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Human peripheral blood lymphocytes culture medium and application thereof

A technology of lymphocytes and liquid culture medium, applied in the field of human peripheral blood lymphocyte culture medium, can solve the problems of inability to achieve lymphocyte growth and proliferation, mitotic expression, interference with cultured lymphocyte growth, lack of reasonable and effective ratio, etc., to increase electrolytes Diffusion and movement, increase cell permeability, and vigorous cell growth

Active Publication Date: 2009-10-07
湖州尧浩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the existing medium formula used in conjunction with this natural medium and synthetic medium can make some cells survive, it cannot achieve better growth and proliferation of lymphocytes and perform mitotic expression, and lacks reasonable and effective components. However, the natural medium belongs to animal serum and there are many unfavorable factors that interfere with the growth of cultured lymphocytes, so it cannot directly and effectively simulate the environment in human blood, and the stability of the reagent is not good.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 culture medium

[0030] The culture medium was formulated according to the formula in the table below.

[0031] The preparation method of culture medium is: 1. under aseptic condition, the RPMI 1640 dry powder of synthesis is weighed on the electronic balance and placed in the post-sterilized container, add double distilled water to dilute and mix well; 2. adopt Pasteurization (pasteurization 3. Add PHA to the culture medium; 4. Add HA to the culture medium; 5. Add CPPs to the culture medium; .7. Use a pH meter to measure the pH value.

[0032] 1#

[0033] After the culture medium was prepared, it was sterilized by negative pressure filtration, and divided into 10ml culture bottles with 5ml of culture medium in each bottle, sealed and refrigerated (-20°C) for later use.

Embodiment 2

[0034] Example 2 Comparison of cell culture medium lymphocyte conversion rate and lymphocyte division index

[0035] The medium prepared in Example 1 was used.

[0036] Comparative medium formula: RPMI 1640 or 199 84.9%, calf serum 10%, PHA 3%, heparin 2%, double antibody 0.1%.

[0037] Training method:

[0038] Blood collection, inoculation and culture: Disinfect the bottle cap with 2.5% iodine and 75% alcohol, burn it with an alcohol lamp flame, extract 0.5-1ml of peripheral blood from the patient under sterile conditions, add about 20 drops of medium to each bottle, and shake evenly , try to avoid contact between the culture and the bottle cap; culture in a 36°C incubator for 72 hours, and shake gently once every 12 hours or so to promote cell growth and proliferation;

[0039] Cell culture: Add colchicine about 2 hours before harvesting, the final concentration is 0.02ug / ml culture solution, shake well and place in the incubator to continue culturing to inhibit the cells...

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Abstract

The invention relates to a human peripheral blood lymphocytes culture medium and the application thereof. The human peripheral blood lymphocytes culture medium of the invention comprises: RPMI1640 liquid culture medium 77-81.9%, bovine blood 10-14.9%, PHA 4-9%, non-sulfated glycosaminoglycan 2-5%, CPPs 2-5% and Double-resist 0.05-0.1%. The culture medium provided in the invention achieves a high lymphocyte conversion rate, a high lymphocyte split index and a good reagent stability; can obtain a human chromosome figure which can provide a firm basis to chromosome core type analysis and clinic diagnosis.

Description

technical field [0001] The invention relates to a human peripheral blood lymphocyte culture medium and its application. Background technique [0002] The small lymphocytes in human peripheral blood are almost in the GI phase (GO phase), and generally do not divide anymore. When phytohemagglutinin PHA is added to the culture medium, these small lymphocytes are stimulated to transform into lymphocytes. Mother cells then enter mitosis, so after short-term culture, colchicine treatment, hypotonicity and fixation, some mitotic cells can be obtained. This method has been widely used in clinical medicine, virology, pharmacology, genetic toxicity and so on. Studies have confirmed that many factors are closely related to the production of cell culture, and the common reasons are: [0003] (1) Pollution-free environment: The non-toxic and sterile culture environment is the primary condition to ensure the survival of cells. When cells are cultured in vitro, compared with in vivo cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08
Inventor 崔贻芬
Owner 湖州尧浩生物科技有限公司
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