Lymphocyte culture medium with stable pH value and preparation method and application thereof

A technology of lymphocytes and culture medium, applied in the field of cell biology, can solve problems such as unfavorable medium preservation and pH value increase, and achieve the effect of being conducive to long-term preservation and stabilizing pH value

Active Publication Date: 2015-10-21
广州达晖生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sodium bicarbonate buffer system is easily affected by carbon dioxide in the environment. As the storage time prolongs, the pH value increases, which is not conducive to the storage of the medium.

Method used

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  • Lymphocyte culture medium with stable pH value and preparation method and application thereof
  • Lymphocyte culture medium with stable pH value and preparation method and application thereof
  • Lymphocyte culture medium with stable pH value and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: the culture medium effect comparison of different formulations

[0017] 1. Prepare basal medium with added serum: 10.5g 1640 basal medium, 0.1g PHA, 200ml calf serum, dilute to 1L with ultrapure water.

[0018] 2. Prepare medium 1: take 1 portion of basal medium, add 2g / L NaHCO 3 .

[0019] 3. Preparation medium 2: take 1 portion of basal medium and add 2g / L Na 2 HPO 4 , 1g / LKH 2 PO 4 .

[0020] 4. Prepare medium 3: Take 1 part of medium 2, add 9g / L sodium glycerophosphate.

[0021] 5. Prepare medium 4: Take 1 portion of medium 2 and add 6g / L HEPES.

[0022] 6. Preparation of medium 5: take basal medium 2, add 3g / L sodium glycerophosphate and 3g / L HEPES.

[0023] 7. The above culture medium was adjusted to pH 7.2 with hydrochloric acid / sodium hydroxide solution or flushed with carbon dioxide.

[0024] 8. Sterilize by filtration with a 0.22 μm membrane filter.

[0025] 9. Peripheral blood lymphocyte culture and chromosome preparation:

[0026] (1...

Embodiment 2

[0048] Example 2: pH Change Determination

[0049] Place the above medium 1, medium 2, medium 3, and medium 4 at 37°C, at room temperature, and at 4°C, and test the change of pH value. Table 3 is a comparison of the pH stability performance of various formulation media.

[0050] table 3

[0051]

[0052]

[0053]The results in the table show that the pH value of the non-sodium bicarbonate buffer system is smaller than that of the sodium bicarbonate buffer system, and the pH value of the sodium glycerophosphate / HEPES double buffer system is the most stable. It can be stored for 4 months at 4°C. constant.

Embodiment 3

[0054] Embodiment 3: the preparation of preferred lymphocyte culture medium of the present invention

[0055] 1. Materials: HEPES, Na 2 HPO 4 、KH 2 PO 4 Provided by Shanghai Sangong Engineering Biology;

[0056] Calf serum, provided by Hangzhou Sijiqing;

[0057] Sodium glycerophosphate, provided by Sigma;

[0058] 1640 dry powder, provided by GIBCO.

[0059] 2. Equipment: pH meter, stirrer.

[0060] 3. Method for preparing 1L lymphocyte culture medium:

[0061] Weigh each component according to the mass: PRMI-1640 dry powder 10.45g medium, 0.1gPHA, 2gNa 2 HPO 4 , 1gKH 2 PO 4 , 3g sodium glycerophosphate, 3g HEPES, add 500ml ultrapure water, 200ml calf serum; hydrochloric acid / sodium hydroxide solution or flush with carbon dioxide to adjust the pH to 7.2; finally dilute to 1L with ultrapure water and filter with a 0.22μm filter membrane Sterilize.

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Abstract

The invention provides a culture medium with a stable pH value. The culture medium is composed of a 1640 base culture medium, a 100 mg/L phytohemagglutinin (PHA), a calf serum with the volume percentage of 20% and a 5-50 mM sodium glycerophosphate/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) dual-buffering system. The sodium glycerophosphate/HEPES dual-buffering system is composed of a phosphate buffer solution, sodium glycerophosphate and 4-HEPES, wherein the phosphate buffer solution contains Na2HPO4 and KH2PO4. The invention also provides a preparation method of the culture medium and an application of the culture medium in lymphocyte culture. The pH value of the culture medium is not affected by gas composition in the environment, and the pH value during the lymphocyte culture also can be stabilized; and long-term preservation of the culture medium is facilitated.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a lymphocyte culture medium with stable pH value and its preparation method and application. Background technique [0002] Genetic diseases refer to diseases caused by changes in genetic material or controlled by disease-causing genes. Genetic diseases involve many disciplines, including obstetrics and gynecology - infertility, eugenics, endocrine disorders; hematology - chronic myelogenous leukemia, acute myelogenous leukemia, myelodysplastic syndrome, acute lymphocytic leukemia etc.; Pediatrics - Birth Defects; Oncology; Internal Medicine - Radiation Sickness, Chromosomal Breakage Syndrome, etc., which have the characteristics of congenital, lifelong and familial, many diseases, and high incidence. Common clinical hypertension, diabetes, asthma, cancer, depression, Alzheimer's disease, etc. are related to heredity. [0003] At present, the diagnosis of genetic diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0781C12N5/0783
Inventor 李晓辉胡可存刘强
Owner 广州达晖生物技术股份有限公司
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