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Culture method of bone marrow cells and application of culture method

A culture method and technology of bone marrow cells, applied in the field of cell culture, can solve the problems of diagnosis basis, large deviation of diagnosis results, failure to use bone marrow, etc.

Pending Publication Date: 2020-11-10
UNIMED BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, it can be seen that the cell suspension obtained after centrifugation is rarely enough to meet the demand of the droplet, and the diagnostic results are large and unstable, which cannot be used as the basis for bone marrow diagnosis.

Method used

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  • Culture method of bone marrow cells and application of culture method
  • Culture method of bone marrow cells and application of culture method
  • Culture method of bone marrow cells and application of culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 whole blood culture

[0063] Draw 380μl white blood cell diluent with the sampling gun, then take a small amount of sample with a 1ml syringe and drop 2-3 drops on the plastic paper, draw 20ul of blood; it is used for bone marrow nucleated cell count.

[0064] When counting cells, obtain the average number N of the large grid, then 50÷N=the number of milliliters of bone marrow blood in the 5ml culture medium added, or 3600÷N=the number of drops of bone marrow blood in the 5ml culture medium that should be added (to make the cells The concentration is 1-2×10 6 / ml). The following formula can be used in actual operation: N≤50, inoculate 1.0ml; 50100, 3600÷N=5ml culture should be added The number of drops of bone marrow blood in the base. Inoculate according to the value calculated from the cell count.

Embodiment 2

[0065] Embodiment 2 centrifugation culture

[0066] When the bone marrow diagnosis is three-line decline and the cell count is 3 M solution. Add the final concentration of 20μg / ml colchicine 4h before terminating the culture, harvest after 4h, transfer the bone marrow culture solution into a 15ml centrifuge tube, centrifuge at 1300r / min for 10min, discard the supernatant and harvest the cells. Harvest cells: add 0.075mol / L KCl, mix well and place in a 37°C incubator for 35min. Pre-fixed with methanol: glacial acetic acid (3:1) solution, fixed for 2-3 times and then dropped (or placed in a 4°C refrigerator for dropping the next day), room temperature overnight.

[0067] It can be seen from the drip sheet that the cell suspension obtained after centrifugation is more than that obtained from whole blood culture, which is convenient for the drop sheet; bake the sheet at 75°C for 3H. G banding: 50ml of 0.025% trypsin solution was poured into the dye vat, and the pH of the acid-ba...

Embodiment 3-7 3

[0069] Example 3-7 Optimization of Three Lines Reduced Whole Blood Culture Conditions

[0070] Optimize experimental condition on the basis of embodiment 1, image 3 Take the effect diagram as an example.

[0071] The optimization of specific experimental parameters is as follows:

[0072]

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PUM

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Abstract

The invention belongs to the field of cell culture, and relates to a culture method of bone marrow cells. The method comprises the following steps of collecting a sample: centrifuging the sample, discarding serum, and collecting white blood cells and red blood cells; performing adding to a culture medium: adding the centrifugally-collected cells into the cell culture medium, and performing uniformdistributing; and performing incubating and culturing: putting the bone marrow cells cultured in the culture medium into a cell incubator for culturing. Massive nucleated cells are collected, loss isreduced, and a good split phase is obtained to facilitate clinical analysis. Compared with a whole blood conventional culture method, through the culture method disclosed by the invention, the culture rate and the detection rate are increased, promotion is convenient and easy to realize, and an accurate and effective basis is provided for diagnosis, treatment, prognosis, curative effect monitoring and the like of a blood disease patient.

Description

technical field [0001] The application belongs to the field of cell culture, and relates to a bone marrow chromosomal cell culture method and application thereof. In particular, the culture method of bone marrow cells diagnosed as trilineage decline and nucleated cell count less than 10. Background technique [0002] The culture of bone marrow cells is generally considered to be difficult and the success rate is low; especially when the diagnosis is trilineage decline and the nucleated cell count is less than 10, the harvested chromosomes are thin and poor in shape, and the chromosomes available for analysis are significantly reduced, and division cannot be analyzed phase or even non-cracked phase, eventually leading to culture failure. The diagnosis, treatment, prognosis, and curative effect monitoring of blood disease patients cannot provide accurate and effective basis. [0003] Trilineage reduction refers to the reduction of red blood cells, white blood cells, and plat...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12Q1/68
CPCC12N5/0669C12Q1/68C12N2509/10
Inventor 周燕苹吴涵许海斌赵鹏飞
Owner UNIMED BIOTECH SHANGHAI CO LTD
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