Cryopreservation pretreatment method for adipose-derived stem cells

A technology for cryopreservation of fat stem cells, which is applied in the field of pre-treatment for cryopreservation of fat stem cells, can solve the problems of cell culture methods, cell cryopreservation density not yet targeted optimization, cell pretreatment process has not been paid attention to, etc., to achieve The operation method is simple and easy, the repeatability is strong, and the effect of good stability

Inactive Publication Date: 2019-04-05
PLASTIC SURGERY HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the cryopreservation process for adipose-derived stem cells has not yet been standardized, and the cell pretreatment process has not received much attention. Among them, the cell culture method and cell cryopreservation density for cryopreservation have not been targeted optimization

Method used

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  • Cryopreservation pretreatment method for adipose-derived stem cells
  • Cryopreservation pretreatment method for adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Step 1: Wash 10ml of adipose tissue with PBS buffer solution with a pH of 7 for 3 times, then perform the first centrifugation at a centrifugation rate of 1200rpm for 5min, discard the upper layer of oily substances, the lower layer of swelling fluid and blood, and obtain the middle layer of tissue. The middle layer tissue is suspended in type I collagenase with a mass fraction of 0.02%. The amount of type I collagenase is the same as that of the adipose tissue, which is 10ml, and then put into an oscillator for shaking. The temperature of the oscillator is 37°C. The shaking rate is 100rpm, the shaking time is 60min, and then the second centrifugation is carried out, the second centrifugation speed is 1200rpm, the second centrifugation time is 10min, the supernatant is sucked off, and the second centrifugation cells are obtained Microspheres;

[0035] Step 2: Suspend the secondary centrifuged cell microspheres described in step 1 in 10 ml of PBS buffer solution with a p...

Embodiment 2

[0039] Step 1: Wash 15ml of adipose tissue twice with PBS buffer solution with a pH of 7.3, then perform the first centrifugation at a centrifugation rate of 1200rpm for 5min, discard the upper layer of oily substances, lower layer of swelling fluid and blood, and obtain the middle layer of tissue. The middle layer tissue is suspended in type I collagenase with a mass fraction of 0.02%. The amount of type I collagenase is the same as that of the adipose tissue, which is 15ml, and then put into an oscillator for shaking. The temperature of the oscillator is 37°C. The shaking rate is 100rpm, the shaking time is 60min, and then the second centrifugation is carried out, the second centrifugation speed is 1200rpm, the second centrifugation time is 10min, the supernatant is sucked off, and the second centrifugation cells are obtained Microspheres;

[0040] Step 2: Suspend the secondary centrifuged cell microspheres described in step 1 in 15 ml of PBS buffer solution with a pH of 7.3...

Embodiment 3

[0044] Step 1: Wash 20ml of adipose tissue with PBS buffer solution with a pH of 7.5 for 3 times, then perform the first centrifugation at a centrifugation rate of 1200rpm for 5min, discard the upper layer of oily substances, the lower layer of swelling fluid and blood, and obtain the middle layer of tissue. The middle layer tissue is suspended in type I collagenase with a mass fraction of 0.02%. The amount of type I collagenase is the same as that of the adipose tissue, which is 20ml, and then put into an oscillator for shaking. The temperature of the oscillator is 37°C. The shaking rate is 100rpm, the shaking time is 60min, and then the second centrifugation is carried out, the second centrifugation speed is 1200rpm, the second centrifugation time is 10min, the supernatant is sucked off, and the second centrifugation cells are obtained Microspheres;

[0045] Step 2: Suspend the secondary centrifuged cell microspheres described in step 1 in 20 ml of PBS buffer solution with a...

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Abstract

The invention relates to a cryopreservation pretreatment method for adipose-derived stem cells. The method includes: 1, using PBS buffer liquid to wash fat tissue, performing first-time centrifuging to obtain middle-layer tissue, suspending the middle-layer tissue in an T-type collagenase solution, oscillating, performing second-time centrifuging, and sucking supernate to obtain two-time centrifuging cell microspheres; 2, suspending the two-time centrifuging cell microspheres in the PBS buffer liquid, filtering, taking filtrate to obtain cell liquid after filtering, adding a culture medium into the cell liquid after filtering, and re-suspending to obtain two-time re-suspending cell liquid; 3, inoculating cells in the two-time re-suspending cell liquid into a culture bottle, wherein erythrolysis is not performed; 4, obtaining second-generation cell culture liquid when the cells in the culture bottle are cultured to the second generation, performing third-time centrifuging on the second-generation cell culture liquid, removing supernate, adding a cryopreservation agent to obtain final cell liquid, putting the final cell liquid into a slow-cooling cryopreservation box, putting the slow-cooling cryopreservation box into a refrigerator, and transferring the same in the refrigerator into a liquid nitrogen tank for preservation.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a pre-treatment method for cryopreservation of fat stem cells. Background technique [0002] Adipose stem cells are a group of mesoderm-derived pluripotent adult stem cells obtained from adipose tissue. They have self-renewal and multi-directional differentiation potentials. Adipose stem cells are an important source of stem cells to replace bone marrow stem cells due to their high yield and easy acquisition. Clinical applications such as the treatment of chronic ulcers by ASCs have been reported in many clinical studies. In addition, they have the potential to treat diabetes, cardiovascular diseases, and scars. At present, the main steps in the process of cryopreserving adipose stem cells include cell pretreatment before cryopreservation, suspension of cells and cryopreservation agent, transfer into cryopreservation tubes, slow cooling, and transfer into liquid nitrogen. ...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 李梓菲栾杰王黔穆大力刘春军辛敏强付苏
Owner PLASTIC SURGERY HOSPITAL CHINESE ACAD OF MEDICAL SCI
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