Culturing method of mesenchymal stem cells of menstrual blood

Culturing method of mesenchymal stem cells of menstrual blood

  • CN 104,560,871 A
  • Filed: 12/29/2014
  • Published: 04/29/2015
  • Est. Priority Date: 12/29/2014
  • Status: Active Application
First Claim
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1. prepare a method for menses mescenchymal stem cell, it is characterized in that, comprise the following steps:

  • 1) moon cup is used to gather in female menstrual period the menses of second day or the 3rd day as raw material;

    Being poured into by the menses collected is equipped with in the sterile chamber of conserving liquid, 4 DEG C of preservations;

    2) in 48h, the menses be immersed in menses conserving liquid are transported to laboratory, menses and conserving liquid are fully mixed, be transferred in centrifuge tube, centrifugal is first 800-1000g/5-15min, removing supernatant;

    Add scavenging solution, fully mix, recentrifuge is 300-700g/5-15min, removing supernatant;

    3), after differential centrifugation, add 1%-6% hydroxyethylamyle, fully after mixing, static 10-60 minute, obtain in menses through hemocytoblast;

    4) serum free medium is used to cultivate menses mescenchymal stem cell, wherein P0 covers with to 80% density for menses mescenchymal stem cell, use trysinization, digestion time is 3-5min, in P0 generation, afterwards, cell set time point in the rear 48-72h of operation of going down to posterity goes down to posterity, and more than cell density 80%-90% when requiring to go down to posterity, after going down to posterity, cell is by 3-8 ×

    10 3individual/cm 2density is inoculated, and notes observing the change of substratum pH value in culturing process, adds fresh culture once turn yellow and removes the old substratum of culture vessel moderate, and cell went down to posterity from P0 generation, reached any generation in the middle of P1 ~ P30;

    6) frozen for decidua mescenchymal stem cell of P0 ~ P30;

    every 10 6-10 7cell adds 1ml frozen storing liquid, puts into freezing storing box, proceeds to-80 DEG C of refrigerators, proceeds in-196 DEG C of liquid nitrogen or gas nitrogen and save backup after 12 ~ 24h.

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