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A method for isolating and expanding stem cells after resuscitating umbilical cord tissue and its culture medium

A technology of umbilical cord tissue and stem cells, applied in tissue culture, cell dissociation methods, cell culture active agents, etc., can solve the problems of clinical application limitations of hUCMSCs, poor passaging ability of primary stem cells, and potential safety hazards of heterogeneous serum, etc. Differentiate potential, improve cell resistance to apoptosis, and solve biological safety problems

Active Publication Date: 2021-04-27
吉林国健生命工程科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Because human umbilical cord mesenchymal stem cells (hUCMSCs) have their own unique advantages and characteristics, and are abundant in content and easy to obtain, they are currently the most widely studied human stem cells. The isolation and culture of hUCMSCs is also increasingly convenient and perfect. The conventional method is The obtained human umbilical cord will be preliminarily processed and frozen, and then when necessary, the frozen human is expected to be separated and cultured. Usually, the primary stem cells are mainly obtained by enzymatic digestion or tissue block method. Enzymatic digestion The method can obtain cells in a short time, but the obtained cells have more impurities and can be purified after multiple passages, and the digestion time is difficult to control, which will cause greater damage to the cells; while the tissue block method has a long culture period, and the obtained cells The number is small, and the obtained primary stem cells have disadvantages such as poor passage ability; how to obtain a large number of stable or favorable cells is the difficulty in the cultivation of human umbilical cord mesenchymal stem cells
On the other hand, in the process of cell culture in vitro, most researchers still use classical medium to add fetal bovine serum for stem cell culture. However, there are certain safety risks in xenogeneic serum, which limits the clinical application of hUCMSCs.

Method used

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  • A method for isolating and expanding stem cells after resuscitating umbilical cord tissue and its culture medium
  • A method for isolating and expanding stem cells after resuscitating umbilical cord tissue and its culture medium
  • A method for isolating and expanding stem cells after resuscitating umbilical cord tissue and its culture medium

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Experimental program
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Effect test

Embodiment 1

[0027] A method for isolating and expanding stem cells after resuscitating umbilical cord tissue, said method comprising the steps of:

[0028] 1) Umbilical cord tissue resuscitation: Take out the cryopreservation tube of frozen umbilical cord tissue, place it in a 37°C water bath to thaw, after thawing, wash it with recovery buffer for 3 times, and obtain the recovered umbilical cord tissue;

[0029] 2) Tissue blocks are obtained; the resuscitated umbilical cord tissue obtained in step 1) is cut into 1mm 3 Tissue blocks were digested by adding digestion solution at 15°C for 1 hour, and then digested at 37°C for 10 minutes. The digestion was terminated, the digestion solution and suspended cells were removed, and undigested tissue blocks were obtained, rinsed in sodium chloride injection, and the supernatant was discarded by centrifugation. The remaining tissue pieces were obtained; wherein, the digestive solution contained 0.5 mg / ml type II collagenase, 0.1 mg / ml TrypLE TM ...

Embodiment 2

[0034] A method for isolating and expanding stem cells after resuscitating umbilical cord tissue, said method comprising the steps of:

[0035] 1) Umbilical cord tissue resuscitation: take out the cryopreservation tube of frozen umbilical cord tissue, place it in a 37°C water bath to thaw, after thawing, wash it with recovery buffer for 5 times, and obtain the recovered umbilical cord tissue;

[0036] 2) Tissue blocks are obtained; the resuscitated umbilical cord tissue obtained in step 1) is cut into 1mm 3 Tissue blocks were digested by adding digestive solution at 15°C for 2 hours, then digested at 37°C for 5 minutes, the digestion was terminated, the digestive solution and suspended cells were removed, undigested tissue blocks were obtained, rinsed in sodium chloride injection solution, and the supernatant was discarded by centrifugation. The remaining tissue pieces were obtained; wherein, the digestive solution contained 0.7mg / ml type II collagenase, 0.2mg / ml TrypLE TM S...

Embodiment 3

[0041] 1) Umbilical cord tissue resuscitation: take out the cryopreservation tube of frozen umbilical cord tissue, place it in a 37°C water bath to thaw, after thawing, wash it with recovery buffer 4 times, and obtain the recovered umbilical cord tissue;

[0042] 2) Tissue blocks are obtained; the resuscitated umbilical cord tissue obtained in step 1) is cut into 1mm 3 Tissue blocks were digested at 15°C for 1.5h by adding digestive fluid, then digested at 37°C for 7min, the digestion was terminated, the digestive fluid and suspended cells were removed to obtain undigested tissue blocks, rinsed in sodium chloride injection, and the supernatant was discarded by centrifugation , to obtain the remaining tissue pieces; wherein, the digestive fluid contains 0.6mg / ml type II collagenase, 0.1mg / ml TrypLE TM Sodium chloride injection of express and 0.4mg / ml prolinephthalein amino acid;

[0043] 3) Isolation of primary stem cells: Place the remaining tissue pieces in a culture dish w...

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Abstract

The invention relates to a method for isolating and expanding stem cells after umbilical cord tissue resuscitation and a special culture medium thereof, including the steps of umbilical cord tissue resuscitation, tissue block acquisition, isolation of primary stem cells, and expansion and passage of stem cells. Type II Collagenase, TrypLE TM Sodium chloride injection of express and prolinephthalein amino acids, the medium used is sodium selenate, bovine transferrin, ornithine, 5-7mg / ml lectin, α-tocopheryl succinate monoester , IL‑6, LIF and HGF in DMEM / F12 medium. The isolation and culture method provided by the present invention has short crawling time of human umbilical cord mesenchymal stem cells, short culture period of primary stem cells, high purity of stem cells and good activity, and can be passed down for many times. Potential for high differentiation.

Description

technical field [0001] The invention belongs to the field of cell culture, in particular to a method for isolating and expanding stem cells after resuscitating umbilical cord tissue and a special culture medium thereof. Background technique [0002] Because human umbilical cord mesenchymal stem cells (hUCMSCs) have their own unique advantages and characteristics, and are abundant in content and easy to obtain, they are currently the most widely studied human stem cells. The isolation and culture of hUCMSCs is also increasingly convenient and perfect. The conventional method is The obtained human umbilical cord will be preliminarily processed and frozen, and then when necessary, the frozen human is expected to be separated and cultured. Usually, the primary stem cells are mainly obtained by enzymatic digestion or tissue block method. Enzymatic digestion The method can obtain cells in a short time, but the obtained cells have more impurities and can be purified after multiple ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/12C12N2500/24C12N2500/30C12N2500/32C12N2501/12C12N2501/2306C12N2501/235C12N2501/998C12N2509/00C12N2535/00
Inventor 曹毓琳刘俊江时兆田白志惠
Owner 吉林国健生命工程科学技术有限公司
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