CN115960825A - Preparation method of adipose-derived stem cells - Google Patents

Abstract

The invention discloses a method for preparing adipose-derived stem cells, which belongs to the technical field of stem cell preparation methods, and comprises the steps of collecting 50ml of adipose tissue samples into a collection bottle, spraying alcohol, placing the collection bottle into a biological safety cabinet, standing for more than 5min, preparing a plurality of 5-10 corresponding centrifuge tubes, adding 20ml of physiological saline into each centrifuge tube for later use, sucking lower liquid in the adipose samples in the layered collection bottle after standing by a 25ml pipette, then sucking adipose tissues by a 25ml pipette, adding 20-25ml of isovolumetric adipose tissues into each centrifuge tube, covering the centrifuge tubes, turning the centrifuge tubes upside down for several times, shaking up, wherein the upper layer is a grease layer, the middle layer is an adipose tissue layer, the lower layer is swollen liquid layer, and carrying out balance centrifugation on the adipose tissues after the treatment and separation. The adipose-derived stem cells cultured without serum can improve the activity of the adipose-derived stem cells and promote the proliferation of the adipose-derived stem cells.

Description

Preparation method of adipose-derived stem cells

Technical Field

The invention relates to a stem cell preparation method, in particular to an adipose-derived stem cell preparation method, and belongs to the technical field of stem cell preparation methods.

Background

Adult stem cells are the most promising cell type based on cell therapy in regenerative medicine, human adipose tissue has been introduced as a new source of pluripotent stem cells, these so-called adipose stem cells (ADSCs) are considered as the ideal choice for regenerative therapy applications, their main advantages compared to mesenchymal stem cells derived from other sources (e.g. bone marrow) are that they can be easily and reproducibly acquired using minimally invasive techniques and the incidence of disease is low.

ADSCs are pluripotent and can differentiate into various cell types of the trioblast lineage, including, for example, bone cells, fat cells, nerve cells, vascular endothelial cells, cardiac muscle cells, pancreatic beta cells and liver cells, and are characterized by immunosuppressive properties and low immunogenicity. Their secreted trophic factors potentiate therapeutic and regenerative outcomes in a wide range of applications. Therefore, the therapeutic potential of ADSCs is enormous.

Bone marrow derived MSCs are not suitable for clinical use and in order to find alternative stem cell sources in the early 21 st century, zuk et al introduced a multipotent, undifferentiated, self-renewing progenitor cell population that was isolated from adipose tissue and similar in morphology and phenotype to MSCs, these so-called adipose tissue-derived stem cells (ADSCs) showed differentiation capacity similar to MSCs and expression of specific stem cell markers in vivo, importantly, the easy repeated harvesting of subcutaneous adipose tissue by minimally invasive methods, simple isolation procedures and the quality and proliferative capacity of stem cells that did not decline with patient age summarize the advantages of ADSCs as an alternative clinical cell source and provide significant advantages over bone marrow derived MSCs.

Stem cells, including ADSCs, have become a key element of regenerative medicine therapy due to their ability to differentiate into a variety of different cell lineages.

The method has the problem that the extraction is complex and the extraction purity is not high enough in the process of preparing the adipose-derived stem cells in the prior art.

Therefore, a method for preparing adipose-derived stem cells is designed to solve the problems.

