Composition for culturing lymphocytes of laboratory rats and culture method thereof

A technology of lymphocytes and experimental mice, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve problems such as expensive medium, higher requirements for culture technology, and inability to meet cell culture requirements

Pending Publication Date: 2020-10-30
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cao Hongguo et al. (CN201210195070.8) induced stem cells through the fusion of mouse lymphocytes and embryonic stem cells (ES), so that the lymphocytes proliferated in large quantities, but the medium for stem cell culture was expensive and required higher culture techniques. Some laboratories cannot meet such cell culture requirements

Method used

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  • Composition for culturing lymphocytes of laboratory rats and culture method thereof
  • Composition for culturing lymphocytes of laboratory rats and culture method thereof
  • Composition for culturing lymphocytes of laboratory rats and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Acquisition of Spleen Single Lymphocyte

[0032] (1) Clean SD rats with a body weight of about 300g and / or clean BALB / c mice with a body weight of about 25g, CO 2 Killed by cervical dislocation after suffocation, soaked in alcohol solution with a volume fraction of 75%, flipped SD rats and / or BALB / c mice with tweezers during the soaking process, so that the alcohol solution fully contacted the body surface and hair of the mice, 5 After -10 minutes, the SD rats and / or BALB / c mice were taken out and placed in a sterile tray, the abdomen was wiped dry with sterilized cotton balls, and the spleen was aseptically taken out.

[0033] (2) The removed spleens were placed in 6 cm-diameter petri dishes, which contained 2 mL of RPMI-1640 medium pre-cooled to 4°C. Place two 6cm Petri dishes on a wide-mouthed container filled with crushed ice to maintain the environment of the isolated spleen at 4°C. This experiment should be kept in a low temperature environment (recomm...

Embodiment 2

[0038] Example 2 Spleen Lymphocyte Classification

[0039] (1) Get the single lymphocyte obtained, press 10 with 75% ethanol or 4% paraformaldehyde 6 cells / ml to prepare a lymphocyte suspension and fix overnight at 4°C.

[0040] (2) Take the fixed cells, wash them twice with PBS, remove the fixative completely, add FITC-CD3 and PE-CD19 monoclonal antibodies respectively, stain at room temperature for 30 minutes, wash twice with PBS, and remove unbound fluorescent antibodies .

[0041] (3) Lymphocytes after the above staining, in PBS by 10 5 cells / ml to prepare the cell suspension, and analyze the ratio of CD3+ and CD19+ cells by flow cytometry, representing the percentages of T lymphocytes and B lymphocytes, respectively.

[0042] (4) The test results showed that the ratio of T cells and B cells before spleen lymphocyte culture was (T / B): 0.87±0.09%.

Embodiment 3

[0043] Embodiment 3 culture medium optimization

[0044] The spleen lymphocyte culture medium in this embodiment includes RPMI-1640 medium, and concanavalin A, phytohemagglutinin, β-mercaptoethanol, mannitol, sodium azide, fetal bovine serum, sodium pyruvate, L-glutamine Amides, non-essential amino acids, penicillin-streptomycin and dimethyl sulfoxide and other substances, there is no sequence requirement for the addition of various substances. The ratio of different substances in the following table is used to optimize the medium, and the initial number of cultured cells is 5×10 6 / ml. After culturing for 7 days, count the cells according to the counting method mentioned above, classify the cells according to Example 2, and calculate the ratio of T and B cells. The culture results showed that the amplification factor of combination 5 in Table 1 could reach 10.67 times, and the ratio of T / B lymphocytes was 0.93±0.03%, which was the best combination. There was no significant...

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Abstract

A composition for culturing the spleen lymphocytes of laboratory rats comprises an RPMI-1640 culture medium, and concanavalin A, phytohemagglutinin, beta-mercaptoethanol, mannitol, sodium azide, dimethyl sulfoxide and the like are further added into the RPMI-1640 culture medium. Test results prove that the composition can achieve the purpose of maintaining in-vitro long-term proliferation of rat lymphocytes, the number of lymphocytes required by fundamental researches can be achieved through proper proliferation, and the ratio of T cells to B cells is kept unchanged.

Description

technical field [0001] The invention relates to a cell culture technology, in particular to a composition for culturing spleen lymphocytes of experimental mice, and a method for cultivating lymphocytes thereby improving the proliferation ability of spleen lymphocytes of experimental mice. Background technique [0002] The spleen is an important filtering organ in the blood circulation of animals and the largest lymphatic organ in the body. Among them, about 40% of T lymphocytes and about 60% of B lymphocytes participate in cellular immunity and humoral immunity respectively. The spleen plays a very important role in the establishment of normal immune function and the enhancement of immune function. The in vitro proliferation of spleen immunocompetent cells, especially the in vitro proliferation of lymphocytes, is an important basis for studying the enhancement of the immune function of the body. Rats and mice are the most commonly used experimental animals in biological an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
CPCC12N5/0635C12N5/0636C12N2501/515C12N2501/2302C12N2501/998C12N2500/44C12N2500/35C12N2500/62C12N2501/999C12N2500/32
Inventor 李超李娅南何凌云阮烨娇王亚楠郭晓令毛百萍
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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