CN107557331B - Method for separating and culturing human adipose-derived stem cells - Google Patents
Method for separating and culturing human adipose-derived stem cells Download PDFInfo
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Abstract
The invention relates to a method for separating and culturing human adipose-derived stem cells, which comprises the following steps: (1) digesting the lipoaspirate by using a mixed digestive enzyme solution consisting of collagenase I, collagenase II and ACCUTASE, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing the mixed cells by using a lymphocyte separation solution; (2) and (3) adopting a serum-free culture medium to culture the adipose-derived stem cells. On one hand, the invention can rapidly and effectively dissociate cells from adipose tissues, thereby not only improving the yield of stem cells and shortening the time for separating the cells, but also keeping the activity of the cells to the maximum extent; on the other hand, the culture medium has definite components, does not contain any exogenous serum component, can obviously improve the adherence capability and the proliferation capability of the adipose-derived stem cells, is beneficial to the in vitro amplification and maintenance of the dryness of the adipose-derived stem cells, and has good application prospect.
Description
Technical Field
The invention belongs to the field of cell separation and culture, and particularly relates to a method for separating and culturing human adipose-derived stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a branch of Stem Cells, and are Cells with self-replicating and multi-directional differentiation capability, and widely exist in various tissues, such as bone marrow, umbilical cord blood and umbilical cord tissue, placenta tissue, and adipose tissue. Mesenchymal stem cells have three significant characteristics: 1. mesenchymal stem cells cultured in vitro are adherently grown; 2. the mesenchymal stem cells highly express CD73, CD90 and CD105, and do not express markers such as CD31, CD34, CD45, HLA-DR, CD14, CD19 and CD11 b; 3. under a proper stimulation factor, the mesenchymal stem cells can be differentiated into cells of various tissues such as osteoblasts, adipocytes and nerve cells.
Adipose-Derived Stem Cells (ADSCs) are Stem Cells with the potential for multi-directional differentiation isolated from Adipose tissue. Research proves that the adipose-derived stem cells can be differentiated into various types of cells such as fat cells, cardiac muscle cells, nerve cells and the like under specific culture conditions. The adipose-derived stem cells have low immunogenicity, so that the transplanted allogeneic adipose-derived stem cells cannot cause strong immune rejection, and favorable conditions are provided for allogeneic transplanted adipose-derived stem cells. The adipose-derived stem cells are widely available, adipose tissues can be obtained by using methods of liposuction or fat excision, and the use of the adipose-derived stem cells does not have ethical problems. The adipose-derived stem cells have strong amplification capacity in vitro, and a large amount of adipose-derived stem cells can be obtained by an in vitro culture method. The adipose-derived stem cells are widely applied to the industries of beauty treatment and plastic shaping such as breast enlargement, wrinkle removal and the like, and play more and more roles in the medical field.
The following problems occur in the preparation and culture processes of adipose-derived stem cells: 1. during the process of separating the adipose-derived stem cells, some mixed cells can be mixed, and the adherence and growth of the adipose-derived stem cells can be influenced. 2. In the process of separating the adipose-derived stem cells, if the enzymolysis time is too long, the adipose-derived stem cells are damaged. 3. Most scientific research or medical institutions still use animal serum to culture the adipose-derived stem cells, but the quality of the serum in different batches is very different, and the components and functions of the serum cannot be kept consistent; furthermore, animal sera contain low levels of substances that inhibit cell growth, as well as potential viral and mycoplasma contamination. 4. There is some differentiation of the adipose stem cells during the culture process.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for separating and culturing human adipose-derived stem cells, which can rapidly and effectively dissociate the cells from adipose tissues on one hand, thereby not only improving the yield of the stem cells and shortening the time for separating the cells, but also keeping the activity of the cells to the maximum extent; on the other hand, the culture medium has definite components, does not contain any exogenous serum component, can obviously improve the adherence capability and the proliferation capability of the adipose-derived stem cells, is beneficial to the in vitro amplification and maintenance of the dryness of the adipose-derived stem cells, and has good application prospect.
