CN108220230B - Method for separating and culturing human adipose-derived stem cells - Google Patents
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Abstract
The invention discloses a method for separating and culturing human adipose-derived stem cells, which comprises the following steps: step 1: digesting the lipoaspirate by using Liberase digestive enzyme solution, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing the hybrid cells by using lymphocyte separation liquid to obtain human adipose-derived stem cells; step 2: adopting a serum-free culture medium to culture the human adipose-derived stem cells, wherein the serum-free culture medium is added with the following components: recombinant human epidermal growth factor; recombinant human basic fibroblast growth factor; recombinant human transforming growth factor-beta; recombinant human platelet-derived growth factor-BB; a recombinant human stem cell factor; reduced glutathione; coenzyme A; biotin; MEM vitamin solution; MEM amino acid solution; MEM optional amino acid solutions; (ii) a GlutaMAX additive; insulin-transferrin-selenium solution; gentamicin. The invention can obviously improve the yield and the activity of the adipose-derived stem cells and obtain more adipose-derived stem cells with proliferative capacity.
Description
Technical Field
The invention relates to the separation and culture of stem cells, in particular 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 mesenchymal Stem Cells, also called Adipose Stem Cells (Adipose Stem Cells), are Stem Cells with multipotential differentiation potential 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 conventional adipose-derived stem cell isolation method has some problems: 1. collagenase is derived from bacterial extracts, with the possibility of residual bacterial components, and endotoxin in collagenase may non-specifically activate stem cells or produce some other negative effect; the quality of collagenase varies from batch to batch, which also affects the yield of stem cells. There is therefore a need to find a better alternative to collagenase. 2. The concentration of the digestive enzyme and the digestion time have an effect on the yield and activity of the adipose-derived stem cells, so that it is necessary to optimize the method for isolating the adipose-derived stem cells. 3. Some 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 invention aims to provide a method for separating and culturing human adipose-derived stem cells, which aims to solve the problems of the existing adipose-derived stem cell separation method.
In order to achieve the above object, the present invention provides a method for isolating and culturing human adipose-derived stem cells, comprising the steps of:
step 1: digesting lipoaspirate by using a Liberase MNP-S (GMP Grade, purchased from Roche corporation) digestive enzyme solution, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing hybrid cells by using lymphocyte separation liquid to obtain human adipose-derived stem cells;
step 2: and (2) culturing the human adipose-derived stem cells obtained in the step (1) by adopting a serum-free culture medium, wherein the serum-free culture medium is added with the following components:
recombinant human epidermal growth factor with final concentration of 10-50 ng/ml;
the final concentration of the recombinant human basic fibroblast growth factor is 10-50 ng/ml;
recombinant human transforming growth factor-beta with a final concentration of 1-50 ng/ml;
recombinant human platelet-derived growth factor-BB with a final concentration of 10-50 ng/ml;
the final concentration of the recombinant human stem cell factor is 1-100 ng/ml;
reduced glutathione with the final concentration of 1-5 mM;
coenzyme A with a final concentration of 1-50 mug/ml;
biotin with a final concentration of 1-50 mug/ml;
gentamicin at a final concentration of 1-100 mug/ml, and,
the following components produced by Saimer Feishel scientific Inc.: minimum Essential Medium Vitamin Solution (MEM) Solution at a final concentration of 1 × (cat # 11120052); a final 1 XMEM Amino acid Solution (MEM Amino Acids Solution, cat # 11130051); a final 1 XMEM Solution of nonessential Amino Acids (MEM Non-Essential Amino Acids Solution, 11140050); GlutaMAX additive (GlutaMAX Supplement, cat # 35050061) at a final concentration of 1 ×; Insulin-Transferrin-Selenium solution (Insulin-Transferrin-Selenium solution, cat # 41400045) at a final concentration of 1 ×.
In the method for separating and culturing the human adipose-derived stem cells, in the step 1, the Liberase digestive enzyme solution is prepared by using a serum-free medium.
In the method for separating and culturing human adipose-derived stem cells, in step 1, the volume ratio of the digestive enzyme solution to the lipoaspirate is 1: 1.
In the method for separating and culturing the human adipose-derived stem cells, in the step 1, the digestion temperature is 37 ℃ and the digestion time is 10-30 minutes.
In the method for separating and culturing human adipose-derived stem cells, in step 1, the digestive enzymes are neutralized by an equal volume of Serum-free medium containing 10% Fetal Bovine Serum (FBS).