Disclosure of Invention

The invention mainly aims to provide a method for preparing adipose-derived stem cells, which comprises the steps of collecting 50mL of adipose tissue samples into a collection bottle, spraying alcohol, placing the collection bottle into a biological safety cabinet, standing for more than 5min, preparing a plurality of 5-10 corresponding centrifuge tubes, adding 20mL of physiological saline into each centrifuge tube for standby, sucking the lower liquid layer in the layered collection bottle after standing by a 25mL pipette, sucking the adipose tissues by a 25mL pipette, adding 20-25mL of the same volume of adipose tissues into each centrifuge tube, covering the centrifuge tube, turning the centrifuge tube upside down for several times, shaking up, balancing and centrifuging the adipose tissues separated by the above steps, wherein the balancing is realized by using an electronic balance, the centrifugal rotation is 1500rpm, the centrifugation is performed for 5min, the adipose tissues in the third step are centrifuged, centrifuging, separating the upper layer of grease and the lower layer of cleaning solution, sucking adipose tissue into a clean centrifuge tube with 25mL pipette, adding equal amount of adipose tissue into each tube, keeping away from white fascia tissue, adding equal amount of normal saline not more than 45mL into the centrifuge tube with adipose tissue, covering with a cover, turning upside down, shaking, centrifuging for the second time, sucking out adipose grease and lower layer of cleaning solution, keeping away from white fascia tissue, sucking out adipose tissue into a clean centrifuge tube with 25mL pipette, putting the cleaned adipose tissue into a 50mL centrifuge tube, finely shearing the tissue with a bent tip for about 300 times, shearing 1mL of the sheared tissue to weight 0.8g, keeping the amount of adipose tissue 15-25mL, adding normal saline to total volume of 40mL, mixing, dissolving 30mg of collagenase I in 10mL of PBS solution, 0.22um filter sterilized and 2-fold diluted to obtain collagenase I with concentration of 1mg/ml, adding the washed fat to 1mg/ml collagenase I in a ratio of 1:1, placing the mixture in a constant-temperature shaking culture bed, digesting the mixture for 40 minutes at 37 ℃ and 130rpm, centrifuging the mixture for 10 minutes at 1300rpm after digestion is finished, removing supernatant, precipitating adipose-derived stem cells at the bottom, resuspending, washing and centrifuging the mixture for 2 times by PBS (phosphate buffer solution), repeating the ten steps, transferring the supernatant to a 50mL centrifuge tube after centrifugation, and adding 1.5-2.0 mL of the supernatant into a freezing tube for sample retention.

The purpose of the invention can be achieved by adopting the following technical scheme:

a method for preparing adipose-derived stem cells comprises the following steps:

the method comprises the following steps: collecting 50ml adipose tissue samples into a collection bottle, spraying alcohol, placing into a biological safety cabinet, and standing for more than 5min;

preparing a plurality of 5-10 corresponding centrifugal tubes, and adding 20ml of physiological saline into each centrifugal tube for later use;

step two: sucking the lower layer liquid in the fat sample in the layered collection bottle after standing by using a 25ml pipette, discarding, sucking the fat tissue by using the 25ml pipette, adding 20-25ml of the fat tissue with the same volume into each centrifuge tube, covering the centrifuge tube, turning the centrifuge tube upside down for several times, and shaking up;

wherein the upper layer is a grease layer, the middle layer is a fat tissue layer, and the lower layer is a swelling liquid layer;

step three: balancing and centrifuging the adipose tissues after the treatment and separation;

wherein the balancing adopts an electronic balance for balancing, the centrifugal rotation is 1500rpm, and the centrifugation is carried out for 5min;

step four: centrifuging the adipose tissues in the third step, dividing the adipose tissues into an upper layer, a middle layer and a lower layer after centrifugation, sucking out the grease on the upper layer and the cleaning solution on the lower layer, discarding the grease on the upper layer and the cleaning solution on the lower layer, sucking the adipose tissues into a clean centrifugal tube by using a 25ml pipette, adding the same amount of adipose tissues into each tube, and avoiding white fascia tissues;

step five: adding equal volume of physiological saline into the centrifuge tube with adipose tissue not more than 45ml, covering the cover, turning upside down for several times, shaking up, and centrifuging for the second time;

step six: after centrifugation, the fatty oil and the lower-layer cleaning solution are sucked out, the white fascia tissue is avoided, and the fatty tissue is sucked out into a clean centrifugal tube by a 25ml pipette;

step seven: putting the cleaned adipose tissues into a 50mL centrifuge tube, finely shearing the minced tissues by using a bent-tip end for about 300 times, wherein 1mL of the minced tissues is 0.8g in weight and 15-25mL in weight, adding normal saline till the total volume is 40mL, and uniformly mixing;

step eight: dissolving 30mg of collagenase I in 10ml of PBS solution, filtering and sterilizing at 0.22um, and diluting by 2 times to obtain collagenase I with the concentration of 1 mg/ml;

step nine: the washed fat was added to 1mg/ml collagenase I in a ratio of 1:1, placing the mixture in a constant-temperature shaking culture bed, and digesting the mixture for 40 minutes at 37 ℃ and 130 rpm;

step ten: centrifuging at 1300rpm for 10 minutes after digestion is finished, removing supernatant, precipitating at the bottom to be adipose-derived stem cells, and carrying out resuspension washing and centrifugation for 2 times by using PBS (phosphate buffer solution);

repeating the above ten operations, transferring the supernate to a 50mL centrifuge tube after centrifugation, and adding 1.5-2.0 mL of supernate into a freezing tube for sample retention.