The invention relates to a method for separating and culturing human adipose-derived stem cells, which comprises the following steps:
(1) digesting the lipoaspirate by using a mixed digestive enzyme solution consisting of collagenase I, collagenase II and ACCUTASE, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing the mixed cells by using a lymphocyte separation solution;
(2) adopting a serum-free culture medium to culture the adipose-derived stem cells, wherein the serum-free culture medium is added with the following components:
recombinant human insulin: 1-10 mug/ml;
recombinant human epidermal growth factor: 10-50 ng/ml;
recombinant human basic fibroblast growth factor: 10-50 ng/ml;
1-50 ng/ml of recombinant human transforming growth factor- β;
recombinant human platelet-derived growth factor-BB: 10-50 ng/ml;
hydrocortisone: 0.5-10 mug/ml;
vitamin C: 10-100 mug/ml;
reduced glutathione: 1 to 5 mM;
recombinant human transferrin: 0.5-10 mug/ml;
ethanolamine: 1-10 mug/mL;
l-glutamine: 1 to 10 mM;
coenzyme A: 1-50 mug/ml;
recombinant human thrombin: 1-10U/ml;
gentamicin: 1-100 mug/ml;
sodium selenite: 1-100 ng/ml;
biotin: 1-50 μ g/ml.
The mixed digestive enzyme solution in the step (1) consists of 0.1-0.4% (m/v) of collagenase I, 0.1-0.4% (m/v) of collagenase II and 1/4-1/2 × ACCUTASE (STEMCELL technologies).
The volume ratio of the mixed digestive enzyme solution to the lipoaspirate in the step (1) is 1: 1.
The digestion temperature in the step (1) is 37 ℃, and the digestion time is within half an hour.
The neutralizing digestive enzyme in the step (1) adopts the culture medium containing 10% FBS in the same volume.
The filtration in the step (1) is specifically implemented by filtering with filter screens with diameters of 100 micrometers and 40 micrometers respectively.
Coating a culture flask with 1-10 mug/mL of recombinant human fibronectin before culturing the adipose-derived stem cells in the step (2).
The flasks were coated overnight at 4 ℃.
The serum-free medium in the step (2) is DMEM-F12.
TrypLE is used in the cell passage process after the adipose stem cells are cultured in the step (2)TMThe cells are digested.
Specifically, first, a solution containing 0.3% (m/v) collagenase I and 0.3% (m/v) collagenase II was prepared in a serum-free DMEM-F12 medium, and the mixture of the two collagenase solutions and ACCUTASE (STEMCELLTechnologies) were mixed at a volume of 1: 1; the resulting mixed digestive enzyme solution contained 0.15% collagenase I, 0.15% collagenase II, and 1/2 × ACCUTASE.
The lipoaspirate washed with physiological saline and the mixed digestive enzyme solution were mixed at a volume of 1:1, and digested in a shaker at a constant temperature of 37 ℃ for about 15 minutes at a rotation speed of 100 rpm. After digestion was complete, the digestive enzymes were neutralized by adding an equal volume of medium containing 10% FBS. The cell pellet was then collected by centrifugation at 400g for ten minutes.
Resuspend the cells in saline and pass the cell suspension through filters 100 microns and 40 microns in diameter, in order to obtain a single cell suspension. To remove further contaminating cells, the cell suspension was added above the lymphocyte separation medium, centrifuged at 400g for 30 minutes, and the cell layer between the uppermost liquid layer and the separation medium was collected directly.
The cells were washed twice with saline, centrifuged, the supernatant was discarded, the cells were resuspended in fresh medium, and the cells were cultured in a flask. After 24 hours of culture, the medium was changed and cells that did not adhere to the cell were washed away with physiological saline. During the subsequent culture, the liquid is changed or passaged according to the actual growth condition of the cells.
The serum-free medium for the adipose-derived stem cells provided by the invention contains various growth factors and nutrients, which can promote the normal growth and metabolism of the stem cells under the serum-free culture condition:
fibronectin is an extracellular matrix protein that mediates adhesion between cells, both cellular and extracellular. Coating the flasks with fibronectin promotes better cell adherence. The present invention uses recombinant human fibronectin coated culture flasks.
