CN111621473A - Preparation method of novel human adipose-derived stem cell preparation - Google Patents
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Abstract
The invention discloses a preparation method of a novel human adipose-derived stem cell preparation, which comprises the following steps: s1: carrying out physical examination on a donor, and then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia; s2: digesting the obtained fat in 0.2-0.4% mixed collagenase and 0.25% Trypsin-EDTA digestive juice respectively, adding Trypsin inhibitor, and uniformly mixing to terminate digestion; then centrifuging, washing the bottom layer cells by PBS, and filtering to obtain primary adipose-derived stem cells; s3: culturing the primary adipose-derived stem cells to obtain a large amount of adipose-derived stem cells; s4: and (5) detecting and freezing. The adipose-derived stem cells finally prepared by the preparation method have high survival rate, uniform cell shape and size, and safe and reliable use, and the possibility of allergy and infection of exogenous pathogens in the use process is eliminated.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a preparation method of a novel human adipose-derived stem cell preparation.
Background
Stem cells are cells having self-replicating ability and multi-directional differentiation potential, and can be differentiated into various human cells such as fat, cartilage, osteogenic, myoblast, and the like. The research of autologous stem cell therapy mostly focuses on bone marrow stem cells, but the bone marrow stem cells have large material taking wound, weak cell proliferation, small cell number in preparations and limited treatment times. Adipose-derived stem cells (ADSCs) have the advantages of abundant raw materials, convenient material acquisition, good cell proliferation, autografting, low immunogenicity and the like, can well avoid the ethical problem of the traditional embryonic stem cells, overcome the defect of rare bone marrow stem cell sources, are quickly and widely used in the medical beauty industry after being discovered in 2001, are gradually favored by other indications, and are widely applied to the field of regenerative medicine research at present; the us FDA approved clinical trials of adipose stem cells on heart failure patients in 2012; stem cell drugs are approved for the market in korea for the treatment of anal fistula, a complication of complex crohn's disease.
In the preparation process of the adipose-derived stem cell preparation, the content of stem cells in raw material adipose and the success rate of separation and purification can be directly influenced by the difference of adipose-derived sources and treatment technologies, the existing adipose-derived stem cell preparation has no systematic quality monitoring system for adipose sources (namely, the donor is subjected to physical examination), and the prepared reagent can bring the possibility of pollution, such as HIV, hepatitis B, HCV and the like; after the fat is mechanically treated, the fat is separated and purified by adopting an enzyme digestion method, and the fat is digested by adopting a collagenase digestion method, so that the method is mild, the damage to cells can be well reduced, but the digestion capability is weak, redundant surface adipose tissues cannot be completely digested, the cells are not completely dispersed, the cells are easy to aggregate into larger cell masses, the loss is caused, and the recovery rate of the SVF cells is reduced. In addition, most of the existing stem cell primary culture methods adopt a fetal bovine serum matched double antibody culture method, the risk of contaminating exogenous viruses and pathogenic factors exists when bovine serum is used in cell culture, the reproducibility of products and experimental results is poor due to the inconsistency of biological activity and factors among different batches of bovine serum, and the bovine serum remaining in the products easily causes anaphylactic reaction of an inoculator to serum.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a novel human adipose-derived stem cell preparation, which can solve the problems of large cell damage, low separation and purification rate, low cell adherence rate and low proliferation rate of an inoculator caused by residual heterologous protein or antibiotics in the culture process in the prior art in the fat digestion process.
