CN102586180A - Separation, storage culture and amplification method of pouch fat interstitial cell - Google Patents
Separation, storage culture and amplification method of pouch fat interstitial cell Download PDFInfo
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- CN102586180A CN102586180A CN2011100075146A CN201110007514A CN102586180A CN 102586180 A CN102586180 A CN 102586180A CN 2011100075146 A CN2011100075146 A CN 2011100075146A CN 201110007514 A CN201110007514 A CN 201110007514A CN 102586180 A CN102586180 A CN 102586180A
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Abstract
The invention relates to a separation and storage method of a pouch fat interstitial cell, which comprises the following steps of: cutting to obtain a pouch fat tissue by adopting a surgical knife, extracting an interstitial tissue of the pouch fat tissue and separating the cell by using an adherent method. The culture and amplification method of the pouch fat interstitial cell comprises the following steps of: carrying out large-scale tissue culture by adopting a paper carrier technology, freezing and storing by using a definite and non-specific genetic component and preserving the cell for a long time. Through the separation and storage method, the pouch interstitial cell can be extracted, massively cultured and preserved for a long time; and the cultured interstitial cell can be used for promoting the healing of skin wound, the healing of diabetic ulcer, the repair of myocardial injury and the repair of hepatic injury.
Description
Technical field
The present invention relates to the separation store method of human body pouch fat mesenchymal cell and the cultivation amplification method of pouch fat mesenchymal cell.
Background technology
The extraction and the application of pouch fat mesenchymal cell are new problems, do not see relevant report at present as yet.
Summary of the invention
The object of the invention is exactly the deficiency to prior art; And amplification method is preserved and is cultivated in the separation that a kind of pouch fat mesenchymal cell is provided; It can extract pouch mesenchymal cell and prolonged preservation and cultivation in a large number, turns out and comes the reparation that mesenchymal cell can be used for promoting union of wounded skin, the healing of promotion diabetic ulcer, the reparation that promotes myocardial damage, promotion liver dysfunction.
Technological solution of the present invention is following:
1, the separation store method of pouch fat mesenchymal cell, 1), under clean work station, the pouch fatty tissue is shifted out from sterile chamber, put into sterile vessel;
2), adding is aseptic and osmotic pressure that contain fungistat is the phosphate buffer soln of 270-330mOsm/kg, after the flushing, shifts out phosphate buffer soln gently;
3), fast tissue is shredded, reject yl moiety as far as possible, be all 1-2mm until tissue block length and width height with operating scissors;
4) add the resuspended tissue block of phosphate buffer soln, once more, centrifugal 5 minutes of 200g-300g discards the upper strata phosphate buffer soln, and it is subsequent use to choose the lower sediment tissue block;
5), get 6 holes-24 well culture plate, one piece of piece tissue block is at the most implanted with the ophthalmology tweezer in every hole, adds with deckglass to cover, and adds a small amount of adherent culture base to wetting bottom surface;
6), calculate by 24 orifice plates, every hole adds has prepared mescenchymal stem cell nutrient solution 0.3-0.5ml completely, at 36-38 ℃ of 1%-5%CO
2After cultivating 48h under the condition, every hole adds 0.3-0.5ml again and has prepared mescenchymal stem cell nutrient solution completely, and amount was changed liquid once in per 3 days half;
7), tissue block implanted about 3 days, when visible when the spindle shape cell that is dispersed in is arranged around the tissue block, amount was changed liquid once in per 3 days half;
8) primary cell of, cultivating to every hole increases to 70%～80% and merges;
9), shift out the substratum in the culture plate, every hole adds 1-1.5ml sterile phosphate buffered soln, draws the soft flushing culture layer of phosphate buffer soln with suction pipe, removes phosphate buffer soln, repeats once;
10), every hole adds the 0.2ml Digestive system, makes abundant infiltrations back jog culturing bottle, about 1-3 minute, the translucent cellular layer of visual inspection bottle wall when seeing when space, fine needle hole occurring, discarded Digestive system, and added the termination of 1ml perfect medium and digest;
11), draw cell suspension with suction pipe, after the soft flushing culture layer, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
12), get 300g, under 4 ℃ of temperature condition, behind the centrifugal 5min, abandoning supernatant;
13), adopt the differential adherent method, discard the attached cell in 30 minutes, attached cell continues adherent culture, discards cell not adherent in 1 hour;
14), to adopt density be the lymphocyte separation medium of 1.073g/ml-1.077g/ml for the attached cell that filters out, adopts density gradient centrifugation centrifugal;
15), cell that layering screen is selected is with 10ml mescenchymal stem cell substratum piping and druming mixing, imports 2 T75 culturing bottles into, supplies 5ml mescenchymal stem cell substratum in each culturing bottle, under 37 ℃, 5%CO2 condition, cultivate;
16), passage cell grows to inferior converging state;
17), use the frozen fatty mesenchymal cell of frozen storing liquid of the DMSO 99.8MIN. component that contains 7%-10%.
