JPS62126971A - Mouse natural killer cell strain - Google Patents

Abstract

PURPOSE:The titled cell strain, obtained by establishing specific mouse lymphatic cells, dependent on interleukin 2, in agreement with normal natural killer cells in properties the aspect of properties and functions and having stable properties. CONSTITUTION:A retro-virus, e.g. Kirstein mouse sarcoma virus (Ki-MSV), etc., is transformed in an animal cell, e.g. mouse fibroblastic cell, etc., and a suspension of the transformed animal cells is inoculated into subcutaneous tissues of the back part of a mouse. When about 2-3 weeks are passed, lamphatic tissues of the mouse lymphatic cells are excised and finely cut to give mouse lymphatic cells, e.g. mouse splenic lymphocytes, etc., which are then mixed with a culture medium containing interleukine 2 and cultivated in a CO2 incubator to afford the titled cell strain having the same surface marker as normal natural killer and cytotoxic ability with good reproducibility.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a natural killer cell line established from mouse lymphoid cells.

(Prior Art) The host defense system of mammalian mammals can be broadly divided into two types: acquired immunity and natural resistance. acquisition (q immunity is B cells and T
whereas innate resistance is borne in part by a class of cells, the various peripheral blood mononuclear cells. This population, called null cells, has hematopoietic progenitor cells, natural killer (NK) activity, antibody-dependent cytotoxicity, and a wide range of tumor cells, virus-infected cells, and undifferentiated cells derived from normal tissues. These cells are toxic. Morphologically, NK cells are large granular lymphocytes (LGLs), but they express cell surface markers characteristic of both cells and monocytes.

Recently, as the importance of NK cells in cancer immunity has become clearer, many laboratories in the financial world are conducting vigorous research into the properties and differentiation of NK cells, and their ability to kill tumor cells. There is. However, because NKl cells are a very small population of lymphocytes, it is difficult to understand their detailed characteristics. To elucidate this, there is an urgent need for N-specific proliferation of cells and cloning of NK cells. Using these NK cells, NK
It is believed that advancing research on cell-derived cytotoxic factors and cancer immunity will greatly contribute to the development of cancer immunotherapy.

On the other hand, there have already been several reports on the cloning of NK-like cells using various condition media (CM), purification, and interleukin-2 (IL-2), but their stability and the normality of their surface markers have not been confirmed. Abnormalities with NK cells have become a problem.

[Problem that the invention seeks to solve]

The purpose of the present invention is to obtain a NK cell line that is identical in properties and functions to normal NK cells and has stable properties.

[Means for solving problems]

The present invention relates to IL cells established from mouse lymphoid cells sensitized with retrovirus-transformed animal cells.
-2-dependent murine NK cell line with substantially the same surface markers and cytotoxic ability as normal NK cells.

The virus used in the present invention includes Rous sarcoma virus, simian sarcoma virus, Fujisei sarcoma virus, Y73 sarcoma virus, Gardner-Rashid feline sarcoma virus,
UR2 avian sarcoma virus, McDonough sarcoma virus, avian erythroblastosis virus, Abelson murine leukemia virus, Shichiro Ni-murine sarcoma virus, 3611 murine sarcoma virus, holiclass endotheliomatosis virus, Harvey murine sarcoma virus, Kirsten mouse Sarcoma virus, MC29 myelocytosis virus, FBJ osteosarcoma virus, and SKi 9 sarcoma virus (--MSV) are preferably used.

Methods for transforming animal cells using retroviruses include, for example, the method of Aaronson et al.
Aronson & W. P. Rowe.

Virolooy 42.9-19 (1970)).

Mouse fibroblasts are preferably used as animal cells depending on the relationship with the retrovirus used.

Mouse lymphoid cells include various lymph node-derived cells,
Although there are tile cells, bone marrow cells, thymocytes, peritoneal exudate liver-derived lymphocytes, and peripheral blood lymphocytes, tile cells are preferably used in the present invention.

Sensitization is usually carried out as follows.

First, a retrovirus-transformed cell suspension is prepared, for example, from 1×10e to 1×1011IIB cells/−
Prepare at a concentration of As a liquid for suspending cells, Dulbecco, Matskoi, Hamm or RPMI-1640 medium, Hank's buffered saline (HBSS), phosphate buffered saline (PBS), etc. can be used.

The cell suspension thus made into 111ml is usually 0.05ml
~0.1 d/mouse: Inoculate subcutaneously on the back of the mouse.

