CN111617105A - Preparation method of adipose-derived stem cell multi-cell active factor freeze-dried powder - Google Patents
Preparation method of adipose-derived stem cell multi-cell active factor freeze-dried powder Download PDFInfo
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- CN111617105A CN111617105A CN201910267822.9A CN201910267822A CN111617105A CN 111617105 A CN111617105 A CN 111617105A CN 201910267822 A CN201910267822 A CN 201910267822A CN 111617105 A CN111617105 A CN 111617105A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
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- Developmental Biology & Embryology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
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- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention discloses a method for preparing adipose-derived stem cell multi-cell active factor freeze-dried powder, which comprises the following steps: s1: collecting human adipose tissues; s2: primary isolation, culture and passage of adipose-derived stem cells; s3: amplifying mesenchymal stem cells in a large scale and collecting supernatant; s4: preparing a concentrated stock solution of the stem cell active factor; s5: and (5) preparing freeze-dried powder. The freeze-dried powder of the multi-cell active factors prepared by the preparation method is safe and reliable to use, has no adverse reaction, high yield, contains a large amount of cell active factors, has good activity and easy preservation, and can remarkably promote the healing of deep cell tissues of damaged skin and fade scars and acne marks.
Description
Technical Field
The invention relates to the technical field of biological medicines, and in particular relates to a preparation method of adipose-derived stem cell multi-cell active factor freeze-dried powder.
Background
Adipose-derived stem cells (ADSCs) are currently widely used in the field of regenerative medicine research. Compared with bone marrow stem cells and umbilical cord stem cells, the adipose-derived stem cells have the advantages of abundant raw materials, convenient material acquisition, strong cell paracrine function and the like, particularly have strong paracrine function, about 800 cell growth factors exist in a human body, 765 cell growth factors can be secreted by the ADSC, and the ADSC is superior to other mesenchymal stem cells such as bone marrow, umbilical cord and the like and adult mesenchymal stem cells.
The excellent paracrine function of adipose stem cells was soon discovered and widely used in the cosmetology industry and is gradually favored by other indications. The secreted cell active factors comprise Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (b-FGF), Nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), stem cell growth factor (HGF), insulin-like growth factor (IGF), interleukin 1(IL-1), interleukin 11(IL-11), Stem Cell Factor (SCF) and the like, can induce the proliferation and migration of cells, slow down apoptosis, repair damaged tissue cells, promote angiogenesis, inhibit inflammation to regulate the immune state of an organism, can also promote the proliferation and differentiation of nerve stem cells, improve the peripheral nerve function, can well promote wound repair on the excellent repair capacity of a microenvironment, and avoid scar formation.
The cell active factors are added into the beauty cosmetics, so that the beauty cosmetics not only have the moisturizing and whitening effects of common cosmetics, but also can repair damaged skin, eliminate skin wrinkles, shrink pores, improve complexion and the like, and are more and more concerned by people. However, when the conventional culture method is used for culture, the activity of stem cells is low, the content of secreted cell active factors is low, the yield is low, the storage time is short, and the cell culture supernatant liquid needs to be collected for multiple generations, so that the efficiency is low, and the uniformity of products is difficult to ensure.
Therefore, there is an urgent need to develop a method for preparing lyophilized powder of adipose-derived stem cell multi-cellular active factor, which can prepare lyophilized powder of multi-cellular active factor with high yield, good activity, high content of cell active factor and good homogeneity.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a preparation method of adipose-derived stem cell multi-cell active factor freeze-dried powder, and the prepared multi-cell active factor freeze-dried powder has high yield, good activity, high content of cell active factors in the product and good uniformity.
In order to solve the problems, the invention adopts the following technical scheme:
a method for preparing multi-cell active factor freeze-dried powder of adipose-derived stem cells comprises the following steps:
s1: collecting human adipose tissues;
s2: primary isolation, culture and passage of adipose-derived stem cells;
s3: amplifying mesenchymal stem cells in a large scale and collecting supernatant;
s4: preparing a concentrated stock solution of the stem cell active factor;
s5: and (5) preparing freeze-dried powder.
Further, the specific process of the human adipose tissue collection in step S1 is as follows: carrying out physical examination on a donor, then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia, and filtering grease and water from the obtained fat through a 500-micron filtering hole to obtain light yellow fat.