Said fungistat is that the benzalkonium chloride of 0.002%-0.01% is configured among the PBS, and every milliliter of PBS contains kantlex 20-100 milligram; The prescription of PBS is dissolved in the tri-distilled water about 900ml for taking by weighing NaCl8g, KCl0.2g, Na2HPO412H2O3.489g, KH2PO40.2g, adjusts pH value between the 7.3-7.4 with HCl or NaOH.
Pouch fat mesenchymal cell is cultivated amplification method, 1), with vaccinated fat mesenchymal stem cell, under 37 ℃, 5%CO2 condition, cultivate and reach 70%～80% and merge,
2), shift out the substratum in the culturing bottle, every bottle adds after 5ml sterile phosphate buffered soln draws phosphate buffer soln and wash culture layer repeatedly with suction pipe, removes phosphate buffer soln, repeats once;
3), add the 3ml Digestive system, make abundant infiltrations back jog culturing bottle, about 1-3 minute, the translucent cellular layer of visual inspection bottle wall when seeing when space, fine needle hole occurring, discarded Digestive system, and added the termination of 5ml perfect medium and digest;
4), draw cell suspension with suction pipe, wash culture layer repeatedly after, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
5), 300g, 4 ℃, centrifugal 5 minutes, abandoning supernatant;
6), with sedimentary cell with mescenchymal stem cell solid support medium piping and druming mixing, add in the paper carrier culturing bottle, under 37 ℃ of conditions, roll screen and cultivated 3-4 days, prepare frozen;
7), equilibrate to 4 ℃ to cells frozen storing liquid;
8), Digestive system and fresh culture are placed in the room temperature, or shake;
9), with 75% ethanol disinfection medium bottle outer wall, be placed on the super clean bench;
10), the paper carrier culturing bottle is taken out, at the bottom of 75% ethanol cotton balls sterilizing bottle;
11), inhale with suction pipe and to abandon old substratum, change suction pipe, add equivalent cell washing liquid, jiggle and make it cover whole culturing bottle, inhale and abandon cell washing liquid, change suction pipe, repeated washing once;
12), add the 25ml cell dissociation buffer, rotation paper carrier culturing bottle digest 2 minutes, Digestive system is drawn in piping and druming, stops digesting;
13) add the 25ml cell dissociation buffer, once more, rotation paper carrier culturing bottle digested 2 minutes, and Digestive system is drawn in piping and druming, stops digestion.
14), merge Digestive system, 300g, 4 ℃, centrifugal 5 minutes, collect the part supernatant, do bacterium, detection of mycoplasma;
15), discard all the other supernatants, cell precipitation suspends with an amount of 4 ℃ of cells frozen storing liquids;
16), cell counting, adjustment cell cryopreservation density is to 106/ml, cell suspension collected add in the 1.8ml freeze pipe, labeled cell batch and density are in tube wall external fixation position.