In the method for separating and culturing the human adipose-derived stem cells, in the step 1, the filtering is specifically performed by filtering with filter screens with diameters of 100 microns and 40 microns in sequence.
In the method for isolating and culturing human adipose-derived stem cells, in step 2, the human adipose-derived stem cells are cultured in a flask coated with 1 to 10. mu.g/mL of recombinant human fibronectin and 1 to 10. mu.g/mL of hyaluronic acid for the first time, and then only in a flask coated with 1 to 10. mu.g/mL of recombinant human fibronectin for the subsequent time.
The method for separating and culturing human adipose-derived stem cells, wherein the culture flask is coated overnight at 4 ℃.
In the method for separating and culturing human adipose-derived stem cells, in step 2, the serum-free medium is DMEM-F12.
In the method for separating and culturing the human adipose-derived stem cells, in the step 2, TrypLE produced by seimer feishell scientific and technological company is used for the generation of the human adipose-derived stem cellsTMSelect digests cells.
The serum-free culture medium for the human adipose-derived stem cells, provided by the invention, contains various growth factors and nutrients, so that the normal growth and metabolism of the human adipose-derived stem cells under the serum-free culture condition can be promoted:
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.
Hyaluronic acid, also known as uronic acid, has a large polysaccharide consisting of two disaccharide units, D-glucuronic acid and N-acetylglucosamine. Hyaluronic acid is capable of maintaining stem cells in an undifferentiated state, and the use of hyaluronic acid in cell culture is advantageous for maintaining the stem cells in a dry state.
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-beta, platelet-derived growth factor-BB and stem cell factor are all growth factors with the ability to promote cell proliferation and division, and the combination of these factors has been shown to significantly promote the proliferation of mesenchymal stem cells and enhance the differentiation capacity of 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.
Coenzyme A is a coenzyme for acetylation in the body and plays a very important role in the metabolism of sugars, lipids and proteins.
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.
The GlutaMAX supplement is a high-grade cell culture supplement that provides L-glutamine, which is required for cell growth, but is more stable than L-glutamine.
The insulin-transferrin-selenium solution provides insulin, transferrin and selenium, specifically sodium selenite, and has the following functions:
insulin can improve the anabolic capacity of cells and stimulate the growth of cells.
Transferrin is the major transferrin protein in cells, which binds iron ions, reduces its toxicity and provides the iron element for cellular metabolism.
Sodium selenite is an essential trace element in cell growth, and plays an antioxidant role in cell metabolism.
Gentamicin is a broad spectrum antibiotic.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, Liberase digestive enzyme is used to replace traditional collagenase, and only 10 minutes are needed to dissociate cells in adipose tissues at 37 ℃ and under the condition of 0.3WU/mL Liberase, so that time is relatively saved, and the damage to the cells is smaller. Compared with the traditional collagenase digestion method, the method for separating the adipose-derived stem cells can obviously improve the yield and the activity of the stem cells; as can be seen from the results of CFU-F experiments, the cells isolated by the method of the present invention contain more adipose-derived stem cells with proliferation capacity, which means that the adipose-derived stem cells isolated by the method of the present invention may have better therapeutic effect. In addition, the Liberase among different batches has high consistency, and the components of the Liberase better meet the safety requirement of clinical application than collagenase, so the Liberase is a good digestive enzyme for separating adipose-derived stem cells, and the Liberase with higher safety is used for replacing the collagenase, which is a good technical improvement. The dissociated adipose-derived stem cells are further purified by using lymphocyte separating medium, and experiments prove that the adipose-derived stem cells of the P1-P10 generation have high purity.
(2) In the aspect of stem cell culture, the serum-free culture medium is used for replacing the traditional serum-containing culture medium, so that the method is safer and more ethical. Various growth factors and nutrient elements are added into the culture medium, so that the adherence of cells can be effectively promoted, and the proliferation capacity of the cells can be remarkably improved. In the serum-free culture medium, GlutaMAX with better stability is used for replacing L-glutamine, and various vitamins and amino acid components are added, so that excellent nutritional conditions are provided for the growth of stem cells; and the dryness of the stem cells can be well maintained, and the osteogenesis experiments show that the fat stem cells of the P10 generation still have good differentiation potential.
(3) The culture bottle coated with fibronectin is matched with a serum-free culture medium, so that stem cells can grow in an adherent manner under the serum-free condition; the newly prepared stem cells are put into the culture bottle coated by fibronectin and hyaluronic acid, so that the stem cells can be quickly recovered from the stress state in the preparation process, and the adherence of the stem cells can be promoted more quickly and better.