As mice, BALB/C, C57BL/6, C3
H/He 1DBA/2, CBA/J, BDF+, C
DF+, B6CF+, etc. are used. After vaccination, about 2
Excise the lymphoid tissue of the mouse through ~3 holes and cut it into small pieces to obtain lymphoid cells.

The lymphoid cells were collected at a final concentration of 50-500 U/Id.
Add 1x105 to 1x1 to the medium containing L-2 so that
The mixture was mixed to a density of 0.07 cells/d, and culture was started at 37°C in a carbon dioxide gas incubator. As a medium, for example, RPM
I-1640 medium etc. can be used. After starting the culture and the cell concentration has increased, add L-2 with a maximum IS degree of 10 to 200 U/1e(7) every few days, and
Feed for 3 weeks. At that time, the remaining cells are collected by centrifugation, mixed again with the above-mentioned medium, and culture is continued. The culture is then scaled up as the cells grow.

By culturing in this manner for about 90 days, NK cell lines can be obtained with good reproducibility.

The NK cell line of the present invention obtained as described above has the following characteristics.

(A) Cell morphology and characteristics: ■ It is LGL that has many azurophilic granules in the cytoplasm.

■ Surface markers are 7hy1'', Lytl-2-, Asialo GM +°, T2O0゛, FCRo.

■ Rat concanavalin A (Con△>-CM (IL
-2 activity) and human recombinant l L-2 (r
-h I L2>dependent growth.

■ It successfully killed NK-sensitive tumor cells, RL♂1 and YΔC-1 cells, and virus-infected cells, HeLa-measle, but did not kill NK-resistant EL-4.

(B) Cultured cell density: 1X10' cells/10-medium (The medium is RPMI-1640 medium 8d.

Fetal bovine serum (Fe2) 1m and rat ConA-CM
lie (200U I L-2) or r-
hlL-21d (2000U IL-2). ) (C) Subculture: Subculture is possible without limit.

When cultured in a 37° C. carbon dioxide gas incubator using 1xio' cells/10 relatives medium, subculture is usually performed every 2 to 3 days.

(D) Storage: Sufficiently suspend 5×10ε cells in a mixture of 10% dimedyl sulfoxide and 90% beef tallow serum, then program.
Freeze at -80°C using a freezer. This is -19
It can be stored permanently by placing it in liquid nitrogen at 6°C.

EXAMPLES Hereinafter, the present invention will be briefly and specifically explained with reference to Examples.

〔Example〕

k'.

Peel with trypsin and centrifuge wash once with PBS. After washing, resuspend in PBS at 2×10′ cells/d. The cell solution thus prepared is subcutaneously inoculated on the back of a BALB/C mouse (4W, male) at 0.1 m/mouse. Three weeks after inoculation, the spleen was removed from the mouse.
Obtain finely chopped tile cells (lymphocytes). This tile lymphocyte 2
00U/ai! r-hlL2.10%FC8 and 2x
RPM>16 with 10'M2-mercaptoethanol
2X10' cells/lll1 in 40 medium! Mix with sea urchin and start culturing in a carbon dioxide incubator at 37°C. One week after starting the culture, add 100U every 2.3 days.
/lll1 of r-hlL-2 is added and cultured for 3 weeks. At that time, the remaining cells were collected by centrifugation, mixed with the above-mentioned medium again, and culture continued.

The culture was then scaled up as the cells grew. By culturing in this way for 90 days, the NK cell line (
SPB2-4> was established. Similarly, C57BL/
B6SP-2 from 6 mice and D from DBA/2 mice.
SP-2 was established.

Table 1 summarizes the results of examining the properties of these cell lines.

Further, the results of investigating the functional characteristics of 5P82-4 are shown in FIGS. 1, 2, and 3.

Below, remaining white Table 1

[Brief explanation of drawings]

Figures 1, 2 and 3 do not show the functional characteristics of SPB:2-4, the NK cell line of the present invention. Applicant: Toshi Co., Ltd. Cell count (/mJ) ■-Q - XX X X Cell count (
/m/)

Claims (2)

[Claims] (1) Mouse natural killer that is interleukin-2 dependent and has substantially the same surface markers and cytotoxic ability as normal natural killer cells, which is established from mouse lymphoid cells sensitized with retrovirus-transformed animal cells. cell line. (2) The cell line according to claim (1), wherein the retrovirus is Kirsten mouse sarcoma virus, the animal cells are mouse fibroblasts, and the mouse lymphoid cells are mouse splenic lymphocytes.