A kind of stem cell cryopreservation medium

A technique for cryopreservation and stem cells, applied in the biological field, can solve the problems of inactive cells, insufficient types, and insufficient types of Caspase inhibitors, so as to improve the survival rate, have good application value, and facilitate preservation and application.

Active Publication Date: 2022-03-22
样美生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the current research, the types of Caspase inhibitors are not rich enough, and there are not enough types to choose from, and zVAD-fmk itself is not completely inactive on cells, making it urgent to study Capase inhibitors

Method used

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  • A kind of stem cell cryopreservation medium
  • A kind of stem cell cryopreservation medium
  • A kind of stem cell cryopreservation medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The preparation of embodiment 1 Caspase monoclonal antibody

[0023] Animal immunization: 5 mg of purified recombinant human Caspase-3 protein (BioVision, Cat. No. 1083-5) was fully mixed with an equal volume of Freund's complete adjuvant, and BALB / c mice were subcutaneously injected with 50 μg of the fusion protein at multiple points. 4 weeks after the initial immunization, multi-point subcutaneous injections were given on the back to boost the immunization, and 4 weeks later, the immunization was boosted again, each time using 50 μg of immunogen per mouse. 8-10 days after the third immunization, blood was collected from the tail vein to measure the serum antibody titer by ELISA. Four days before fusion, a mouse with the highest antibody titer was selected for booster immunization by intraperitoneal injection of 150 μg antigen without adjuvant. Cell fusion and screening: Aseptically take splenocytes from BALB / c mice for booster immunity, use 50% polyethylene glycol as...

Embodiment 2

[0037] Example 2 Cryopreservation experiment of umbilical cord mesenchymal stem cells

[0038] The cells were divided into three groups, and the following 3 different compositions of cryopreservation solutions were added respectively (the formulas in each group are in volume ratio).

[0039] Group A uses cryopreservation solution (mixed by IMDM medium, fetal bovine serum and dimethyl sulfoxide at 7:2:1)

[0040] Adjust cell density to 1x10 5 / ml, stored in liquid nitrogen according to the following procedure: 15 min at 4°C, 2 h at -20°C, overnight at -80°C, and finally frozen at -196°C. After the cells were frozen at -196°C for 4 weeks, they were immediately placed in a water bath at 40°C, thawed within 2 minutes, and washed with IMDM medium for later use (conventional cryopreservation group).

[0041] B Use cryopreservation solution (mixed by IMDM medium, fetal calf serum and dimethyl sulfoxide at 7:2:1, add zVAQ-fmk (30 μmol / L) to adjust the cell density to 1×10 5 / ml, st...

Embodiment 3

[0047] Induction and differentiation experiment of cells after cryopreservation in embodiment 3

[0048] Induced differentiation of umbilical cord blood mesenchymal stem cells into chondrocytes: Three groups of resurrected cells were taken after cryopreservation, and the density was 5X10 7 L- 1 Inoculate in a 24-well culture plate (with a sterile cover glass built into the well), and change the medium on the second day, and half of the culture wells are used to induce chondrocyte culture medium (DMEM with 15% fetal bovine serum, containing a final concentration of 100pg / L insulin Like growth factor 1) for culture, change the culture medium once every 3 days, and induce for 12 days.

[0049] Collect the cell clusters of chondrogenic induction 12 days respectively, aspirate and discard the medium, wash lightly twice with PBS, add an appropriate amount of RNAisoplus, and blow and beat repeatedly to fully lyse the cells. cDNA was synthesized by reverse transcription after extrac...

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Abstract

The invention relates to a stem cell cryopreservation liquid, which comprises a Caspase monoclonal antibody, and the monoclonal antibody has better ability of binding Caspase protein. By adding corresponding antibodies to the cryopreservation solution, the survival rate of cells after cryopreservation can be effectively improved, which is beneficial to the preservation and application of umbilical cord mesenchymal stem cells, and has good application value.

Description

technical field [0001] The invention relates to the field of biology, in particular to a stem cell cryopreservation solution. Background technique [0002] The content of hematopoietic stem cells and mesenchymal stem cells in umbilical cord blood is relatively rich, and the mesenchymal stem cells can be induced to differentiate into various tissue cells in vitro under certain conditions, which is beneficial to the repair of diseased or damaged tissue cells, and is widely used in clinical practice. It is used in hematological malignancies, cartilage and bone repair and regeneration, repair of damaged nerves, and treatment of liver diseases. [0003] In recent years, umbilical cord blood stem cell transplantation has gradually become one of the important means for clinical treatment of various malignant and non-malignant hematological diseases. Previous studies believed that the SCL gene of stem cell leukemia and TAL1 fusion gene of acute T lymphocytic leukemia played an impo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00
CPCA01N1/0221
Inventor 王京陈帅
Owner 样美生物科技(北京)有限公司
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