Said Digestive system prescription is for taking by weighing trypsinase 0.1g-0.25, and adding PH is the 100ml PBS of 7.0-7.4, stirs, and it is dissolved fully, is placed in after packing after the filtration sterilization in 4 ℃ of refrigerators to get final product.
Beneficial effect of the present invention is: it can extract pouch mesenchymal cell and prolonged preservation and cultivation in a large number, turns out and comes the reparation that mesenchymal cell can be used for promoting union of wounded skin, the healing of promotion diabetic ulcer, the reparation that promotes myocardial damage, promotion liver dysfunction.
Embodiment:
Embodiment: 1, the separation store method of pouch fat mesenchymal cell, 1), under clean work station, the pouch fatty tissue is shifted out from sterile chamber, put into sterile vessel;
2), adding is aseptic and osmotic pressure that contain fungistat is the phosphate buffer soln of 270-330mOsm/kg, after the flushing, shifts out phosphate buffer soln gently;
3), fast tissue is shredded, reject yl moiety as far as possible, be all 1-2mm until tissue block length and width height with operating scissors;
4), tissue block adds and to add phosphate buffer soln once more, centrifugal 5 minutes of 200g-300g discards the upper strata phosphate buffer soln, it is subsequent use to choose the lower sediment tissue block;
5), get 6 holes-24 well culture plate, one piece or many pieces of tissue block are implanted with the ophthalmology tweezer in every hole, add with deckglass to cover, and add a small amount of adherent culture base to wetting bottom surface;
6), calculate by 24 orifice plates, every hole adds has prepared mescenchymal stem cell nutrient solution 03-0.5ml completely, at 36-38 ℃ of 1%-5%CO
2After cultivating 48h under the condition, every hole adds 0.3-0.5ml again and has prepared mescenchymal stem cell nutrient solution completely, and amount was changed liquid once in per 3 days half;
7), tissue block implanted about 3 days, when visible when the spindle shape cell that is dispersed in is arranged around the tissue block, amount was changed liquid once in per 3 days half;
8) primary cell of, cultivating to every hole increases to 70%～80% and merges;
9), shift out the substratum in the culture plate, every hole adds 1-1.5ml sterile phosphate buffered soln, draws the soft flushing culture layer of phosphate buffer soln with suction pipe, removes phosphate buffer soln, repeats once;
10), every hole adds the 0.2ml Digestive system, makes abundant infiltrations back jog culturing bottle, generally digestion time was controlled within 3 minutes; The translucent cellular layer of visual inspection bottle wall; When seeing when space, fine needle hole occurring, discard Digestive system, and add the 1ml perfect medium and stop digestion;
11), draw cell suspension with suction pipe, after the soft flushing culture layer, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
12), get 300g, under 4 ℃ of temperature condition, behind the centrifugal 5min, abandoning supernatant;
13), adopt the differential adherent method, discard the attached cell in 30 minutes, attached cell continues adherent culture, discards cell not adherent in 1 hour;
14), to adopt density be the lymphocyte separation medium of 1.073g/ml-1.077g/ml for the attached cell that filters out, adopts density gradient centrifugation centrifugal;
15), cell that layering screen is selected is with 10ml mescenchymal stem cell substratum piping and druming mixing, imports 2 T75 culturing bottles into, supplies 5ml mescenchymal stem cell substratum in each culturing bottle, under 37 ℃, 5%CO2 condition, cultivate;
16), passage cell grows to inferior converging state;
17), use the frozen fatty mesenchymal cell of frozen storing liquid of the DMSO 99.8MIN. component that contains 7%-10%.
Said fungistat is that the benzalkonium chloride of 0.002%-0.01% is configured among the PBS, and every milliliter of PBS contains kantlex 20-100 milligram; The prescription of PBS is dissolved in the tri-distilled water about 900ml for taking by weighing NaCl8g, KCl0.2g, Na2HPO412H2O3.489g, KH2PO40.2g, adjusts pH value between the 7.3-7.4 with HCl or NaOH.
