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Culture medium for lymphocyte culture and application thereof

A technology of lymphocyte and culture medium, applied in the field of culture medium for lymphocyte culture, can solve the problems of high price, limit wide application, increase the probability of allergic reaction, etc., and achieve the effect of guaranteed effect.

Active Publication Date: 2013-11-27
SHENZHEN PREVENTION & TREATMENT CENT FOR OCCUPATIONAL DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of using animal serum are as follows: the quality of animal serum is unstable between batches; the introduction of foreign proteins and anti-cell antibodies inhibits the function of lymphocytes; when used in cellular immunotherapy, there is a potential risk of spreading diseases, and foreign proteins increase the risk of allergic reactions probability
[0004] In view of the disadvantages of animal serum, serum-free medium has emerged in recent years. By adding additional cytokines, hormones, antioxidants, etc. to replace serum, commercial serum-free medium suitable for various cell cultures has emerged, but it is expensive. The selling price limits its wide application

Method used

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  • Culture medium for lymphocyte culture and application thereof
  • Culture medium for lymphocyte culture and application thereof
  • Culture medium for lymphocyte culture and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, measure lymphocyte transformation rate

[0020] Preparation of Medium-1: Add mixed nucleotides (mixture of 5'-AMP, 5'-CMP, 5'-GMP and 5'-TMP), PHA, heparin sodium and Genta in PRMI-1640 culture medium Mycin, so that the concentration of each nucleotide in the mixed nucleotides is 20mmol / L, the concentration of PHA is 160μg / mL, the concentration of heparin sodium is 60IU / mL, and the concentration of gentamicin is 0.1mg / mL .

[0021] Preparation of Medium-2: Add AB type human blood plasma, mixed nucleotides (mixture of 5'-AMP, 5'-CMP, 5'-GMP and 5'-TMP) to PRMI-1640 culture medium, PHA, heparin sodium and gentamicin, so that the plasma concentration is 5% (volume ratio), the concentration of each nucleotide in the mixed nucleotide is 20mmol / L, the concentration of PHA is 160μg / mL, and the concentration of heparin sodium The concentration is 60IU / mL, and the concentration of gentamicin is 0.1mg / mL.

[0022] Preparation of Medium-3: Add AB type human blood ...

Embodiment 2

[0029] Embodiment 2, measure the division index of lymphocyte

[0030] Prepare eight culture media in turn, the only difference from the eight culture media in Example 1 is the addition of colchicine (to make the cells in the mid-division phase, which is convenient for counting the division index) and make the concentration 7.2×10 -8 mmol / mL.

[0031] Take 2×10 6 The lymphocytes of volunteers with blood type AB were inoculated in 4 mL of various culture media respectively, and placed at 37 ° C, 5% CO 2 Cultivate in the incubator for 52 hours, transfer the culture into a 15mL centrifuge tube, add 4mL of hypotonic solution (0.075mol / L KCl aqueous solution) and shake well, let it stand for 3min; add 1mL of fixative solution (mixture of methanol: acetic acid = 3:1 solution; volume ratio), shake well, centrifuge at 1200rpm / min for 5 minutes, discard the supernatant; add 8mL fixative solution, shake well, let stand for 30min, centrifuge at 1200rpm / min for 5 minutes, suck off the s...

Embodiment 3

[0032] Embodiment 3, peripheral blood lymphocyte micronucleus analysis

[0033] Lymphocytes were isolated from the peripheral blood of 30 volunteers (with random blood types).

[0034] Preparation of Medium-I: Add AB type human blood plasma, mixed nucleotides (mixture of 5'-AMP, 5'-CMP, 5'-GMP and 5'-TMP) to PRMI-1640 culture medium, PHA, heparin sodium and gentamicin, so that the plasma concentration is 10% (volume ratio), the concentration of each nucleotide in the mixed nucleotide is 5mmol / L, the concentration of PHA is 160μg / mL, and the concentration of heparin sodium The concentration is 60IU / mL, and the concentration of gentamicin is 0.1mg / mL.

[0035] Preparation of medium-Ⅱ: Add fetal bovine serum, PHA, heparin sodium and gentamicin to PRMI-1640 culture medium, so that the concentration of fetal bovine serum is 20% (volume ratio), and the concentration of PHA is 160μg / mL , the concentration of heparin sodium is 60IU / mL, and the concentration of gentamicin is 0.1mg / mL...

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Abstract

The invention discloses a culture medium for lymphocyte culture and application thereof. The culture medium for lymphocyte culture provided by the invention comprises a basic medium, mixed deoxymononucleotides and human blood plasma.

Description

technical field [0001] The invention relates to a medium for lymphocyte culture and its application. Background technique [0002] In vitro lymphocyte culture is not only the traditional research method of cytogenetics, but also the basis for the realization of emerging technologies such as autologous cell immunotherapy. [0003] In order to simulate the growth environment of lymphocytes, in addition to maintaining physiological osmotic pressure and pH value, the medium needs to add various nutritional factors required for cell growth, such as inorganic salts, carbohydrates, amino acids, vitamins, trace elements, etc. The addition of animal serum has always been necessary for the in vitro culture of lymphocytes. Fetal bovine serum and newborn bovine serum are commonly used. Serum plays an important role in traditional lymphocyte culture methods. In addition to the role of nutrient enrichment, it is more important It contains growth factors, hormones and some trace nutrients...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
Inventor 惠长野李智民张文高朝贤陈郁筠黄先青陈钰婷张健杰
Owner SHENZHEN PREVENTION & TREATMENT CENT FOR OCCUPATIONAL DISEASES
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