Further, the primary isolation, culture and passage of the adipose-derived stem cells in step S2 are specifically performed as follows:
(1) washing the fat obtained in the step S1 for multiple times by PBS (phosphate buffer solution) until the washing liquid is clear and transparent, the fat is in clean light yellow, then putting the obtained fat 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 ℃, and then adding a Trypsin inhibitor to uniformly mix and terminate 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 to obtain primary adipose-derived stem cells;
(2) adding the obtained primary adipose-derived stem cells into a serum-free culture medium for resuspension, then inoculating the primary adipose-derived stem cells into a cell culture bottle coated with a Laminin-521 solution, culturing the primary adipose-derived stem cells in a 5% CO2 culture box at 37 ℃ for 2 days, removing the culture solution, replacing a fresh serum-free culture medium, continuously culturing the cell culture bottle in a 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 the cells by using 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.
Further, the mass expansion of the mesenchymal stem cells and the collection of the supernatant in step S3 are specifically performed as follows:
(1) washing and centrifuging a large amount of adipose-derived stem cells obtained in the step S2 for multiple times by PBS, then inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, carrying out subculture in a 5% CO2 incubator at 37 ℃, and digesting and passaging by 0.125% Trypsin-EDTA digestive juice when the cells grow and fuse to 80-90%;
(2) selecting P3-P8 generation cells, inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, culturing the cells under the gas concentration of 21% O2, 5% CO2 and 74% N2 for 8 hours after inoculation, then gradually reducing the oxygen concentration to 10% and continuing culturing for 8 hours, gradually reducing the oxygen concentration to 5% again and continuing culturing for 8 hours, wherein the cell growth is fused to 90%, recovering the supernatant and storing the supernatant at 4 ℃, replacing a paracrine special culture medium and reducing the oxygen concentration to 2% and continuing culturing for 24 hours, collecting the supernatant every 24 hours, replacing a fresh paracrine special culture medium once, and collecting the supernatant for 5 times in total.
Further, the preparation process of the stem cell active factor concentrated stock solution in step S4 is as follows:
(1) filtering the supernatant collected in the step S3 by a 0.45-micrometer filter membrane in sequence to remove cell debris, and filtering and sterilizing by a 0.22-micrometer filter membrane; then carrying out sterile detection, mycoplasma detection, endotoxin detection, protein concentration and key cytokine concentration detection on the collected supernatant;
(2) and then, pressurizing and concentrating the qualified supernatant through a 100KDa filter membrane to obtain a stem cell active factor concentrated stock solution for later use.
Further, the preparation process of the lyophilized powder in step S5 is as follows: adding freeze-drying auxiliary materials into the stem cell active factor concentrated stock solution obtained in the step S4, adjusting the protein concentration of the stem cell active factor to 5-10 mu g/mL, uniformly mixing, quickly subpackaging into penicillin bottles, carrying out freeze-drying treatment under certain conditions, and then capping and sealing to obtain the multi-cell active factor freeze-dried powder of the adipose-derived stem cells; the freeze-drying auxiliary materials comprise mannitol with the final concentration of 40-50 wt%, oligopeptide-1 with the final concentration of 12-16 wt%, acetyl hexapeptide-8 with the final concentration of 12-16 wt%, octyl glycol with the final concentration of 12-16 wt%, sodium hyaluronate with the final concentration of 0.5-1 wt%, citric acid with the final concentration of 0.04-0.045 wt% and sodium citrate with the final concentration of 0.7-0.8 wt%.
Further, the freeze-drying treatment process specifically operates as follows: rapidly cooling to-50 deg.C, vacuumizing and maintaining for 1 hr; then, the temperature is raised to-40 ℃, and the temperature is kept for 1 hour after the temperature is stabilized; raising the temperature to-30 ℃, stabilizing and keeping for 2 hours; continuously heating to-25 ℃, and keeping for 2 hours after the temperature is stable; raising the temperature to-20 ℃, stabilizing and keeping for 2 hours; raising the temperature to-15 ℃, stabilizing and keeping for 2 hours; raising the temperature to-10 ℃, stabilizing and keeping for 2 hours; finally, the temperature is raised to 20 ℃, and the temperature is kept for 12 hours after the temperature is stabilized.
