Method for ultralow-temperature cryopreservation and resuscitation of mesenchymal stem cells

A technology of mesenchymal stem cells and stem cells, applied in the field of ultra-low temperature cryopreservation and recovery of mesenchymal stem cells, can solve the problems of reduced number of bone marrow mesenchymal stem cells, failure to collect, liquid nitrogen entry, etc., to reduce pollution and explode tubes during recovery risk, ensure the effect of freezing, and reduce the effect of liquid nitrogen entering

Inactive Publication Date: 2019-05-03
和携科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, bone marrow-derived mesenchymal stem cells are the most widely used, but there are the following problems: with aging, the number of bone marrow mesenchymal stem cells decreases significantly, and the ability of proliferation and differentiation declines significantly; the preparation process is not easy to quality control; transplantation Immunological reaction may be caused when the allogeneic donor is given; the patient will be damaged when the material is collected, and it cannot be collected when the patient has bone marrow disease, even if it is a healthy donor, too much bone marrow cannot be extracted
[0005] Usually, fetal bovine serum (FBS) should be used for cryopreservation of stem cells, otherwise the recovery rate of cells is low; although FBS can provide necessary nutrients for mesenchymal stem cells, FBS is an animal-derived substance, and its composition is relatively low. Complicated, there are potential risks in clinical application, and it also increases the chance of cell contamination
In addition, most of the cryopreserved cells are stored in ordinary liquid nitrogen tanks. Since the cryopreservation tubes are immersed in liquid nitrogen for a long time, it is inevitable that liquid nitrogen will enter the cryopreservation tubes. On the one hand, this increases the risk of contamination of the cryopreserved cells. On the other hand, there is also the risk of tube explosion due to careless operation when resuscitating cells

Method used

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  • Method for ultralow-temperature cryopreservation and resuscitation of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Acquisition of small pieces of umbilical cord tissue

[0031] 1) Take the treated umbilical cord of a healthy newborn born by cesarean section at full term, remove the ligated ends of the umbilical cord, and use ophthalmic scissors to cut the 10-15cm long middle section into a 2-3cm long umbilical cord tissue section, and use 1% double antibody (penicillin-streptomycin mixed solution) saline solution was repeatedly rinsed the umbilical cord tissue to remove blood stains.

[0032] 2) Dissect the above-mentioned umbilical cord tissue section, and remove the arterial and venous blood vessels (2 arteries and 1 vein) and epidermal tissue to obtain the umbilical cord Wharton's jelly tissue section, rinse repeatedly with saline containing 1% double antibody, Cut it into pieces of Wharton's jelly tissue about 3mm×3mm×1mm.

Embodiment 2

[0033] Example 2: Adhesive culture of Wharton's jelly tissue pieces and acquisition of primary mesenchymal stem cells

[0034] 1) According to 1 piece / cm 2 Shred to 4~10mm 3 Human umbilical cord Wharton's jelly tissue pieces of the same size were inoculated into cell culture dishes with a diameter of 10 cm and placed on the wall, and about 30-50 pieces of the tissue pieces were added to each culture dish, and the tissue pieces were air-dried for about 15 minutes to reduce the Moisture, strong adhesion to the wall;

[0035] 2) Gently add 7 mL of complete medium consisting of basal medium (α-MEM Cornig) and serum substitute (Helios GMP grade) to each Petri dish (which contains 3% volume ratio of serum substitute), do not Blow up the tissue block and then place it in 37°C, 5% CO 2 Incubation in an incubator; 5 mL of fresh complete medium was replaced after 5 days of culture, and the culture was continued for 5-9 days, during which time a new complete medium was replaced every ...

Embodiment 3

[0038] Example 3: Subculture of mesenchymal stem cells

[0039] 1) The mesenchymal stem cells (the primary human umbilical cord mesenchymal stem cells prepared in Example 2 were used for the first expansion and culture, that is, the P0 generation) were measured by a cell counter and the cell viability was calculated by trypan blue staining , the above-mentioned mesenchymal stem cells were inoculated into T175 cell culture flasks, and the initial seeding density was about 6×10 3 / cm 2 , the total volume is 25mL / bottle;

[0040] 2) Place the cell culture flask at 37°C, 5% CO 2 Cultivate in the incubator for 3-4 days. When the confluence of the cells reaches 80%-90%, discard the culture medium in the culture flask, wash it twice with normal saline, and then add 7 mL of TrypLE to each culture flask to fully digest the cells. About 3-5 minutes to digest;

[0041] 3) After the pseudopodia of the cells are completely retracted and the cells become round, add 7 mL of the complete me...

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Abstract

The invention provides a serum-free cryopreservation solution for mesenchymal stem cells and a method for ultralow-temperature cryopreservation and resuscitation of the mesenchymal stem cells by usingthe cryopreservation solution. Compared with a fetal calf serum containing cryopreservation solution and a cryopreservation and resuscitation method employing the fetal calf serum containing cryopreservation solution in the prior art, the serum-free cryopreservation solution and the method have the advantages that through subjecting the mesenchymal stem cells to cryopreservation by using the serum-free cryopreservation solution provided by the invention, fetal calf serum is not added during cryopreservation, and thus, risk possibly caused by introduction of heterogeneous proteins is avoided;and through adopting sealing operations and a programmed cooling box and storing cells by a gas-phase liquid-nitrogen tank, the entry of liquid nitrogen during cryopreservation is effectively reducedwhile a cryopreservation effect is ensured, so that risk of pollution during cryopreservation and tube burst during resuscitation is lowered, the motility rate of the mesenchymal stem cells after resuscitation reaches 90% or more finally, and the requirements of the mesenchymal stem cells in clinical application can be met.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a cryopreservation and recovery method of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in the early stages of development, with the ability of self-renewal and multidirectional differentiation. Mesenchymal stem cells can be used clinically to solve a variety of blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, partial knee meniscus resection injury repair, autoimmune diseases, etc. has development prospects. [0003] Mesenchymal stem cells were first discovered in the bone marrow, and later found to exist in many tissues in the process of human development and development. At present, bone marrow-derived mesenchymal stem cells are the most widely used, but there are the following problems: with ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0775
Inventor 赵进军谷广其杨桂花赵宇飞李张鹏
Owner 和携科技有限公司
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