Preferably, 1mL of tissue after clipping in step seven weighs 0.8g.

Preferably, after the ninth step, the mixture is centrifuged at 1300rpm for 10 minutes after digestion, the supernatant is removed, the bottom sediment is adipose-derived stem cells, PBS is resuspended, washed and centrifuged for 2 times, 1ml of adipose tissue is inoculated into a T175 cell culture flask for inoculation and culture according to the adipose tissue obtained after washing, 22ml of culture medium is added into each T175 culture flask, the culture label is marked, the mixture is shaken evenly and placed into a constant temperature incubator at 37 ℃ and 5 percent CO2 for static culture.

Preferably, the method also comprises the steps of changing the liquid of the cells, and pouring the old liquid into a 50mL centrifugal tube by inclining a bottle mouth;

adding new F-M-N medium T175 into 22mL, adding 12mL into T75 flask, adding 5mL into T25 flask, tightening bottle cap to make medium flat on the culture surface, adding 5% CO2, and culturing at 37 deg.C in incubator;

the liquid changing period is adjusted according to different cell conditions.

Preferably, the liquid is changed under the aseptic operation condition and the cross contamination among cells is prevented, the operation of only operating the same donor cell each time, and the pipette is only used for sucking the same liquid;

the liquid changing period is adjusted according to different cell conditions;

observing the cell morphology every day, and mastering the standard whether the cells are healthy or not: healthy cells are full in shape and good in refractivity, and can be passaged when growing compactly.

Preferably, the method also comprises the following steps of trypsinization after cell liquid change:

the method comprises the following steps: sucking out the old culture solution in a 50mL centrifuge tube, washing the culture flask with 10mL0.9% sodium chloride injection, sucking out 0.9% sodium chloride injection, and adding 2.5mL recombinant pancreatin solution;

wherein, the added culture medium inoculated before the culture process of the F-M-N culture medium is an old culture medium, namely culture stock solution;

step two: observing the digested cells under an inverted microscope, wherein if cytoplasm is retracted, the cells are not connected into slices;

step three: adding 5mL of old culture solution to terminate digestion, blowing and beating dispersed cells, repeatedly blowing and beating the digested cells by using a pipette to remove walls and disperse the cells;

step four: sucking the cell suspension into a 50mL centrifuge tube, and adding 0.9% sodium chloride injection to 40mL;

step five: and (3) turning the centrifugal tube upside down and mixing uniformly, putting the centrifugal tube into a centrifugal machine, balancing, centrifuging for 3 minutes at 2000 rpm, removing supernate, slightly poking the bottom of the centrifugal tube to break up cells, and adding a proper amount of fresh culture medium to prepare cell suspension.

Preferably, cell passage is also included after cell exchange, and the specific steps are as follows:

the method comprises the following steps: taking 0.2mL of the uniformly mixed cell suspension into a 0.6mLEP tube, and counting in a counter;

step two: determining the passage amount according to the counting result, taking a corresponding amount of culture bottles, and marking;

step three: adding 22mLF-M-N culture medium;

step four: sucking the cell suspension according to the volume of the cell suspension, subpackaging the cell suspension into a culture bottle, screwing down a bottle cap to enable the cell suspension to be tiled on a culture surface, and uniformly mixing the cell suspension in a infinity form on a table;

step five: the culture bottle is put into a CO2 incubator for culture.

