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Method for separating living cell and constructing cell bank by means of tissue homogenate method

A technology for tissue homogenization and tissue cells, applied in chemical libraries, combinatorial chemistry, library creation, etc., can solve problems such as time-consuming, high requirements for technicians’ operating skills, and cumbersome operating procedures, so as to ensure high-quality quality and improve construction Efficiency, simple operation effect

Active Publication Date: 2013-12-25
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the following characteristics: 1) only need to cut the tissue into 1-1.5mm 3 The operation is relatively simple; 2) the cultivation of cell clusters takes several days to achieve the separation of cells, which takes a long time; 3) the long-term cultivation of cells has a higher possibility of contamination
[0014] It can be seen that the current solid tissue cell separation method for building cell banks has a cumbersome operation process, takes a long time, completely relies on manual operation, and requires high operating skills for technicians.
In addition, chemical enzymatic hydrolysis introduces exogenous reagents from animals, which will increase the risk of stem cell drug treatment in public cell banks

Method used

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  • Method for separating living cell and constructing cell bank by means of tissue homogenate method
  • Method for separating living cell and constructing cell bank by means of tissue homogenate method
  • Method for separating living cell and constructing cell bank by means of tissue homogenate method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Using the tissue homogenate method to separate placental lobular cells and construct a cell bank

[0048] The method for placental lobular tissue homogenate comprises the following steps:

[0049] (1) Placental tissue cleaning: The placental tissue is processed in a biological safety cabinet. According to the size of the placenta, an appropriate amount of PBS buffer solution containing 1% double antibody is used to wash the placental tissue until the residual blood on the surface of the placental tissue is washed clean. blood clots;

[0050] (2) Pre-treatment of placental lobule tissue: Use surgical scissors to cut placental lobule from the placental tissue obtained in step (1), transfer the placental lobule to a petri dish and cut it into small pieces, put 60-90 grams of placental lobule tissue Put the block into the homogenization container of the tissue cell processor PlacentaPro, add 50-80 ml of PBS and seal the processing cup;

[0051] (3) Homogenizati...

Embodiment 2

[0060] Embodiment 2: Comparative experiment of placenta lobule tissue homogenate processing and manual processing

[0061] The comparison experiment was divided into 8 groups. With reference to the method of Example 1, a fully automatic tissue processing experiment was carried out. Refer to the method described in the patent CN201210292509.9 (publication number CN102807966A) to carry out the manual processing (digestion) experiment. The placental tissue was obtained from 8 different donors, and the processed volume was between 60-90 grams. The comparison experiment results are shown in Table 2.

[0062]

Fully automatic processing

Manual processing (digestion)

test

Extracted cell number / g

Extracted cell number / g

1

3.8×10 6 ／g

2.0×10 6 ／g

2

3.9×10 6 ／g

0.79×10 6 ／g

3

2.3×10 6 ／g

1.0×10 6 ／g

4

2.9×10 6 ／g

1.1×10 6 ／g

5

2.6×10 6 ／g

1.4×10 6 ／g

6

2.7×10 6 ／g

...

Embodiment 3

[0065] Example 3: Primary growth of cells isolated from placental lobule tissue by homogenization

[0066] (1) Preparation before cell culture: Add erythrocyte lysate (Roche) at a ratio of 1 part of cell suspension to 2-3 parts of erythrocyte lysate, incubate at 15-25°C for 10-15 minutes, and put it in a centrifuge Centrifuge at 1400rpm for 10 minutes, remove the supernatant, observe the lysis of red blood cells, and repeat this step for lysis if necessary. After the lysis is complete, add PBS to resuspend the cells and take a small sample for cell counting, put it into a centrifuge and centrifuge at a speed of 1400rpm for 10 minutes to wash the cells, remove the supernatant, and add mesenchymal stem cell medium (15% FBS+1 %L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to resuspend cells, and 2-5×10 4 / cm 2 Placenta-derived nucleated cells were inoculated into culture flasks at a density of .

[0067] (2) Primary cell culture: put the culture bottle into C0 2The concentration i...

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PUM

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Abstract

The invention discloses a method for separating the tissue cell of a fetal appendage and constructing a cell bank by means of a tissue homogenate method, so that the process for constructing the cell blank is simple in steps, time-saving, and capable of achieving easy quality control and standardization. The method disclosed by the invention comprises the following steps of (1) cleaning the tissue of the fetal appendage; (2) carrying out homogenate pretreatment on the tissue; (3) carrying out homogenate treatment on the tissue to separate a cell (cell cluster); (4) filtering the tissue and collecting the cell (cell cluster) after the homogenate; (5) detecting the cell (cell cluster); (6) cryopreserving the separated cell (cell cluster) and constructing the cell bank. Compared with the method in the prior art, the method disclosed by the invention has the advantages of simple operation, short time consumption, no introduction of an exogenous reagent, easiness for standardization, easy quality control, great cell gain and the like, and is of great significance in improving the construction efficiency of the fetal appendage cell bank and obtaining the stem cell which is high in quality and applied to treatment.

Description

technical field [0001] The invention relates to a method for constructing a fetal appendage tissue cell bank, more specifically a physical method for separating cells from the fetal appendage tissue to construct a cell (cluster) bank. Background technique [0002] Fetal appendage tissue is mainly composed of placental lobules, placental base, decidua basalis, amniotic membrane and umbilical cord tissue. Originating from the extraembryonic mesoderm during embryonic development, the placenta is composed of mesenchyme, blood vessels and trophoblast cells. It is the earliest hematopoietic organ of the fetus and contains a large amount of mesenchyme. The latest research shows that the placenta contains mesenchymal stem cells, placental sub-totipotent stem cells and abundant hematopoietic stem cells. The isolation and culture of these pluripotent stem cells from the placenta will open up a new and rich source for experimental research and clinical application. [0003] Placental ...

Claims

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Application Information

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IPC IPC(8): C40B50/06
Inventor 许晓椿肖海蓉
Owner BOYALIFE
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