Pouch fat mesenchymal cell is cultivated amplification method, 1), with vaccinated fat mesenchymal stem cell, under 37 ℃, 5%CO2 condition, cultivate and reach 70%～80% and merge,
2), shift out the substratum in the culturing bottle, every bottle adds after 5ml sterile phosphate buffered soln draws phosphate buffer soln and wash culture layer repeatedly with suction pipe, removes phosphate buffer soln, repeats once;
3), add the 3ml Digestive system, make abundant infiltrations back jog culturing bottle, general digestion time was controlled within 3 minutes, the translucent cellular layer of visual inspection bottle wall when seeing when space, fine needle hole occurring, discards Digestive system, and adds the 5ml perfect medium and stop digesting;
4), draw cell suspension with suction pipe, wash culture layer repeatedly after, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
5), 300g, 4 ℃, centrifugal 5 minutes, abandoning supernatant;
6), with sedimentary cell with mescenchymal stem cell solid support medium piping and druming mixing, add in the paper carrier culturing bottle, under 37 ℃ of conditions, roll screen and cultivated 3-4 days, prepare frozen;
7), equilibrate to 4 ℃ to cells frozen storing liquid;
8), Digestive system and fresh culture are placed in the room temperature, or shake;
9), with 75% ethanol disinfection medium bottle outer wall, be placed on the super clean bench;
10), the paper carrier culturing bottle is taken out, at the bottom of 75% ethanol cotton balls sterilizing bottle;
11), inhale with suction pipe and to abandon old substratum, change suction pipe, add equivalent cell washing liquid, jiggle and make it cover whole culturing bottle, inhale and abandon cell washing liquid, change suction pipe, repeated washing once;
12), add the 25ml cell dissociation buffer, rotation paper carrier culturing bottle digest 2 minutes, Digestive system is drawn in piping and druming, stops digesting;
13) add the 25ml cell dissociation buffer, once more, rotation paper carrier culturing bottle digested 2 minutes, and Digestive system is drawn in piping and druming, stops digestion.
14), merge Digestive system, 300g, 4 ℃, centrifugal 5 minutes, collect the part supernatant, do bacterium, detection of mycoplasma;
15), discard all the other supernatants, cell precipitation suspends with an amount of 4 ℃ of cells frozen storing liquids;
16), cell counting, adjustment cell cryopreservation density is to 106/ml, cell suspension collected add in the 1.8ml freeze pipe, labeled cell batch and density are in tube wall external fixation position.
Said Digestive system prescription is for taking by weighing trypsinase 0.1g-0.25, and adding PH is the 100ml PBS of 7.0-7.4, stirs, and it is dissolved fully, is placed in after packing after the filtration sterilization in 4 ℃ of refrigerators to get final product.