The invention has the beneficial technical effects that:

the invention provides a method for preparing adipose-derived stem cells, which comprises the steps of collecting 50mL of adipose tissue samples into a collection bottle, spraying alcohol, placing the collection bottle into a biological safety cabinet, standing for more than 5min, preparing a plurality of 5-10 corresponding centrifuge tubes, adding 20mL of physiological saline into each centrifuge tube for later use, sucking the lower layer liquid in the layered fat samples in the collection bottle after standing by a 25mL pipette, sucking the adipose tissues by a 25mL pipette, adding 20-25mL of the same volume of adipose tissues into each centrifuge tube, covering a cover, turning the tubes upside down for a plurality of times, shaking up uniformly, wherein the upper layer is a grease layer, the middle layer is an adipose tissue layer, the lower layer is a swelling liquid layer, balancing and centrifuging the adipose tissues after the processing and separation, wherein the balancing is performed by an electronic balance, the centrifugal rotation is 1500rpm, the centrifugation is performed for 5min, and the adipose tissues in the third step are centrifuged, centrifuging, separating the upper layer from the middle layer, removing the upper layer of grease and the lower layer of cleaning solution, sucking adipose tissues into a clean centrifuge tube by using a 25mL pipette, adding an equal amount of adipose tissues into each tube to avoid white fascia tissues, adding equal volume of physiological saline into the centrifuge tube with the adipose tissues, the total volume of which is not more than 45mL, covering a cover, turning upside down for several times, shaking up, centrifuging for the second time by using the above operation, sucking out the adipose tissues and the lower layer of cleaning solution after centrifuging, sucking out the adipose tissues to avoid the white fascia tissues by using the 25mL pipette, putting the cleaned adipose tissues into a 50mL centrifuge tube, shearing and finely shearing the tissues by using a bent tip for about 300 times, shearing 1mL of the sheared tissues to be 0.8g, uniformly mixing 15-25mL of the adipose tissues, adding the physiological saline to the total volume of 40mL, dissolving 30mg of collagenase I in 10mL of PBS solution, 0.22um filter sterilized and 2-fold diluted to obtain collagenase I with concentration of 1mg/ml, adding the washed fat to 1mg/ml collagenase I in a ratio of 1:1, placing the mixture in a constant-temperature shaking culture bed, digesting the mixture for 40 minutes at 37 ℃ and 130rpm, centrifuging the mixture for 10 minutes at 1300rpm after digestion is finished, removing supernatant, precipitating adipose-derived stem cells at the bottom, resuspending, washing and centrifuging the mixture for 2 times by PBS (phosphate buffer solution), repeating the ten steps, transferring the supernatant to a 50mL centrifuge tube after centrifugation, and adding 1.5-2.0 mL of the supernatant into a freezing tube for sample retention.

Drawings

FIG. 1 is a photograph of primary adipose-derived stem cells isolated and photographed under a 4X-ray microscope according to a preferred embodiment of a method for preparing adipose-derived stem cells according to the present invention;

FIG. 2 is a 4 Xmicroscope photograph of isolated and extracted P1 generation adipose-derived stem cells according to a preferred embodiment of the adipose-derived stem cell preparation method of the present invention;

FIG. 3 is a photograph of isolated and extracted P2 generation adipose-derived stem cells observed under a 4 Xmirror, according to a preferred embodiment of a method for preparing adipose-derived stem cells according to the present invention;

FIG. 4 is a photograph of isolated and extracted P3 generation adipose-derived stem cells observed under a 4 Xmirror for a preferred embodiment of a method for preparing adipose-derived stem cells according to the present invention;

FIG. 5 is a photograph of isolated and extracted P4 generation adipose-derived stem cells observed under a 4X mirror, according to a preferred embodiment of a method for preparing adipose-derived stem cells according to the present invention.

Detailed Description

In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

As shown in fig. 1 to fig. 5, the method for preparing adipose-derived stem cells provided in this embodiment includes the following steps:

the method comprises the following steps: collecting 50ml adipose tissue samples into a collection bottle, spraying alcohol, placing into a biological safety cabinet, and standing for more than 5min;

preparing a plurality of 5-10 corresponding centrifugal tubes, and adding 20ml of physiological saline into each centrifugal tube for later use;

step two: sucking lower layer liquid in a fat sample in a layered collection bottle after standing by using a 25ml pipette, discarding, sucking adipose tissues by using the 25ml pipette, adding 20-25ml of adipose tissues with the same volume into each centrifugal tube, covering the centrifugal tube, turning upside down for several times, and shaking up;

