Serum-free stem cell freezing medium and stem cell freezing method

A cryopreservation method and stem cell technology, applied in the field of serum-free stem cell cryopreservation solution and stem cell cryopreservation, can solve the problems of large differences in cryopreservation protection effects, uncontrollable effects, and poor universality, so as to maintain the vitality and characteristics of stem cells , The effect of eliminating interference and influence

Inactive Publication Date: 2019-03-08
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are still some deficiencies in common stem cell cryopreservation solutions: 1) high specificity, poor universality, and the cryopreservation effect of the same cryopreservation solution on stem cells of different types or different species is quite different; 2) there are The components of serum cryopreservation solution are complex, prone to interference, and the effect is uncontrollable; 3) Some cryopreservation solutions are not suitable for long-term cryopreservation, and the cell viability decreases significantly with the extension of cryopreservation time

Method used

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  • Serum-free stem cell freezing medium and stem cell freezing method
  • Serum-free stem cell freezing medium and stem cell freezing method
  • Serum-free stem cell freezing medium and stem cell freezing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Separation, identification and survival rate detection of stem cells

[0038]Isolation of human tissue stem cells (taking tumor tissue as an example): Place the obtained tumor tissue in a sterile box, add 500ml of normal saline, and transport it to a GMP laboratory. Under sterile conditions, tear off the amniotic membrane of the placenta, cut the chorion of the placenta with scissors, and cut it into strips of about 1 cm, squeeze it with tweezers, and at the same time rinse it repeatedly with normal saline until it becomes clear, and squeeze out the blood and water with tweezers After that, weigh with an electronic balance to obtain the wet weight of the chorion. Then add an appropriate amount of normal saline, and use a hand-held electric homogenizer to process it into fine particles (about the size of rice grains), rinse it again and again with normal saline until it is clear, and then continue to use the homogenizer to process it until it is meaty (slightly...

Embodiment 2

[0044] Example 2 Cryopreservation, recovery and proliferation of stem cells

[0045] Preparation of freezing solution:

[0046] (1) DMEM / F12 (2.5×) stock solution

[0047] Dissolve the DMEM powder that can be used to prepare 1L medium and the bagged F12 powder that can be used to prepare 1L medium.

[0048] (2) RPMI 1640 (2×) stock solution

[0049] Dissolve RPMI 1640 powder, which can be used to prepare 1L of medium, in distilled water and stir evenly, with a final volume of 500ml. Filter sterilize and store at 4°C.

[0050] (3) HEPES (1M) stock solution

[0051] Dissolve 23.8g of HEPES in distilled water and stir evenly, the final volume is 100ml. Filter sterilize and store at 4°C.

[0052] (4) Glucose (30% w / v) stock solution

[0053] Weigh 30g of glucose powder and dissolve in distilled water to a final volume of 100ml. Filter sterilize and store at 4°C.

[0054] (5) bFGF (10μg / ml) stock solution

[0055] Basic fibroblast growth factor (bFGF), each tube contains ...

Embodiment 3

[0074] Example 3 Stem cell activity detection and differentiation ability detection

[0075] Treatment of Stem Cells:

[0076] Aspirate the cell culture medium, add 100gL (containing 10% CCK-8 reagent solution) viability assay solution to each well, place in a 37°C incubator and incubate for 3h, then measure the D value at a wavelength of 450nm.

[0077] Activity detection and differentiation ability detection of human neural stem cells after cryopreservation:

[0078] Detect and compare the growth activity of human neural stem cells recovered after cryopreservation. For specific results, see image 3 , Figure 4 , Table 2, and Table 3, the results show that this formula cryopreservation solution has a good protective effect on neural stem cells. The culture observation results of human neural stem cells after resuscitation also show that the stem cells after cryopreservation and resuscitation still have good proliferation and differentiation ability, see Figure 5 , Ima...

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Abstract

The invention provides a serum-free stem cell freezing medium and a stem cell freezing method. The freezing medium comprises the following raw materials by volume percentage: 8-12% of DMSO (dimethylsulfoxide), 83-87% of serum-free culture medium and 3-8% of other ingredients. The freezing medium can keep activity and characteristics of stem cells in a relatively long freezing time and has a good freezing and protection effect on the stem cells of different kinds and from different species sources.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a serum-free stem cell cryopreservation solution and a stem cell cryopreservation method. Background technique [0002] Stem cell therapy is a new type of treatment for complex diseases such as organ failure, tumors and genetic diseases. Stem cells are also one of the hot spots in the research of disease mechanism and pharmacology. Stem cells have broad application prospects in cell therapy and regenerative medicine, so their applications in scientific research and clinical practice are becoming more and more extensive. However, most of the stem cells currently used clinically are cultured in vitro and preserved at low temperature. Studies have shown that the biological effect of stem cells in the early stages is more obvious due to the late stem cells with more passages. In addition, some people think that due to the high proliferative activity of stem cells, lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 谭龙
Owner TIANJIN MEDICAL UNIV
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