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B lymphocyte in vitro culture system and applications thereof

An in vitro culture system and technology of B lymphocytes, applied in the direction of cell culture active agents, animal cells, tissue culture, etc., can solve the problems of long development cycle, easy loss, unstable coding antibody gene, etc., and achieve high biological activity effect

Active Publication Date: 2020-08-11
优睿赛思(武汉)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is still widely used in many fields such as scientific research, clinical diagnosis and drug development, its limitations are obvious, mainly in: 1) the development cycle is long, generally takes 4-6 months; 2) The gene encoding the antibody in the obtained hybridoma cells is unstable: after long-term propagation, it is easy to lose, and loses the function of secreting antibodies; 3) It is not universal: the development of myeloma cells is time-consuming and laborious. Rats and rabbits have corresponding myeloma cells, and rabbit myeloma cells are still in the patent protection period, and other companies are not available except Abcam

Method used

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  • B lymphocyte in vitro culture system and applications thereof
  • B lymphocyte in vitro culture system and applications thereof
  • B lymphocyte in vitro culture system and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Detection of interleukin levels secreted by trophoblast cells after gamma ray treatment

[0040] On the first day, the HuT78 cells used as trophoblasts were mixed at 0.3X 10 6 The density is in the basal medium of RPMI-1640+10% fetal bovine serum, and placed in an incubator with 5% CO2 concentration at 37°C for cultivation.

[0041] On the second day, phorbol-12-tetradecanoyl-13-acetate (Phorbol Myristate Acetate, PMA) was added to a final concentration of 10 ng / ml, and culture was continued for 24 hours.

[0042] On the third day, HuT78 cells were irradiated with 4500 RAD of gamma rays. After treatment, according to the concentration required by the trophoblasts (2.5X 10 5 cells / ml) to adjust the HuT78 cells and continue to culture.

[0043] After culturing for 24 hours, 48 ​​hours, 7 days and 10 days, the culture supernatants were collected respectively, and the IL-2 molecule sandwich ELISA detection kit (purchased from Abotec Biotechnology, catalog numbe...

Embodiment 2

[0046] Embodiment 2 rabbit B lymphocyte culture system

[0047] This embodiment provides a culture system for rabbit B lymphocytes expressing rabbit IgG molecules on a single culture surface in a 96-well U-bottom cell culture plate, including: trophoblast cells treated with gamma ray irradiation, IL- 21. Human CD40L trimeric protein (purchased from Kaijia Biotechnology, catalog number: CDL-HM140) and basal medium. Among them, rabbit B lymphocytes expressing rabbit IgG molecules on the surface are isolated from rabbit spleen cells (the method is the same as the technical scheme in patent number ZL20191012091.4). Trophoblast cells after gamma ray irradiation treatment (treatment conditions under 3 conditions in the same embodiment 1), concentration is 2.5 × 10 5 cells / ml. The final concentration of IL-21 was 50 ng / ml, and IL-21 was human interleukin 21 protein. The human CD40L trimer protein concentration was 0.75ug / ml. The basal medium is RPMI-1640 medium containing 15% fet...

Embodiment 3

[0069] Embodiment 3 alpaca B lymphocyte culture system

[0070] This embodiment provides a culture system for alpaca B lymphocytes expressing alpaca IgG molecules on a single culture surface in a 96-well U-shaped bottom cell culture plate, including: trophoblast cells ( Same as the treatment condition of 3 in Example 1), IL-21, human CD40L trimeric protein and basal medium. Among them, the alpaca B lymphocytes expressing alpaca IgG molecules on the surface are isolated from the peripheral blood lymphocytes of alpacas (the method is the same as the technical scheme in the patent No. ZL20191012091.4), and the trophoblast after gamma ray irradiation treatment cells at a concentration of 2.5X 10 5 cells / ml. The final concentration of IL-21 was 50 ng / ml. IL-21 is human interleukin 21 protein. The human CD40L trimer protein concentration was 0.75ug / ml. The basal medium is RPMI-1640 medium containing 15% fetal bovine serum and 1% penicillin / streptomycin; the culture conditions a...

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Abstract

The invention belongs to the technical field of primary cell culture, and especially relates to a B lymphocyte in vitro culture system and applications thereof. The culture system utilizes a T lymphocyte line capable of secreting interleukin molecules such as IL-2 as trophoblastic cells so as to provide micro interleukin molecules required by the growing development of B lymphocyte; and trimer CD40L molecules expressed in vitro through a recombinant manner and having high biological activity are added as important molecules so as to further stimulate the growth, reproduction and development ofthe B lymphocyte. The system can also perform gamma ray irradiation treatment on the trophoblastic cells, so that the pre-treated trophoblastic cells can lose the ability of cell reproduction but still has the ability of protein synthesis and secretion within 7-14 days. The provided system can effectively culture the B lymphocyte derived from rabbits and alpacas in vitro, and can utilize the method to develop corresponding monoclonal antibodies.

Description

technical field [0001] The invention belongs to the technical field of primary cell culture, and in particular relates to an in vitro culture system and application of B lymphocytes. Background technique [0002] With the development of modern biology, scientists have learned that antibodies in humans or animals are produced and secreted by B lymphocytes in the spleen or peripheral blood lymphocytes and plasma cells developed therefrom. Therefore, if a single B lymphocyte can be obtained, a uniform and specific antibody to a specific antigenic substance, that is, a monoclonal antibody, can be obtained from it. [0003] Since the B lymphocytes capable of producing corresponding antibodies cannot reproduce infinitely in vitro, the current development of monoclonal antibodies mainly relies on the hybridoma technology created by molecular biologists G.J.F. Koehler and C. Milstein in 1975. The main principle of this technology is to develop a myeloma cell (such as mouse myeloma ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/10C12P21/08
CPCC07K16/00C07K2317/14C07K2317/20C07K2317/22C07K2317/569C12N5/0635C12N2501/2302C12N2501/2321C12N2501/52C12N2502/99
Inventor 娄阳吴海
Owner 优睿赛思(武汉)生物科技有限公司
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