The recombinant human insulin can improve the anabolic capacity of cells and stimulate the growth of the cells.
The epidermal growth factor is a growth factor with multiple functions and has strong mitogenic action on cells.
Basic fibroblast growth factor, transforming growth factor- β and platelet-derived growth factor-BB are growth factors that promote cell proliferation and division, and the combination of these three factors has been shown to significantly promote the proliferation of mesenchymal stem cells and enhance the differentiation capacity of stem cells.
Hydrocortisone is a glucocorticoid and has the effects of promoting gluconeogenesis and increasing protein catabolism.
Vitamin C is an antioxidant, can protect cells from being threatened by free radicals, is also involved in the metabolism of the cells, and can remarkably promote the proliferation of various mesenchymal stem cells.
Reduced glutathione is a tripeptide compound containing Sulfhydryl (SH), and has important physiological activities of activating an oxidation-reduction system, activating SH enzyme, detoxifying and the like in a human body. Reduced glutathione is also involved in the tricarboxylic acid cycle and sugar metabolism, acting as a coenzyme.
Transferrin is the major transferrin protein in cells, which binds iron ions, reduces its toxicity and provides the iron element for cellular metabolism.
Ethanolamine is involved in phospholipid metabolism and is an essential nutrient for cell growth.
L-glutamine is an important energy source for cell growth, participates in the synthesis and metabolism of protein and lipid, and can also improve the antioxidant capacity of cells. L-glutamine is not stable enough and is supplemented before the preparation of the culture medium.
Coenzyme A is a coenzyme for acetylation in the body and plays a very important role in the metabolism of sugars, lipids and proteins.
The thrombin can promote the secretion of fibronectin by the mesenchymal stem cells, thereby enhancing the adherence of the mesenchymal stem cells and promoting the proliferation of the cells.
Gentamicin is a broad spectrum antibiotic.
Sodium selenite is an essential trace element in cell growth, and plays an antioxidant role in cell metabolism.
Biotin, also known as vitamin H, is a water-soluble vitamin and also belongs to the vitamin B group. It is an essential substance for the synthesis of vitamin C, an essential substance for the normal metabolism of fats and proteins.
Advantageous effects
(1) The mixed digestive juice used in the invention is an optimized digestive enzyme combination, and the cells in the adipose tissues can be dissociated in only 15 minutes by using the mixed digestive juice, which is obviously less than the time consumed by the conventional enzymolysis method. The method not only can improve the yield of the stem cells, but also can maintain the activity of the stem cells to the maximum extent.
(2) The clinical grade stem cell culture first avoids the use of animal serum because animal-derived serum carries the risk of pathogen contamination and is complex in composition and subject to large batch-to-batch variation. The invention uses serum-free culture medium to culture stem cells, and adds a plurality of growth factors and nutrient elements into the culture medium, which can effectively promote cell adherence and obviously improve the proliferation capability of the cells, and has good clinical application value and potential.
(3) Conventional culture methods use pancreatin to digest the cells. Although the efficiency of the pancreatin digestion of cells is high, the cells are damaged greatly if the digestion is not well controlled. Furthermore, pancreatin is generally derived from porcine or bovine tissue, and the activity of pancreatin is terminated with fetal bovine serum, so that animal components are introduced during cell culture. TrypLETMIs a genetically engineered enzyme, and contains no animal-derived components. TrypLETMCan effectively and milder dissociate adherent cells, thus the cells are not easy to be damaged in digestion, and fine cells are well ensuredThe viability of the cells. And TrypLETMThe activity of the compound (A) is not required to be stopped by fetal calf serum, and only physiological saline or a culture medium is required to be diluted.