In order to solve the problems, the invention adopts the following technical scheme:
a preparation method of a novel human adipose-derived stem cell preparation comprises the following steps:
s1: human adipose tissue collection: carrying out physical examination on a donor, and then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia;
s2: preparing primary adipose-derived stem cells: putting the fat obtained in the step S1 into 0.2% -0.4% mixed collagenase, uniformly shaking and digesting for 30-40 minutes at the temperature of 37 ℃, then adding 0.25% Trypsin-EDTA digestive juice (the final concentration is 0.0625%), uniformly shaking and digesting for 10-15 minutes at the temperature of 37 ℃, adding a Trypsin inhibitor, uniformly mixing and stopping digestion; centrifuging the mixture at 900g for 10 min, sucking the upper mixed solution, washing the bottom cells with PBS, and filtering through a 100-mesh screen; centrifuging the filtrate for 10 minutes at the rotating speed of 210g, and sucking and removing supernatant to obtain primary adipose-derived stem cells;
s3: culturing primary adipose-derived stem cells: adding the primary adipose-derived stem cells obtained in the step S2 into a serum-free culture medium for resuspension, counting the cells, inoculating the cells into a cell culture bottle coated with a Laminin-521 solution according to the density of more than 3 x 104/cm2, culturing the cells in a 5% CO2 culture box at 37 ℃ for 2 days, removing the culture solution, replacing the fresh serum-free culture medium, continuously culturing the cells in the 5% CO2 culture box at 37 ℃, and replacing the fresh serum-free culture medium every two days; when the growth and fusion of the adipose-derived stem cells reach more than 80%, digesting with Trypsin-EDTA digestive juice, and carrying out passage according to the ratio of 1:3 to obtain a large amount of adipose-derived stem cells;
s4: detection and cryopreservation: counting and detecting the adipose-derived stem cells obtained in the step S3, and judging that the cells are enough to reach the qualified standard; and (4) after the detection is qualified, adding the adipose-derived stem cells obtained in the step S3 into the cell freezing solution for resuspension, then loading the cells into a cell freezing tube for freezing and storing.
Further, in the step S1, in the process of obtaining fat by using the liposuction with the tumescence method, the maximum negative pressure is 430mmHg, and the obtained fat is filtered through the filtering holes of 300-.
Further, the step S1 and the step S2 further include preprocessing the collected fat, specifically: and (5) washing the fat obtained in the step (S1) for multiple times by using PBS (phosphate buffer solution) until the washing liquid is clear and transparent, and the fat is in clean light yellow.
Further, the serum-free medium in step S3 includes the following components: alpha-MEM, 20% human platelet lysate, 105U/LIF, 5-10ng/mL b-FGF, 5-10ng/mL GF-beta and 500-1000IU/L heparin sodium.
Further, the cell freezing medium in the step S4 is composed of the following components: 10% DMSO, 40% HPL serum replacement and α -DMEM.
Further, the specific operation of the cryopreservation in the step S4 is to store the cell cryopreservation tube at 4 ℃ for 30 minutes, store the cell cryopreservation tube at-20 ℃ for 2 hours, store the cell cryopreservation tube at-80 ℃ for more than one week, and then store the cell cryopreservation tube in a liquid nitrogen tank for a long time.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, fat is obtained by adopting a swelling liposuction method and is filtered through a filtering hole of 800 mu m with the size of 300-; in the process of preparing the primary adipose-derived stem cells, after collagenase digestion, a low-concentration Trypsin-EDTA (Trypsin-EDTA) solution is adopted for pre-incubation, so that adipose tissue blocks can be digested more effectively, the recovery rate of SVF is increased, and the single enzyme digestion can be reduced by incubating the low-concentration Trypsin-EDTA solution in a short time and matching with mixed collagenase digestion with a proper concentration, so that the activity of the recovered cells is improved; the method has the advantages that the serum substitute of human-derived components is adopted to be matched with a basic culture medium in the process of culturing the primary adipose-derived stem cells, adhesion factors, growth factors and the like are added, adverse factors brought by serum can be reduced, cell proliferation is promoted, the cell culturing conditions are more stable, meanwhile, the Laminin-521 coating is adopted to replace gelatin coating, the anchorage rate of cells is effectively improved, and the cell amplification efficiency is increased.
Drawings
The invention is described in further detail below with reference to specific embodiments and with reference to the following drawings.
FIG. 1 is a cell morphology map of the adipose stem cells prepared in example 1;
FIG. 2 shows immunophenotypic measurements of the adipose stem cells prepared in example 1.