Claims (4)
1. the separation store method of pouch fat mesenchymal cell is characterized in that:
1), under clean work station, the pouch fatty tissue is shifted out from sterile chamber, put into sterile vessel;
2), adding is aseptic and osmotic pressure that contain fungistat is the phosphate buffer soln of 270-330mOsm/kg, after the flushing, shifts out phosphate buffer soln gently;
3), fast tissue is shredded, reject yl moiety, until the about 2mm * 2mm of tissue block size * 2mm with operating scissors as far as possible;
4) add the resuspended tissue block of phosphate buffer soln, once more, centrifugal 5 minutes of 200g-300g discards the upper strata phosphate buffer soln, and it is subsequent use to choose the lower sediment tissue block;
5), get 6 holes-24 well culture plate, one piece of piece tissue block is at the most implanted with the ophthalmology tweezer in every hole, adds with deckglass to cover, and adds a small amount of adherent culture base to wetting bottom surface;
6), calculate by 24 orifice plates, every hole adds has prepared mescenchymal stem cell nutrient solution 0.3-0.5ml completely, at 36-38 ℃ of 1%-5%CO
2After cultivating 48h under the condition, every hole adds 0.3-0.5ml again and has prepared mescenchymal stem cell nutrient solution completely, and amount was changed liquid once in per 3 days half;
7), tissue block implanted about 3 days, when visible when the spindle shape cell that is dispersed in is arranged around the tissue block, amount was changed liquid once in per 3 days half;
8) primary cell of, cultivating to every hole increases to 70%～80% and merges;
9), shift out the substratum in the culture plate, every hole adds 1-1.5ml sterile phosphate buffered soln, draws the soft flushing culture layer of phosphate buffer soln with suction pipe, removes phosphate buffer soln, repeats once;
10), every hole adds the 0.2ml Digestive system, makes abundant infiltrations back jog culturing bottle, about 1-3 minute, the translucent cellular layer of visual inspection bottle wall when seeing when space, fine needle hole occurring, discarded Digestive system, and added the termination of 1ml perfect medium and digest;
11), draw cell suspension with suction pipe, after the soft flushing culture layer, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
12), get 300g, under 4 ℃ of temperature condition, behind the centrifugal 5min, abandoning supernatant;
13), adopt the differential adherent method, discard the attached cell in 30 minutes, attached cell continues adherent culture, discards cell not adherent in 1 hour;
14), to adopt density be the lymphocyte separation medium of 1.073g/ml-1.077g/ml for the attached cell that filters out, adopts density gradient centrifugation centrifugal;
15), cell that layering screen is selected is with 10ml mescenchymal stem cell substratum piping and druming mixing, imports 2 T75 culturing bottles into, supplies 5ml mescenchymal stem cell substratum in each culturing bottle, under 37 ℃, 5%CO2 condition, cultivate;
16), passage cell grows to inferior converging state;
17), use the frozen fatty mesenchymal cell of frozen storing liquid of the DMSO 99.8MIN. component that contains 7%-10%.
2. the separation store method of pouch fat mesenchymal cell according to claim 1, it is characterized in that: fungistat is that the benzalkonium chloride of 0.002%-0.01% is configured among the PBS, every milliliter of PBS contains kantlex 20-100 milligram; The prescription of PBS is for taking by weighing NaCl8g, KCl0.2g, Na
2HPO
412H
2O3.489g, KH
2PO
40.2g, be dissolved in the tri-distilled water about 900ml, adjust pH value between the 7.3-7.4 with HCl or NaOH.
3. pouch fat mesenchymal cell is cultivated amplification method, it is characterized in that:
1), with vaccinated fat mesenchymal stem cell, at 37 ℃, 5%CO
2Cultivation reaches 70%～80% fusion under the condition,
2), shift out the substratum in the culturing bottle, every bottle adds after 5ml sterile phosphate buffered soln draws phosphate buffer soln and wash culture layer repeatedly with suction pipe, removes phosphate buffer soln, repeats once;
3), add the 3ml Digestive system, make abundant infiltrations back jog culturing bottle, about 1-3 minute, the translucent cellular layer of visual inspection bottle wall when seeing when space, fine needle hole occurring, discarded Digestive system, and added the termination of 5ml perfect medium and digest;
4), draw cell suspension with suction pipe, wash culture layer repeatedly after, the collecting cell suspension in sterile chamber, mixing, sampling cell counting;
5), 300g, 4 ℃, centrifugal 5 minutes, abandoning supernatant;
6), with sedimentary cell with mescenchymal stem cell solid support medium piping and druming mixing, add in the paper carrier culturing bottle, under 37 ℃ of conditions, roll screen and cultivated 3-4 days, prepare frozen;
7), equilibrate to 4 ℃ to cells frozen storing liquid;
8), Digestive system and fresh culture are placed in the room temperature, or shake;
9), with 75% ethanol disinfection medium bottle outer wall, be placed on the super clean bench;
10), the paper carrier culturing bottle is taken out, at the bottom of 75% ethanol cotton balls sterilizing bottle;
11), inhale with suction pipe and to abandon old substratum, change suction pipe, add equivalent cell washing liquid, jiggle and make it cover whole culturing bottle, inhale and abandon cell washing liquid, change suction pipe, repeated washing once;
12), add the 25ml cell dissociation buffer, rotation paper carrier culturing bottle digest 2 minutes, Digestive system is drawn in piping and druming, stops digesting;
13) add the 25ml cell dissociation buffer, once more, rotation paper carrier culturing bottle digested 2 minutes, and Digestive system is drawn in piping and druming, stops digestion.