wherein the upper layer is a grease layer, the middle layer is a fat tissue layer, and the lower layer is a swelling liquid layer;

step three: balancing and centrifuging the adipose tissues after the treatment and separation;

wherein the balancing adopts an electronic balance for balancing, the centrifugal rotation is 1500rpm, and the centrifugation is carried out for 5min;

step four: centrifuging the adipose tissues in the third step, dividing the adipose tissues into an upper layer, a middle layer and a lower layer after centrifugation, sucking out the grease on the upper layer and the cleaning solution on the lower layer, discarding the grease on the upper layer and the cleaning solution on the lower layer, sucking the adipose tissues into a clean centrifugal tube by using a 25ml pipette, adding the same amount of adipose tissues into each tube, and avoiding white fascia tissues;

step five: adding equal volume of physiological saline into the centrifuge tube with adipose tissue not more than 45ml, covering the cover, turning upside down for several times, shaking up, and centrifuging for the second time;

step six: after centrifugation, the fatty grease and the lower layer cleaning solution are sucked out in the same way, the white fascia tissue is avoided, and the fatty tissue is sucked out into a clean centrifuge tube by a 25ml pipette;

step seven: putting the cleaned adipose tissues into a 50mL centrifuge tube, finely shearing the minced tissues by using a bent-tip end for about 300 times, wherein 1mL of the minced tissues is 0.8g in weight and 15-25mL in weight, adding normal saline till the total volume is 40mL, and uniformly mixing;

step eight: dissolving 30mg of collagenase I in 10ml of PBS solution, filtering and sterilizing at 0.22um, and diluting by 2 times to obtain collagenase I with the concentration of 1 mg/ml;

step nine: the washed fat was added to 1mg/ml collagenase I in a ratio of 1:1, placing the mixture in a constant-temperature shaking culture bed, and digesting the mixture for 40 minutes at 37 ℃ and 130 rpm;

step ten: centrifuging at 1300rpm for 10 minutes after digestion is finished, removing supernatant, precipitating at the bottom to be adipose-derived stem cells, and carrying out resuspension washing and centrifugation for 2 times by using PBS (phosphate buffer solution);

repeating the tenth operation of the steps, transferring the supernate to a 50mL centrifuge tube after centrifugation, and adding 1.5-2.0 mL of supernate into a freezing tube for sample retention.

In this example, 1mL of tissue after clipping in step seven weighs 0.8g.

In this example, after digestion was completed after step nine, the cells were centrifuged at 1300rpm for 10 minutes, the supernatant was removed, the bottom was precipitated as adipose stem cells, PBS was resuspended, washed and centrifuged 2 times, 1ml of adipose tissue was inoculated into a T175 cell culture flask for inoculation and culture, and 22ml of medium was added to each T175 flask, labeled with a culture tag, shaken, and placed in a 37 ℃ C., 5% CO2 incubator for static culture.

In this embodiment, the method further comprises changing the liquid for the cells, and pouring the old liquid into a 50mL centrifuge tube by inclining the bottle mouth;

adding new F-M-N medium T175 into 22mL, adding T75 into 12mL, adding T25 into 5mL, screwing the bottle cap to make the medium flat on the culture surface, placing into 5% CO2, and culturing at 37 deg.C incubator;

the liquid changing period is adjusted according to different cell conditions.

In the embodiment, the pipette is only used for sucking the same liquid under the sterile operation condition and preventing the cross contamination among cells during liquid changing, and only the operation of the same donor cell is operated each time;

the liquid changing period is adjusted according to different cell conditions;

observing the cell morphology every day, and mastering the standard whether the cells are healthy or not: healthy cells are full in shape and good in refractivity, and can be passaged when growing compactly.