Detailed Description
Example 1
The embodiment provides a preparation method of a novel human adipose-derived stem cell preparation, which comprises the following steps:
s1: human adipose tissue collection: carrying out HIV, HBV and HCV physical examination on a donor, then obtaining fat by adopting a liposuction method with a swelling method under the state of local anesthesia, wherein the maximum negative pressure is 430mmHg, the collection part is a thigh part, and the obtained fat is filtered through a 500-micron filtering hole to remove grease and moisture to obtain light yellow fat;
s2: preparing primary adipose-derived stem cells: pretreating the fat obtained in the step S1, and washing for multiple times by using PBS (phosphate buffer solution) until the washing liquid is clear and transparent, and the fat is in clean light yellow; putting the fat subjected to multiple cleaning into a mixed solution of 0.2-0.4% of type I collagen protease and type VI collagen protease which have the same volume with the fat, uniformly vibrating and digesting the mixed solution at 37 ℃ for 30-40 minutes, then adding one third of Trypsin-EDTA digestive juice (the final concentration is 0.0625%) with the volume of 0.25%, uniformly vibrating and digesting the mixed solution at 37 ℃ for 10-15 minutes, and adding a Trypsin inhibitor to uniformly mix the mixture to stop digestion; centrifuging the mixture at 900g for 10 min, sucking the upper mixed solution, washing the bottom cells with PBS, and filtering through a 100-mesh screen; centrifuging the filtrate for 10 minutes at the rotating speed of 210g, sucking and removing supernatant, and removing undigested fibrous tissues and impurities to obtain primary adipose-derived stem cells;
s3: culturing primary adipose-derived stem cells: adding the primary adipose-derived stem cells obtained in the step S2 into a serum-free culture medium for resuspension, counting the cells, inoculating the cells into a cell culture bottle coated with Laminin Laminin-521 solution according to the density of more than 3 x 104/cm2, culturing for 2 days in a 37 ℃ and 5% CO2 culture box, removing the culture solution, replacing the fresh serum-free culture medium, continuously culturing the cell culture bottle in the 37 ℃ and 5% CO2 culture box, and replacing the fresh serum-free culture medium once every two days; when the growth and fusion of the adipose-derived stem cells reach more than 80%, digesting with Trypsin-EDTA digestive juice, and carrying out passage according to the ratio of 1:3 to obtain a large amount of adipose-derived stem cells;
s4: detection and cryopreservation: counting and detecting the adipose-derived stem cells obtained in the step S3, and judging that the cells are enough to reach the qualified standard; and after the detection is qualified, adding the adipose-derived stem cells obtained in the step S3 into a cell freezing solution for resuspension so that the final concentration of the cell suspension is 1 × 106/mL, subpackaging the cell freezing solution into a cell freezing tube, placing the cell freezing tube at a corresponding position of a freezing box, storing the freezing box at 4 ℃ for 30 minutes at-20 ℃ for 2 hours at-80 ℃ for more than one week, and then transferring the cell freezing solution into a liquid nitrogen tank for long-term storage.
Wherein, the serum-free culture medium in the step S3 comprises the following components: alpha-MEM, 20% human platelet lysate, 105U/LIF (leukocyte inhibitory factor), 5-10ng/mLb-FGF (basic fibroblast growth factor), 5-10 ng/mLTGF-beta (transforming growth factor-beta) and 500-1000IU/L heparin sodium, wherein TGF-beta and b-FGF can effectively support the growth and undifferentiated state of adipose-derived stem cells and prevent the differentiation of the cells; the heparin sodium can promote the growth of the adipose-derived stem cells, inhibit the differentiation of the adipose-derived stem cells and protect the cells; the platelet lysate is a serum substitute and provides various necessary factors and nutrient substances for the growth of cells; LIF inhibits the multipotential differentiation of stem cells and maintains the dryness.
Wherein, the cell freezing solution in the step S4 comprises the following components: 10% DMSO, 40% HPL serum replacement and α -DMEM.
Example 2
Screening of adipose tissue harvesting sites
In this example, based on example 1, in the process of collecting adipose tissues of a human body, fat was obtained by liposuction with the tumescent method at the thigh, the back and the abdomen of a donor, and the appearance and SVF cell count of the collected adipose tissues were tested, and the experimental results are shown in table 1.
