Method of recovering cell on microvector from passive outer space carrier system

Method of recovering cell on microvector from passive outer space carrier system

  • CN 100,494,354 C
  • Filed: 12/08/2004
  • Issued: 06/03/2009
  • Est. Priority Date: 12/08/2004
  • Status: Active Grant
First Claim
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1. method that reclaims cell on the microcarrier from passive space treatment system is characterized in that:

  • may further comprise the steps;

    (1) extracts nutrient solution out in the frozen pipe that microcarrier and mammal cell are housed that from passive space treatment system, returns, change the space perfect medium, contain 789.85 milliliters of DMEM, 200 milliliters of import foetal calf serum, content during the space perfect medium is every liter and be the cultivation of 0.15 milliliter of 10 milliliters of the glutamine of every milliliter of 0.03 gram and hydrocortisone that content is 5 milligrams every milliliter Be placed on 5%CO 2, hatched 15-20 hour or hatched in the multiplication phase in 37 ℃

    of incubators at the mammal cell;

    (2) the space perfect medium in the frozen pipe of sucking-off adds 2 milliliters of serum free mediums then and cleans microcarrier;

    (3) treat the serum free medium in the frozen pipe of sucking-off after the complete sedimentation of microcarrier;

    (4) add 1 milliliter 0.25% pancreatin and the mixed solution of 0.2%EDTA again in frozen pipe, jog 2-4 minutes digests the cell on the microcarrier;

    (5) 20% blood serum medium that contains that adds 2 milliliters takes mixed solution in the neutralization procedure (4);

    (6) solution in the frozen pipe is put upside down mixing, place frozen pipe, microcarrier descends on one side in pipe, on one side from frozen pipe the sucking-off supernatant liquor, and this supernatant liquor placed centrifuge tube;

    (7) add 3 milliliters 20% blood serum medium that contains again in frozen pipe, put upside down and shake frozen pipe 40-60 times, microcarrier descends in pipe on one side, on one side from frozen pipe the sucking-off supernatant liquor, and this supernatant liquor placed the described centrifuge tube of step (6);

    (8) repeating step (2)-(7) is 2 to 3 times, and the supernatant liquor that obtains is placed the described centrifuge tube of step (6);

    (9) supernatant liquor in the above-mentioned centrifuge tube is carried out centrifugal treating 5-8 minute, centrifugal speed is 1000-1200rpm, and at room temperature collecting cell precipitates then;

    (10) with 20% blood serum medium that contains that adds 0.25 milliliter in the cell precipitation of collecting, mixing is got 0.01 milliliter of counting;

    (11) according to the count results of step (10), cell precipitation is extracted in requirement according to 20-30 cell of one 96 orifice plate discharging, and after the routine that adds the space perfect medium account for cumulative volume 90% and account for cumulative volume 10% in this cell precipitation cultivates the supernatant liquor of retaining behind this cell and dilute, bed board.

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