14), merge Digestive system, 300g, 4 ℃, centrifugal 5 minutes, collect the part supernatant, do bacterium, detection of mycoplasma;
15), discard all the other supernatants, cell precipitation suspends with an amount of 4 ℃ of cells frozen storing liquids;
16), cell counting, adjustment cell cryopreservation density is to 106/ml, cell suspension collected add in the 1.8ml freeze pipe, labeled cell batch and density are in tube wall external fixation position.
4. pouch fat mesenchymal cell according to claim 3 is cultivated amplification method; It is characterized in that: said Digestive system prescription is for taking by weighing trypsinase 0.1g-0.25g; Adding PH is the 100ml PBS of 7.0-7.4; Stir, it is dissolved fully, be placed in after packing after the filtration sterilization in 4 ℃ of refrigerators and get final product.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100075146A CN102586180A (en) | 2011-01-14 | 2011-01-14 | Separation, storage culture and amplification method of pouch fat interstitial cell |
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|---|---|---|---|
| CN2011100075146A CN102586180A (en) | 2011-01-14 | 2011-01-14 | Separation, storage culture and amplification method of pouch fat interstitial cell |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849597A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Scale production process of adipose-derived stem cells |
| CN107164326A (en) * | 2017-06-28 | 2017-09-15 | 北京再生生物科技研究院有限公司 | A kind of method of the neural precursor in 3D culture autologous fats MSCs sources |
| CN107384857A (en) * | 2017-08-02 | 2017-11-24 | 广州中科博雅干细胞科技有限公司 | The cultural method of autologous fat mescenchymal stem cell and the culture medium used |
| CN109423479A (en) * | 2017-09-05 | 2019-03-05 | 江苏齐氏生物科技有限公司 | A kind of rat marrow macrophage isolation and culture method |
| CN109566604A (en) * | 2019-01-31 | 2019-04-05 | 北京华龛生物科技有限公司 | A kind of method that three-dimensional microcarrier In-situ condensation saves cell |
-
2011
- 2011-01-14 CN CN2011100075146A patent/CN102586180A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849597A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Scale production process of adipose-derived stem cells |
| CN107164326A (en) * | 2017-06-28 | 2017-09-15 | 北京再生生物科技研究院有限公司 | A kind of method of the neural precursor in 3D culture autologous fats MSCs sources |
| CN107164326B (en) * | 2017-06-28 | 2020-04-10 | 北京再生生物科技研究院有限公司 | Method for 3D culture of autologous adipose MSCs (mesenchymal stem cells) derived neural precursor cells |
| CN107384857A (en) * | 2017-08-02 | 2017-11-24 | 广州中科博雅干细胞科技有限公司 | The cultural method of autologous fat mescenchymal stem cell and the culture medium used |
| CN107384857B (en) * | 2017-08-02 | 2020-07-31 | 广州中科博雅干细胞科技有限公司 | Culture method of autologous adipose-derived mesenchymal stem cells and culture medium used in culture method |
| CN109423479A (en) * | 2017-09-05 | 2019-03-05 | 江苏齐氏生物科技有限公司 | A kind of rat marrow macrophage isolation and culture method |
| CN109566604A (en) * | 2019-01-31 | 2019-04-05 | 北京华龛生物科技有限公司 | A kind of method that three-dimensional microcarrier In-situ condensation saves cell |
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Application publication date: 20120718 |