The invention has the following beneficial effects:
(1) in the invention, fat is obtained by adopting a liposuction method with a swelling method, and the obtained fat is filtered through a 500-micron filtering hole, so that liquid and grease in the fat are effectively removed, and the content of SVF (stromal vascular cell population) in the obtained fat raw material is improved;
(2) fat is digested by mixed collagenase and then is pre-incubated by a low-concentration Trypsin-EDTA (Trypsin-EDTA) solution, so that adipose tissue blocks can be more effectively digested, the recovery rate of SVF is increased, and the single enzyme digestion can be reduced to damage cells by short-time incubation of the low-concentration Trypsin-EDTA solution and the mixed collagenase digestion with proper concentration, and the activity of the recovered cells is improved;
(3) the cell culture bottle is coated by adopting a Laminin-521 solution, so that the anchorage rate and the proliferation rate of cells are increased, and the growth of the cells is facilitated; the multilayer cell culture bottle is adopted to replace a conventional single-layer cell culture bottle, so that the utilization rate of the incubator is improved, the large-scale production is realized, the production cost is reduced, and the uniformity of a finally prepared product is ensured; in the process of cell culture, a serum substitute of human-derived components is adopted to be matched with a basic culture medium, and adhesion factors, growth factors and the like are added, so that adverse factors caused by serum can be reduced, cell proliferation is promoted, and the cell culture conditions are more stable;
(4) the oxygen content is adjusted at different stages of cell hypoxia culture, so that the rapid amplification of cells is realized, the apoptosis resistance of the cells is obviously enhanced, the paracrine performance of the cells is effectively improved, the content of the cell active factors in the collected supernatant is high, the stability is good, the paracrine special culture medium is matched to induce the cells to secrete more cell active factors while maintaining the growth state of the cells, and the yield of the final product and the content of the cell active factors in the product are improved;
(5) the collected supernatant is pressurized and concentrated, and the effective cell activity factor protein component can be retained to the maximum extent while the redundant components such as high salt, high sugar and the like in the liquid are removed; the freeze-drying auxiliary material is added in the freeze-drying process, and can be matched with the cell active factors to promote the regeneration of subcutaneous blood vessels, secrete protein, promote the healing of deep cell tissues of damaged skin and fade scars and acne marks.
The freeze-dried powder of the multi-cell active factors prepared by the preparation method is safe and reliable to use, has no adverse reaction, high yield, contains a large amount of cell active factors, has good activity and easy preservation, and can remarkably promote the healing of deep cell tissues of damaged skin and fade scars and acne marks.
Detailed Description
Example 1
A method for preparing multi-cell active factor freeze-dried powder of adipose-derived stem cells comprises the following steps:
s1: human adipose tissue collection:
carrying out physical examination on a donor, then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia, and filtering grease and water from the obtained fat through a 500-micron filtering hole to obtain light yellow fat.
S2: primary isolation, culture and passage of adipose-derived stem cells:
(1) washing the fat obtained in the step S1 for multiple times by PBS (phosphate buffer solution) until the washing liquid is clear and transparent, the fat is in clean light yellow, then putting the obtained fat into 0.2-0.4% of mixed collagenase (a mixed solution of type I collagenase and type VI 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 to obtain primary adipose-derived stem cells;
(2) adding the obtained primary adipose-derived stem cells into a serum-free culture medium for resuspension, then inoculating the primary adipose-derived stem cells into a cell culture bottle coated with a Laminin-521 solution, culturing the primary adipose-derived stem cells in a 5% CO2 culture box at 37 ℃ for 2 days, removing the culture solution, replacing a fresh serum-free culture medium, continuously culturing the cell culture bottle in a 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 the cells by using 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.
Wherein, the serum-free culture medium 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.