(4) Cell passaging method in the present inventionUsing TrypLETMSelect replaces traditional pancreatin. If the digestion time of the pancreatin is not well controlled, the pancreatin can cause great damage to cells, and the pancreatin is generally from pig or cattle tissues, the activity of the pancreatin is stopped by fetal calf serum, so that animal components are introduced in the cell culture process. TrypLETMSelect is a genetically engineered enzyme, contains no animal-derived component, TrypLETMThe activity of Select does not need to be stopped by fetal calf serum, and only needs to be diluted by normal saline or culture medium, so TrypLE is usedTMSelect is safer than using traditional pancreatin. TrypLETMSelect can dissociate adherent cells effectively and milder using TrypLETMCell passage by Select will allow stem cells to grow better.
Drawings
FIG. 1 is a schematic diagram comparing cell yields of the method of the present invention and a conventional adipose stem cell-producing method;
FIG. 2 is a schematic diagram comparing cell viability in the method of the present invention and a conventional adipose stem cell preparation method;
FIG. 3 is a comparative schematic view of CFU-F comparing the method of the present invention and the conventional adipose stem cell preparation method;
FIG. 4 is a schematic diagram showing the adipogenic capacity of the adipose-derived stem cells prepared by the method of the present invention at the P10 generation;
fig. 5 is a schematic diagram of the osteogenesis capacity of the adipose-derived stem cells prepared by the method of the present invention at the P10 generation.
Detailed Description
The invention will be further described by the following specific examples in conjunction with the drawings, which are provided for illustration only and are not intended to limit the scope of the invention.
Example 1
Firstly, preparing a digestive enzyme solution:
solutions containing 0.01-0.4WU/mL Liberase digestive enzyme were prepared in serum-free medium (DMEM-F12) and sterilized with a filter membrane having a pore size of 0.2. mu.M.
Secondly, coating a cell culture bottle:
when the serum-free culture medium provided by the invention is used, each culture bottle needs to be coated in advance. The human adipose-derived stem cells obtained by digestion are cultured in a flask coated with 1-10. mu.g/mL of recombinant human fibronectin and 1-10. mu.g/mL of hyaluronic acid for the first time, and only a flask coated with 1-10. mu.g/mL of recombinant human fibronectin is used thereafter. 5 ml of coating solution diluted with physiological saline was added to a T75 flask, and the coating solution was aspirated off before adding the cells.
Thirdly, separating and culturing human 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.
Adipose tissues and the Liberase digestive enzyme solution were mixed at a volume of 1:1, and digested in a constant temperature water bath shaker at 37 ℃ for about 10 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.
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 discarded, the cells were resuspended in fresh serum-free 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 invention cultures human adipose-derived stem cellsIn the process of (3), the following nutrients are added to the serum-free medium: recombinant human epidermal growth factor with the final concentration of 10-50 ng/ml, recombinant human basic fibroblast growth factor with the final concentration of 10-50 ng/ml, recombinant human transforming growth factor-beta with the final concentration of 10-50 ng/ml, recombinant human platelet-derived growth factor-BB with the final concentration of 10-50 ng/ml, recombinant human stem cell factor with the final concentration of 1-100 ng/ml, reduced glutathione with the final concentration of 1-5 mM, coenzyme A with the final concentration of 1-50 mug/ml, biotin with a final concentration of 1-50 mug/ml, MEM vitamin solution with a final concentration of 1x, MEM amino acid solution with a final concentration of 1x, MEM nonessential amino acid solution with a final concentration of 1x, GlutaMAX additive with a final concentration of 1x, insulin-transferrin-selenium solution with a final concentration of 1x and gentamicin with a final concentration of 1-100 mug/ml. 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, and is beneficial to the in vitro amplification and the maintenance of the dryness of the adipose-derived stem cells. During the subsequent culture, the liquid is changed or passaged according to the actual growth condition of the cells. When the cell fusion degree reaches 70-80%, TrypLE is usedTMThe cells were passaged by selecting digestion, and the passage ratio of the cells was 1: 3.
Fourth, the method of the present invention and the conventional method are compared
To compare the difference between the method of isolating and culturing adipose stem cells according to the present invention and the conventional method, the experiment of the present invention used two collagenase methods as controls: 1.5mg/ml collagenase I was digested at 37 ℃ for 30 minutes (C1), and 1mg/ml collagenase I was digested at 37 ℃ for 60 minutes (C2). The yield and viability of cells dissociated from adipose tissue by the three methods were compared in the experiment, and it was found from the yield of cells obtained after digestion of fat that the yield of cells in the present invention was significantly higher than that of C1 and C2, as shown in fig. 1. The method of the present invention has little difference in viability of the cells obtained from C1, but the viability of the cells obtained from C2 is significantly lower than that obtained from the isolation method of the present invention, as shown in FIG. 2.