Drawings
FIG. 1 is a graph showing the growth curves of adipose-derived stem cells in the serum-free medium of example 1 and in a conventional serum-containing culture;
FIG. 2 is a morphological diagram of the adipose-derived stem cells of P1 generation under the condition of using the serum-free medium of example 1.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Firstly, preparing a mixed digestive enzyme solution for digesting fat:
a solution containing 0.3% (M/v) collagenase I and 0.3% (M/v) collagenase II was prepared in serum-free DMEM-F12 medium and sterilized with a filter having a pore size of 0.2. mu.M. The mixture of two collagenases and ACCUTASE solutions (STEMCELL technologies) were mixed at a volume of 1:1, and the resulting mixed digestive enzyme solution contained 0.15% collagenase I, 0.15% collagenase II, and 1/2 × ACCUTASE solution.
Coating a cell culture flask with fibronectin:
when the serum-free medium provided in this example was used, each flask was previously coated with recombinant human fibronectin. Taking a T75 culture flask as an example, the coating process is described as follows: a culture flask of T75 was filled with 5 ml of a 5.0. mu.g/ml recombinant human fibronectin solution diluted with physiological saline and coated overnight at 4 ℃ (about 12 to 16 hours). The fibronectin solution was aspirated away before the cells were added.
Thirdly, separating and culturing the adipose-derived stem cells:
adding the lipoaspirate into a 250 ml centrifuge tube, and then adding an equal volume of normal saline; forcibly shaking the centrifugal tube for several times, and then allowing the centrifugal tube to stand for several minutes, wherein the adipose tissues can float on the physiological saline; the saline is sucked away, fresh saline is added again, and the same operation is repeated for several times until the blood inside the fat is washed away.
The lipoaspirate and the mixed digestive enzyme solution were mixed at a volume of 1:1 and digested in a constant temperature shaker at 37 ℃ for about 15 minutes at a shaker speed of 100 rpm. After digestion was complete, the digestive enzymes were neutralized by adding an equal volume of medium containing 10% FBS. The cell pellet was then collected by centrifugation at 400g for ten minutes.
The cell pellet was resuspended in saline, and the cells were passed through filters 100 microns and 40 microns in diameter, one after the other, to collect individual cells.
20 ml of lymphocyte separation medium Ficoll was added to a clean 50 ml centrifuge tube and the cell suspension was slowly added to the top of the medium. The centrifuge deceleration valve was closed and centrifuged at 400g for 30 minutes to collect the white cell layer between the upper liquid layer and the separation medium.
The cells were resuspended in 2 volumes of physiological saline, centrifuged at 300g for 10 minutes and the supernatant discarded. The cells were washed again with physiological saline, centrifuged, the supernatant was discarded, the cells were suspended with fresh medium, and the cells were cultured in a flask. After 24 hours of culture, the medium was changed and cells that did not adhere to the cell were washed away with physiological saline.
The serum-free culture provided in this example contained 10. mu.g/ml recombinant human insulin, 20ng/ml recombinant human epidermal growth factor, 20ng/ml recombinant human basic fibroblast growth factor, 10ng/ml recombinant human transforming growth factor- β, 20ng/ml recombinant human platelet-derived growth factor-BB, 10ng/ml recombinant human stem cell factor, 0.5. mu.g/ml hydrocortisone, 50. mu.g/ml vitamin C, 2mM reduced glutathione, 5. mu.g/ml recombinant human transferrin, 1. mu.g/ml ethanolamine, 2mM L-glutamine, 50. mu.g/ml coenzyme A, 5U/ml recombinant human thrombin, 10. mu.g/ml gentamycin, 10. mu.g/ml biotin and 5ng/ml sodium selenite, followed byAccording to the actual growth condition of the cells, the culture process of (2) is changed or passaged. When the cell fusion degree reaches 70-80%, TrypLE is usedTMThe cells were passaged by digesting them, and the passage ratio of the cells was 1: 3. Fourthly, the amplification capacity of the serum-free medium and the serum-containing medium of the present example were compared:
the adipose-derived stem cells isolated in this example were cultured in two media, respectively: serum-free medium of this example and conventional serum-containing medium (DMEM-F12+ 10% FBS). At the time of P2 passage, the cells were digested and still suspended in serum-free medium and serum-containing medium, respectively, into a six-well plate. 2 ml of culture medium is filled in a single hole of each six-hole plate, and the number of initial cells is 6000; both groups of cells had 5 six-well plates seeded in parallel. The six well plate was returned to the incubator. Every 2 days, a six-well plate was removed from the incubator, the cells in the plate were digested and counted. The remaining cells were all replaced at day 7 and the cell proliferation experiment continued until day 10.