TABLE 1 Effect of adipose tissue harvesting sites on SVF cell recovery
| Experiment number | Collection site | Shape of adipose tissue | SVF cell count/10 mL |
| 1 | Thigh part | Golden color and uniform fat block size | 1.12E+06 |
| 2 | Waist and back | Golden color and uniform fat block size | 9.32E+05 |
| 3 | Abdomen part | Golden color and uniform fat block size | 8.42E+05 |
The experimental result shows that although the fat of the three parts is similar in shape, the recovery rate of the SVF cells has obvious difference. Among them, thigh fat or low back fat is recommended as much as possible because thigh fat is most preferable, low back fat is next to low back fat, and low back fat is worst.
Example 3
Effect of fat pretreatment method on SVF cell recovery
Different fat processing technologies can directly affect the stem cell content in the raw material fat and the success rate of separation and purification, in this embodiment, in the process of collecting human adipose tissue in step S1, on the basis of example 1, the obtained fat is filtered by four methods of only sedimentation separation, filtration through 300 μm filter pores, filtration through 500 μm filter pores and filtration through 800 μm filter pores, and the number of SVF cells and the number of hybrid cells recovered by the four methods are tested, and the experimental results are shown in table 2.
TABLE 2 Effect of different fat pretreatment methods on SVF cell recovery
The experimental results prove that the SVF cells recovered by the four methods of sedimentation separation and filtration of 300 mu m, 500 mu m and 800 mu m are different in number and the number of the hybrid cells is also different. Wherein, the SVF cell number obtained by the sedimentation separation method is minimum, the hybrid cell number is more, and the SVF cell can be completely purified and eliminated after 2 passages. The filtration method can obtain SVF with high cell purity, and the P1 generation has few mixed cells. Wherein cell recovery increases but the number of contaminating cells increases with increasing filter mesh size. Comprehensive analysis shows that the cost performance of the cells obtained by 500-micron filtration is highest in purity and quantity.
Example 4
Effect of digestion methods on SVF cell recovery
In the preparation method of the novel human adipose-derived stem cell preparation, fat digestion in step S2 is required to protect the activity of adipose-derived stem cells while removing fat and connective tissues as much as possible. The type, concentration and digestion time of the digestion reagent are all strictly required. In this example, 6 different recipes and digestion methods were selected based on example 1, and the number and purity of SVF cells recovered were tested, respectively, and the experimental results are shown in Table 3.
TABLE 3 Effect of digestion methods on SVF cell recovery
The results of the experiments show that the cells recovered by method 5 are superior to several other methods in terms of both cell number and cell purity.
Example 5
Selection of coating Agents
The coating of the cell culture bottle can enhance the adherence rate of the cells and increase the proliferation efficiency. In this example, culture flasks were not coated, gelatin coated, and lamin-521 coated on the basis of example 1, and inoculated proliferation experiments were performed, respectively, and the percentage of adherent cells in the basal area after 24 hours of cell inoculation and the number of cell proliferations after 72 hours were measured, respectively, and the experimental results are shown in table 4.
Table 4 selection of coating reagents
The experimental result shows that the adherence rate and the proliferation rate of the Laminin-521 coating are higher than those of gelatin or samples without coating, and the kit is more suitable for the growth of adipose-derived stem cells.
Example 6
When the adipose-derived stem cells prepared in example 1 were subjected to cell morphology observation, as shown in fig. 1, the cells appeared in a relatively uniform spindle-shaped or spindle-shaped form, grew in a circular or spiral shape, and were uniformly distributed, and the bottom of the flask was about 80% full of cells after 72 hours.
The international society for stem cells has set standards for mesenchymal stem cells, including the immunophenotype of cells by flow assay, which, according to the society, must express CD90/73/105, but not CD14/19/34/45 and HLA-DR. The immunophenotype of the 3 rd generation adipose-derived stem cells prepared in example 1 was examined, and the results are shown in table 5 and fig. 2. The results show that the concentration of the positive markers is higher than 95%, the expression of the negative markers is lower than 5%, and the international stem cell association standard is met.