In this example, the cell exchange solution further comprises the following steps of trypsinization:

the method comprises the following steps: the old culture solution was aspirated into a 50mL centrifuge tube, the flask was rinsed with 10mL0.9% sodium chloride injection and 0.9% sodium chloride injection, and 2.5mL recombinant pancreatin solution was added;

wherein, the added culture medium inoculated before the culture process of the F-M-N culture medium is an old culture medium, namely culture stock solution;

step two: observing the digested cells under an inverted microscope, wherein if cytoplasm is retracted, the cells are not connected into slices;

step three: adding 5mL of old culture solution to terminate digestion, blowing and beating dispersed cells, repeatedly blowing and beating the digested cells by using a pipette to remove walls and disperse the cells;

step four: sucking the cell suspension into a 50mL centrifuge tube, and adding 0.9% sodium chloride injection to 40mL;

step five: and (3) turning the centrifugal tube upside down and mixing uniformly, putting the centrifugal tube into a centrifugal machine, balancing, centrifuging for 3 minutes at 2000 rpm, discarding supernatant, slightly poking the bottom of the centrifugal tube to disperse cells, and adding a proper amount of fresh culture medium to prepare cell suspension.

In this embodiment, cell passaging is also included after cell exchange, and the specific steps are as follows:

the method comprises the following steps: taking 0.2mL of the uniformly mixed cell suspension into a 0.6mLEP tube, and counting in a counter;

step two: determining the passage amount according to the counting result, taking a corresponding amount of culture bottles, and marking;

step three: adding 22mLF-M-N culture medium;

step four: sucking the cell suspension according to the volume of the cell suspension, subpackaging the cell suspension into culture bottles, screwing down bottle caps to enable the cell suspension to be laid on culture surfaces, and uniformly mixing the cell suspension on a table in a mode of drawing infinity;

step five: the culture bottle is put into a CO2 incubator for culture.

The above are only further embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and its concept within the scope of the present invention.

Claims (7)

1. A method for preparing adipose-derived stem cells, which is characterized by comprising the following steps: the method comprises the following steps:

the method comprises the following steps: collecting a 50ml adipose tissue sample into a collection bottle, spraying alcohol, putting into a biological safety cabinet, and standing for more than 5min;

preparing a plurality of 5-10 corresponding centrifugal tubes, and adding 20ml of physiological saline into each centrifugal tube for later use;

step two: sucking lower layer liquid in a fat sample in a layered collection bottle after standing by using a 25ml pipette, discarding, sucking adipose tissues by using the 25ml pipette, adding 20-25ml of adipose tissues with the same volume into each centrifugal tube, covering the centrifugal tube, turning upside down for several times, and shaking up;

wherein the upper layer is a grease layer, the middle layer is a fat tissue layer, and the lower layer is a swelling liquid layer;

step three: balancing and centrifuging the adipose tissues after the treatment and separation;

wherein the balancing adopts an electronic balance for balancing, the centrifugal rotation is 1500rpm, and the centrifugation is carried out for 5min;

step four: centrifuging the adipose tissues in the third step, dividing the adipose tissues into an upper layer, a middle layer and a lower layer after centrifugation, sucking out the grease on the upper layer and the cleaning solution on the lower layer, discarding the grease on the upper layer and the cleaning solution on the lower layer, sucking the adipose tissues into a clean centrifugal tube by using a 25ml pipette, adding the same amount of adipose tissues into each tube, and avoiding white fascia tissues;

step five: adding equal volume of physiological saline into the centrifuge tube with adipose tissue not more than 45ml, covering the cover, turning upside down for several times, shaking up, and centrifuging for the second time;

step six: after centrifugation, the fatty oil and the lower-layer cleaning solution are sucked out, the white fascia tissue is avoided, and the fatty tissue is sucked out into a clean centrifugal tube by a 25ml pipette;

step seven: putting the cleaned adipose tissues into a 50mL centrifuge tube, finely shearing the minced tissues by using a bent-tip end for about 300 times, wherein 1mL of the minced tissues is 0.8g in weight and 15-25mL in weight, adding normal saline till the total volume is 40mL, and uniformly mixing;

step eight: dissolving 30mg of collagenase I in 10ml of PBS solution, filtering and sterilizing at 0.22um, and diluting by 2 times to obtain collagenase I with the concentration of 1 mg/ml;

step nine: the washed fat was added to 1mg/ml collagenase I in a ratio of 1:1, placing the mixture in a constant-temperature shaking culture bed, digesting the mixture for 40 minutes at 37 ℃ and 130 rpm;

step ten: centrifuging at 1300rpm for 10 minutes after digestion is finished, removing supernatant, precipitating at the bottom to be adipose-derived stem cells, and carrying out resuspension washing and centrifugation for 2 times by using PBS (phosphate buffer solution);

repeating the tenth operation of the steps, transferring the supernate to a 50mL centrifuge tube after centrifugation, and adding 1.5-2.0 mL of supernate into a freezing tube for sample retention.

