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General preserving fluid for human umbilical cord, amniotic membrane and placenta samples and preparation method thereof

A technology of preservation solution and human umbilical cord, applied in the field of stem cells, which can solve the problems of low viability and low number of cells

Active Publication Date: 2021-03-12
杜德(江门)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, it takes several hours or several days for the sample to go from the delivery room of the hospital to the GMP laboratory. The sample will be affected by factors such as time, temperature, nutrition, etc., resulting in a small number of cells and low vitality in the sample.

Method used

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  • General preserving fluid for human umbilical cord, amniotic membrane and placenta samples and preparation method thereof
  • General preserving fluid for human umbilical cord, amniotic membrane and placenta samples and preparation method thereof
  • General preserving fluid for human umbilical cord, amniotic membrane and placenta samples and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The present embodiment carries out the preparation of universal preservation solution, comprises the following steps:

[0037] Add 50mL of 20% human albumin (concentration of human albumin is 10g / 50mL) and 1mL of heparin sodium injection into 950mL of DMEM basal medium, mix well, then add 100000U of penicillin, 100mg of streptomycin, 10mg of insulin, 100mg of Vitamin C and 10 μg EGF were stirred and dissolved, and then filtered through a 0.22 μm sterile filter membrane to remove bacteria to obtain a universal preservation solution, marked with the date of manufacture, and stored at 4°C.

[0038] Among them, the concentration of heparin sodium injection is 6250U / mL.

Embodiment 2

[0048] Under sterile conditions, take the 24cm umbilical cords of newborns who are healthy and have no infectious diseases after cesarean section, and divide them evenly into 8 parts, and store them in the general preservation solution of Example 1, normal saline, DMEM medium, sterile containers, and the general control medium respectively. In Preservation Solution 1, Control Universal Preservation Solution 2, Control Universal Preservation Solution 3 and Control Universal Preservation Solution 4, after 24h, 48h, and 72h storage at a constant temperature at 4°C, take the same length of umbilical cord tissue and use collagenase digestion to separate umbilical cord mesenchyme Stem cells were filtered and centrifuged, counted using trypan blue, and the cell viability was calculated.

[0049] see figure 1 , Table 1 and Table 2, figure 1It is the mesenchymal stem cell viability figure after adopting different preservation methods to preserve the umbilical cord for 24h, 48h, and 72...

Embodiment 3

[0056] Under sterile conditions, take an example of a healthy neonatal placenta from a cesarean section without infectious diseases, peel off the placental amnion, divide it into 8 parts, and store them in the general preservation solution of Example 1, normal saline, DMEM medium, In the sterile container, the reference universal preservation solution 1, the reference universal preservation solution 2, the reference universal preservation solution 3 and the reference universal preservation solution 4, after 24h, 48h, and 72h storage at a constant temperature at 4°C, take the amnion tissue of the same quality and digest it with collagenase Amniotic mesenchymal stem cells were separated by the method, filtered and centrifuged, and counted using trypan blue.

[0057] Please refer to Table 3 for the results. The results show that the universal preservation solution in Example 1 has a good preservation effect on amnion, and a large number of mesenchymal stem cells can still be separ...

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Abstract

The invention belongs to the technical field of stem cells, and particularly relates to a universal preserving fluid for human umbilical cord, amniotic membrane and placenta samples and a preparationmethod thereof. In the universal preservation solution, the DMEM basal culture medium provides the most basic nutrition for the mesenchymal stem cells of the sample; insulin can promote the sample mesenchymal stem cells to absorb and utilize sugar in the universal preservation solution, and the cell activity is maintained; the human serum albumin provides dynamic balance for the mesenchymal stem cells and maintains cell osmotic pressure; vitamin C can prevent free radicals generated by long-time oxidation from damaging the mesenchymal stem cells; the EGF can promote the proliferation of the mesenchymal stem cells and maintain the original form of the cells. Experimental results show that the preserving fluid can be used for preserving umbilical cords, amnions and placentas for a long time,the number of obtained mesenchymal stem cells is large, the survival rate is high, and human albumin, insulin, vitamin C and EGF have a synergistic effect on increasing the number of preserved umbilical cord mesenchymal stem cells and increasing the survival rate of the mesenchymal stem cells.

Description

technical field [0001] The invention belongs to the technical field of stem cells, in particular to a universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and a preparation method thereof. Background technique [0002] Mesenchymal stem cells are a type of cells with self-renewal and multi-directional differentiation potential, also known as multipotential stromal cells, or MSCs for short, are a type of pluripotent stem cells belonging to the mesoderm, which mainly exist in connective tissue and organ interstitium. Including: bone marrow, umbilical cord, fat, amniotic membrane, dental pulp, placenta, etc. Under suitable conditions, it can differentiate into various tissue cells such as fat, bone, and cartilage. Compared with MSCs derived from bone marrow, mesenchymal stem cells derived from umbilical cord, amniotic membrane and placenta have no moral and ethical disputes due to their convenience in obtaining materials. The total a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0226A01N1/0215
Inventor 张玲洁吴基伟郑婕
Owner 杜德(江门)生物科技有限公司
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