TABLE 5 immunophenotypic measurements for adipose stem cells in example 1
In the invention, fat is obtained by adopting a swelling liposuction method and is filtered through a filtering hole of 800 mu m with 300-; in the process of digesting fat, the fat is digested by collagenase and then is pre-incubated by adopting a low-concentration Trypsin-EDTA (Trypsin-EDTA) solution, so that fat tissue blocks can be digested more effectively, the recovery rate of SVF is increased, the incubation of the low-concentration Trypsin-EDTA solution in a short time is matched with the digestion of mixed collagenase with proper concentration, the damage of single enzyme digestion to cells can be reduced, and the activity of the recovered cells is improved; the method has the advantages that the serum substitute of human-derived components is adopted to be matched with a basic culture medium in the process of culturing the primary adipose-derived stem cells, adhesion factors, growth factors and the like are added, adverse factors brought by serum can be reduced, cell proliferation is promoted, the cell culturing conditions are more stable, meanwhile, the Laminin-521 coating is adopted to replace gelatin coating, the anchorage rate of cells is effectively improved, and the amplification efficiency of the primary cells is increased.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (6)
1. A preparation method of a novel human adipose-derived stem cell preparation is characterized by comprising the following steps:
s1: human adipose tissue collection: carrying out physical examination on a donor, and then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia;
s2: preparing primary adipose-derived stem cells: putting the fat obtained in the step S1 into 0.2-0.4% of mixed collagenase, uniformly shaking and digesting for 30-40 minutes at the temperature of 37 ℃, then adding 0.25% of Trypsin-EDTA digestive juice, uniformly shaking and digesting for 10-15 minutes at the temperature of 37 ℃, adding a Trypsin inhibitor, uniformly mixing and stopping digestion; centrifuging the mixture at 900g for 10 min, sucking the upper mixed solution, washing the bottom cells with PBS, and filtering through a 100-mesh screen; centrifuging the filtrate for 10 minutes at the rotating speed of 210g, and sucking and removing supernatant to obtain primary adipose-derived stem cells;
s3: culturing primary adipose-derived stem cells: adding the primary adipose-derived stem cells obtained in the step S2 into a serum-free culture medium for resuspension, counting the cells, inoculating the cells into a cell culture bottle coated with a Laminin-521 solution according to the density of more than 3 x 104/cm2, culturing the cells in a 5% CO2 culture box at 37 ℃ for 2 days, removing the culture solution, replacing the fresh serum-free culture medium, continuously culturing the cells in the 5% CO2 culture box at 37 ℃, and replacing the fresh serum-free culture medium every two days; when the growth and fusion of the adipose-derived stem cells reach more than 80%, digesting with Trypsin-EDTA digestive juice, and carrying out passage according to the ratio of 1:3 to obtain a large amount of adipose-derived stem cells;
s4: detection and cryopreservation: counting and detecting the adipose-derived stem cells obtained in the step S3, and judging that the cells are enough to reach the qualified standard; and (4) after the detection is qualified, adding the adipose-derived stem cells obtained in the step S3 into the cell freezing solution for resuspension, then loading the cells into a cell freezing tube for freezing and storing.
2. The method as claimed in claim 1, wherein in step S1, the maximum negative pressure is 430mmHg during fat extraction by liposuction with tumescence method, and the extracted fat is filtered through a filter hole of 300-800 μm to remove oil and water to obtain light yellow fat.
3. The method for preparing the novel human adipose-derived stem cell preparation of claim 1, further comprising the following steps between the step S1 and the step S2: and (5) washing the fat obtained in the step (S1) for multiple times by using PBS (phosphate buffer solution) until the washing liquid is clear and transparent, and the fat is in clean light yellow.
4. The method for preparing the novel human adipose-derived stem cell preparation of claim 1, wherein the serum-free medium in step S3 comprises the following components: alpha-MEM, 20% human platelet lysate, 105U/LIF, 5-10ng/mL b-FGF, 5-10ng/mL GF-beta and 500-1000IU/L heparin sodium.
5. The method for preparing the novel human adipose-derived stem cell preparation of claim 1, wherein the cell freezing medium in step S4 comprises the following components: 10% DMSO, 40% HPL serum replacement and α -DMEM.
6. The method for preparing the novel human adipose-derived stem cell preparation according to claim 1 or 5, wherein the specific procedure of the cryopreservation in step S4 is to store the cell cryopreservation tube at 4 ℃ for 30 minutes, then store the cell cryopreservation tube at-20 ℃ for 2 hours, store the cell cryopreservation tube at-80 ℃ for more than one week, and then store the cell cryopreservation tube in a liquid nitrogen tank for a long time.
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