is not expressed in the 1-N strain.
T) S medium and (r fetal bovine serum at 1.5 or 1
It was inoculated into a medium supplemented with 0% and cultured at 37°C for 20 hours.

After cultivation! Cell numbers were calculated using a blood cell 1139 plate.

The results are shown in Table 3.

Table 3 As a result, there is no difference in jf111II7 concentration or between the serum-added medium and the serum-free medium.

Example 5 Morphological characteristics of KD-1 strain N B s 10 ! KID cells and 1<']” −
] Comparison of the cell lines using electron microscopy was carried out as follows.

Each cell (1,500 r p + 1 m as a precipitate of r+
1) 4-0.1M:'M1.1fJii? & (pl
+7.0)] mIte? After 9UII+, fixed with 1 ml of 2% glucour 'J'ruthehyde-Cj'lil,
3. (1+10 r p rn ) Collect i++ cells by centrifugation for 0 minutes, add 1 ml of f'IILO1O, 1M phosphorous solution (pl+7.0) 3 times to 13L,
/1+ L, ta.

Add 1 ml of 1% osmic acid and incubate for 60 minutes at room temperature.
1, centrifuged in the same column A'1, and diluted with 61.0ml of 0.1 M IJ acidic acid (ptl 7.0). Purified. (HJ) 2111 cells were treated with horse chestnut 1 ethanol series (45%, 70%, 90%.

After dehydration using 100%) and air drying, ultrathin sections were sectioned and stained with uranium acetate under I00 KV.
Electron microscope manufactured by JEOL Ltd., JIMIOL-IQO
Observation was made using a C transmission type. On the other hand, for the scanning method, dehydrated and dried Kllll cells were deposited with gold by the smear method and observed and imaged using the same electron microscope.

Scanning electron microscopy images showed no difference in the cell surface, 1, or rectangular shape of both cells.

The section image obtained by the transmission method shows that the K i'-1 strain has very well-developed tochondria in the cytoplasm, with a size of 0.2 to 1.01 mm (average 0.51 mm and a length of 2 mm). It was found that it had a spherical or filamentous shape of ~5μ.
The fact that the i''-1 strain has a higher number of mido:1 andria indicates that the KT-1 strain has a strengthened energy system.

The N 13 S ] 0-acclimated cells were significantly different from the KT 1 strain in that the presence of numerous lipid droplets was observed.

Example 6゜1 < T-1 strain 5 cells 105 cells/...1 Ii'' PS medium 1 Y medium supplemented with bovine serum 1%, 5%, 10% (N) (
S 1゜5, I [1) and calf fat serum 1%,
The cells were inoculated into 5% and 10% supplemented medium (Fe2L, 5.IO) and cultured at 37°C for 21''. After culturing, 50% of the culture medium was replaced with the same medium, 200 IIΔLJ/ml of Hindu virus was added, and cultured at 37°C for 24 hours.

Interf... ff the results of I'ln production! 4 Not shown in table.

Table 4, I'l'PS 5,680 As a result, the K'r'-1 strain is 11+t? The lower the n concentration, the higher the interferonization) gamma ability (, no 11
The highest interferon production ability was shown in the 11-incubation medium (TIS').

Patent No. 8'1 Applicant (114) V Industrial Technology Director 1.11
Yube

Claims (1)

[Claims] (Does not proliferate in a medium that does not contain 1,1 blood In) Human 1 nonpaenchymal cells are acclimatized and grown in a serum-free medium.-C
11 new H. lintsuma blast cells. (2) Blood-free In medium is added to the basal medium - Nidinone, Strezo-1-mycin, Curtamine, Hepes km tIT
Li liquid. Insulin, transsoerin, ai! 1st L of the desired range = '1llI'I characterized by containing one type of sawdust selected from lenic acid and pyruvic acid and having the + sound jlL+
Human lymphoblast Lp 2m cell described in 3'j. (3) Basal medium is RI) M + - River 40. Ek'kuru MEM, Tarbesokou MEM, NO\J, l
1 in the blade ball with the hitorinno according to claim 2, characterized in that it is separated from the ham F12.
1 cell. (4) Old serum-free medium) M + -1 [i 40
Culture medium 1 with selenite and pyruvate added [! The human lymphoblastoid cell according to claim 2, characterized in that the lymphoblastoid cell is H.I. (5) The human lymphoblastoid cell according to claim 1, characterized in that it has the ability to produce interferon upon virus induction. (6) The human lymphoblastoid cell according to claim 5, characterized in that its ability to produce interferon induced by a virus is sensitive to vitamin λ. (7) Lymphoblast cells that do not proliferate in a serum-free medium are cultured in a medium in which the serum concentration is gradually lowered, and finally cultured in a blood-free medium. ■Acclimation of the cells so that they can grow in a serum-free medium - 1 Lymphoblastoid II cells that can grow in a serum-free medium! l manufacturing method. (8) Serum-free medium contains penisoline, S-I-resol-1-mycin, glutamine, and '\Bestt'A f+T in the basal medium.
j7f1. Ink purine, transferrin, selenite 4, 1
8. The preparation method according to claim 7, characterized in that the medium contains one selected from , and pyruvate. (9) The preparation method according to claim m8, wherein the basal medium is selected from RPMI--1640, C-glu MEM, Darbenokou MEM, Hano, F]O, and Ham F12. 00) Serum-free medium is RI) M I-1640 with benicillin, streptoficin, glutamine, and Hevenu H
The preparation method according to claim 7, characterized in that the medium is a medium to which insulin, transferrin, selenite and pyruvate are added. Human lymphoblastoid cells, which have been adapted to allow blast cells to proliferate in a serum-free medium, are transformed into cells that have the ability to induce interferon by virus induction,
A method for producing iterferon, which comprises culturing it in a medium, accumulating interferon in the C1 culture, and collecting interferon from the culture.