As shown in fig. 1, the proliferation rates of the two groups of cells showed a significant difference, p < 0.01. The proliferation rate of the adipose-derived stem cells in the serum-free medium provided in this example is significantly higher than that in the conventional serum-containing medium.
Claims (9)
1. A method of isolating and culturing human adipose stem cells, comprising:
(1) digesting the lipoaspirate by using a mixed digestive enzyme solution consisting of collagenase I, collagenase II and ACCUTASE, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing the mixed cells by using a lymphocyte separation solution;
(2) culturing adipose-derived stem cells by adopting a serum-free culture medium, wherein the serum-free culture medium is added with the following components of 10 mu g/ml recombinant human insulin, 20ng/ml recombinant human epidermal growth factor, 20ng/ml recombinant human basic fibroblast growth factor, 10ng/ml recombinant human transforming growth factor- β, 20ng/ml recombinant human platelet-derived growth factor-BB, 10ng/ml recombinant human stem cell factor, 0.5 mu g/ml hydrocortisone, 50 mu g/ml vitamin C and 2mM reduced glutathione5 mug/ml recombinant human transferrin, 1 mug/ml ethanolamine, 2mM L-glutamine, 50 mug/ml coenzyme A, 5U/ml recombinant human thrombin, 10 mug/ml gentamycin, 10 mug/ml biotin and 5ng/ml sodium selenite; TrypLE is used in the cell passage process after the adipose stem cells are cultured in the step (2)TMThe cells are digested.
2. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the mixed digestive enzyme solution in the step (1) consists of 0.1-0.4% of collagenase I, 0.1-0.4% of collagenase II and 1/4-1/2 multiplied ACCUTASE.
3. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the volume ratio of the mixed digestive enzyme solution to the lipoaspirate in the step (1) is 1: 1.
4. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the digestion temperature in the step (1) is 37 ℃, and the digestion time is within half an hour.
5. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the neutralizing digestive enzyme in the step (1) adopts the culture medium containing 10% FBS in the same volume.
6. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the filtration in the step (1) is specifically implemented by filtering with filter screens with diameters of 100 micrometers and 40 micrometers respectively.
7. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: coating a culture flask with 1-10 mug/mL of recombinant human fibronectin before culturing the adipose-derived stem cells in the step (2).
8. The method for isolating and culturing human adipose-derived stem cells according to claim 7, wherein: the flasks were coated overnight at 4 ℃.
9. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein: the serum-free medium in the step (2) is DMEM-F12.
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| CN108220230B (en) * | 2018-02-01 | 2022-01-18 | 上海莱馥医疗科技有限公司 | Method for separating and culturing human adipose-derived stem cells |
| CN111420034A (en) * | 2019-01-08 | 2020-07-17 | 上海莱馥医疗科技有限公司 | Mixed stem cell preparation for treating psoriasis and preparation method thereof |
| CN109797136A (en) * | 2019-03-14 | 2019-05-24 | 湖南南华爱世普林生物技术有限公司 | A kind of isolated culture method of human adipose mesenchymal stem cells |
| CN110343661B (en) * | 2019-04-24 | 2021-08-24 | 朗姿赛尔生物科技（广州）有限公司 | Extraction, culture and amplification method of human adipose-derived stem cells |
| CN110531057B (en) * | 2019-07-01 | 2020-11-20 | 启迪禾美生物科技(嘉兴)有限公司 | Method for high-throughput screening of active composition for skin care products |
| CN115261312A (en) * | 2022-08-03 | 2022-11-01 | 湖北万海赛康生命科学有限公司 | Preparation method of mesenchymal stem cells |
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