Extraction method of autologous fat mesenchymal stem cell

Extraction method of autologous fat mesenchymal stem cell

  • CN 103,540,564 A
  • Filed: 10/21/2013
  • Published: 01/29/2014
  • Est. Priority Date: 10/21/2013
  • Status: Active Application
First Claim
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1. an extracting method for autologous fat mescenchymal stem cell, is characterized in that:

  • comprise the steps;

    A) obtain and separated human fat tissue, the method collector'"'"'s fatty tissue by lipectomy or liposuction under toponarcosis at least 100-200mL fat in sealing sterilising vessel;

    Add 100-200mL phosphate buffered saline buffer (PBS) in 37 oc gas bath incubator, with 100rpm convolution concussion, repeats rinsing, each 30 minutes, with transfer pipet by organizing the liquid sucking-off of lower floor, till the liquid clear of institute'"'"'s sucking-off;

    B) collagenase digesting;

    add and the 0.2-0.4mg/mL I-Collagenase Type of organizing same volume, making solution final concentration is 0.1-0.2mg/mL, sealing, was placed in 37 °

    of C constant-temperature tables, with 200rpm concussion digestion 30 minutes;

    C) centrifugal collection;

    add calf serum (Fetal Bovine Serum, FBS), making its ultimate density is 10%, end digestion, the fatty packing having digested is moved on in 50mL centrifuge tube, with centrifugal force 1000 x g speed at room temperature centrifugal 15 minutes, now liquid level is divided into 3 layers, upper strata is yellow oily adipose cell layer, and middle level is fat tissue layer, and lower floor is for containing various kinds of cell layer;

    D) the upper middle level in sucking-off centrifuge tube, removes;

    Retain confluent monolayer cells layer, with the washing of PBS damping fluid, under room temperature with centrifugal force 300xg centrifugal 5 minutes, remove upper strata liquid, in triplicate to remove FBS;

    E) lower confluent monolayer cells is suspended in PBS damping fluid again, the cell that every 100mL fat extracts is suspended in 10mLPBS again;

    Every gram of fat can extract 0.5-1x10 6autologous fat mescenchymal stem cell;

    F) get 100 microlitre autologous fat mescenchymal stem cell samples and do cell counting, carry out cellular elements biological analysis;

    G) with 1-5x10 4/ cm 2density inoculation do cell cultures amplification, the typical morphological feature of autologous fat mescenchymal stem cell is shuttle type, adherent growth, 2-5 is for future use frozen for cell routine;

    Wherein amplification the 5th generation cell passed through Flow cytometry, expressed CD44, CD166, CD105, CD73 and HLA1, but do not express HLA2, CD45, CD14 and CD34;

    To wherein amplification the 5th generation cell carried out Induction of committed differentiation research, autologous fat mescenchymal stem cell in vitro Induction of committed differentiation for becoming scleroblast and chondrocyte.

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