2. The method for preparing adipose-derived stem cells according to claim 1, wherein: in step seven, 1mL of minced tissue weighs 0.8g.

3. The method for preparing adipose-derived stem cells according to claim 2, wherein: and after the ninth step, centrifuging at 1300rpm for 10 minutes after digestion is finished, removing supernatant, precipitating at the bottom to be adipose-derived stem cells, resuspending, washing and centrifuging for 2 times by using PBS, inoculating 1ml of adipose tissue into a T175 cell culture flask for inoculation and culture according to the adipose tissue obtained after washing, adding 22ml of culture medium into each T175 culture flask, marking a culture label, shaking uniformly, and placing into a constant-temperature incubator at 37 ℃ and 5 CO2 for standing and culture.

4. The method for preparing adipose-derived stem cells according to claim 3, wherein: changing the liquid of the cells, and pouring the old liquid into a 50mL centrifugal tube by inclining a bottle mouth;

adding new F-M-N medium T175 into 22mL, adding T75 into 12mL, adding T25 into 5mL, screwing the bottle cap to make the medium flat on the culture surface, placing into 5% CO2, and culturing at 37 deg.C incubator;

the liquid changing period is adjusted according to different cell conditions.

5. The method for preparing adipose-derived stem cells according to claim 4, wherein: when the liquid is changed, under the aseptic operation condition, the cross contamination among cells is prevented, the operation of only operating the same donor cell is performed each time, and the pipette is only used for sucking the same liquid;

the liquid changing period is adjusted according to different cell conditions;

observing cell morphology every day, and mastering the standard whether cells are healthy or not: healthy cells are full in shape and good in refractivity, and can be passaged when growing compactly.

6. The method for preparing adipose-derived stem cells according to claim 5, wherein: the method also comprises the following steps of trypsinization after cell exchange:

the method comprises the following steps: sucking out the old culture solution in a 50mL centrifuge tube, washing the culture flask with 10mL0.9% sodium chloride injection, sucking out 0.9% sodium chloride injection, and adding 2.5mL recombinant pancreatin solution;

wherein, the added culture medium is inoculated before the culture process of the F-M-N culture medium, and is the old culture medium, namely culture stock solution;

step two: observing the digested cells under an inverted microscope, wherein if cytoplasm is retracted, the cells are not connected into slices;

step three: adding 5mL of old culture solution to terminate digestion, blowing and beating dispersed cells, repeatedly blowing and beating the digested cells by using a pipette to remove walls and disperse the cells;

step four: sucking the cell suspension into a 50mL centrifuge tube, and adding 0.9% sodium chloride injection to 40mL;

step five: and (3) turning the centrifugal tube upside down and mixing uniformly, putting the centrifugal tube into a centrifugal machine, balancing, centrifuging for 3 minutes at 2000 rpm, removing supernate, slightly poking the bottom of the centrifugal tube to break up cells, and adding a proper amount of fresh culture medium to prepare cell suspension.

7. The method for preparing adipose-derived stem cells according to claim 6, wherein: cell passage is also included after cell exchange, and the specific steps are as follows:

the method comprises the following steps: taking 0.2mL of the uniformly mixed cell suspension, putting the cell suspension into a 0.6mL LEP tube, and counting the cell suspension in a counter;

step two: determining the passage amount according to the counting result, taking a corresponding amount of culture bottles, and marking;

step three: adding 22mLF-M-N culture medium;

step four: sucking the cell suspension according to the volume of the cell suspension, subpackaging the cell suspension into a culture bottle, screwing down a bottle cap to enable the cell suspension to be tiled on a culture surface, and uniformly mixing the cell suspension in a infinity form on a table;

step five: the culture bottle is put into a CO2 incubator for culture.

Images