To compare the differences in the fibroblast colony-forming units (CFU-F) obtained by the three isolation methodsThe adipose-derived stem cells separated by the three methods are divided into 2 x 104Per cm2The cell number of more than 10 clones was observed and recorded under a microscope after 10 days in a culture flask of T25, and the cells isolated by the method of the present invention contain more adipose-derived stem cells with proliferation capacity, as shown in FIG. 3, which means that the adipose-derived stem cells isolated by the method of the present invention may have better therapeutic effect.
Fifthly, the adipose-derived stem cells separated by the method of the invention keep good purity and differentiation potential in the passage process
The adipose-derived stem cells separated and cultured by the method of the invention are collected, and the detection experiment of a flow cytometer shows that: adipose stem cells maintained good purity in P1, P5, and P10 passages, as shown in table 1.
TABLE 1 marker expression levels of ASCs prepared and cultured according to the methods of this patent
Meanwhile, the differentiation potential of the fat stem cells of the P10 generation is detected, the Oil-Red-O staining method (Red-Oil-O staining) result of figure 4 shows that the fat stem cells of P10 still have obvious fat forming capability, and the silver nitrate staining method (Von Kossa staining) result of figure 5 shows that the fat stem cells of P10 still have obvious bone forming capability.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (9)
1. A method for separating and culturing human adipose-derived stem cells is characterized by comprising the following steps:
step 1: digesting the lipoaspirate by using Liberase digestive enzyme solution, neutralizing the digestive enzyme after digestion, centrifuging, filtering, and further removing the hybrid cells by using lymphocyte separation liquid to obtain human adipose-derived stem cells;
step 2: and (2) culturing the human adipose-derived stem cells obtained in the step (1) by adopting a serum-free culture medium, wherein the serum-free culture medium is added with the following components:
recombinant human epidermal growth factor with final concentration of 10-50 ng/ml;
the final concentration of the recombinant human basic fibroblast growth factor is 10-50 ng/ml;
recombinant human transforming growth factor-beta with a final concentration of 1-50 ng/ml;
recombinant human platelet-derived growth factor-BB with a final concentration of 10-50 ng/ml;
the final concentration of the recombinant human stem cell factor is 1-100 ng/ml;
reduced glutathione with the final concentration of 1-5 mM;
coenzyme A with a final concentration of 1-50 mug/ml;
biotin with a final concentration of 1-50 mug/ml;
gentamicin with a final concentration of 1-100 mug/ml, and,
the following components: MEM vitamin solution at final concentration of 1 ×; MEM amino acid solution at a final concentration of 1 ×; MEM non-essential amino acid solution with final concentration of 1 ×; (ii) a GlutaMAX additive at a final concentration of 1 ×; insulin-transferrin-selenium solution at a final concentration of 1 ×;
in the step 2, the human adipose-derived stem cells are cultured in culture bottles coated with 1-10 mug/mL of recombinant human fibronectin and 1-10 mug/mL of hyaluronic acid for the first time, and then only culture bottles coated with 1-10 mug/mL of recombinant human fibronectin are used for culture.
2. The method for separating and culturing human adipose-derived stem cells according to claim 1, wherein in step 1, the Liberase digestive enzyme solution is prepared using a serum-free medium.
3. The method for separating and culturing human adipose-derived stem cells according to claim 1, wherein in step 1, the volume ratio of the digestive enzyme solution to the lipoaspirate is 1: 1.
4. The method for separating and culturing human adipose-derived stem cells according to claim 1, wherein in step 1, the digestion temperature is 37 ℃ and the digestion time is 10 to 30 minutes.
5. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein in step 1, the digestive enzymes are neutralized with an equal volume of serum-free medium containing 10% FBS.
6. The method for separating and culturing human adipose-derived stem cells according to claim 1, wherein in step 1, the filtration is performed by sequentially using filter screens with diameters of 100 microns and 40 microns.
7. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein the culture flask is coated overnight at 4 ℃.
8. The method for separating and culturing human adipose-derived stem cells according to claim 1, wherein the serum-free medium in step 2 is DMEM-F12.
9. The method for isolating and culturing human adipose-derived stem cells according to claim 1, wherein in step 2, TrypLE is used for the generation of the human adipose-derived stem cellsTMSelect digests cells.
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