S3: large-scale amplification of mesenchymal stem cells and collection of supernatant:
(1) washing and centrifuging a large amount of adipose-derived stem cells obtained in the step S2 for multiple times by PBS, then inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, carrying out subculture in a 5% CO2 incubator at 37 ℃, and digesting and passaging by 0.125% Trypsin-EDTA digestive juice when the cells grow and fuse to 80-90%;
(2) selecting P3-P8 generation cells, inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, culturing the cells under the gas concentration of 21% O2, 5% CO2 and 74% N2 for 8 hours after inoculation, then gradually reducing the oxygen concentration to 10% and continuing culturing for 8 hours, gradually reducing the oxygen concentration to 5% again and continuing culturing for 8 hours, wherein the cell growth is fused to 90%, recovering the supernatant and storing the supernatant at 4 ℃, replacing a paracrine special culture medium and reducing the oxygen concentration to 2% and continuing culturing for 24 hours, collecting the supernatant every 24 hours, replacing a fresh paracrine special culture medium once, and collecting the supernatant for 5 times in total.
Wherein the paracrine special culture medium is prepared by adding 2-4mg/mL of thioglycerol, 0.5-0.8mol/mL of pyridoxine hydrochloride, 5-10ng/mL of coenzyme A and 2-8ng/mL of coenzyme A on the basis of a serum-free culture medium.
S4: preparing a concentrated stock solution of the stem cell active factor:
(1) filtering the supernatant collected in the step S3 by a 0.45-micrometer filter membrane in sequence to remove cell debris, and filtering and sterilizing by a 0.22-micrometer filter membrane; then carrying out sterile detection, mycoplasma detection, endotoxin detection, protein concentration and key cytokine concentration detection on the collected supernatant;
(2) and then, pressurizing and concentrating the qualified supernatant through a 100KDa filter membrane to obtain a stem cell active factor concentrated stock solution for later use.
S5: preparing freeze-dried powder:
adding mannitol with the final concentration of 40-50 wt%, oligopeptide-1 with the final concentration of 12-16 wt%, acetyl hexapeptide-8 with the final concentration of 12-16 wt%, octyl glycol with the final concentration of 12-16 wt%, sodium hyaluronate with the final concentration of 0.5-1 wt%, citric acid with the final concentration of 0.04-0.045 wt% and sodium citrate with the final concentration of 0.7-0.8 wt% into the concentrated stock solution of the dry cell activity factor obtained in the step S4, adjusting the protein concentration of the dry cell activity factor to 5-10 mug/mL, and uniformly mixing; and quickly subpackaging the obtained product into penicillin bottles, carrying out freeze-drying treatment under certain conditions, and then carrying out gland sealing to obtain the multi-cell active factor freeze-dried powder of the adipose-derived stem cells.
The freeze-drying treatment process comprises the following specific operations: rapidly cooling to-50 deg.C, vacuumizing and maintaining for 1 hr; then, the temperature is raised to-40 ℃, and the temperature is kept for 1 hour after the temperature is stabilized; raising the temperature to-30 ℃, stabilizing and keeping for 2 hours; continuously heating to-25 ℃, and keeping for 2 hours after the temperature is stable; raising the temperature to-20 ℃, stabilizing and keeping for 2 hours; raising the temperature to-15 ℃, stabilizing and keeping for 2 hours; raising the temperature to-10 ℃, stabilizing and keeping for 2 hours; finally, the temperature is raised to 20 ℃, and the temperature is kept for 12 hours after the temperature is stabilized.
Example 2
Selection of cell culture flask coating reagent
The coating of the cell culture bottle can enhance the adherence rate of the cells and increase the proliferation efficiency. In the present example, culture flasks were not coated (method 1), gelatin coated (method 2), Laminin-111 coated (method 3) and Laminin-521 coated (method 4) based on example 1, and the number of cells in the subculture process was measured and the cytokine in the finally prepared lyophilized powder was measured according to example 1, and the experimental results are shown in tables 1 and 2.
TABLE 1 Effect of selection of cell culture flask coating Agents on cell proliferation
TABLE 2 Effect of selection of cell culture flask coating Agents on cytokine levels
The experimental results in tables 1 and 2 show that in method 4, the proliferation rate when the cell culture flask is coated with lamin-521 and the content of the cell activity factor in the finally prepared lyophilized powder are higher than those of samples which are not coated (method 1), are coated with gelatin (method 2) or are coated with lamin-111 (method 3).
Example 3
Influence of oxygen concentration on secretion of cell active factors during anaerobic cell culture
In this embodiment, on the basis of embodiment 1, the oxygen concentration in the process of the large-scale expansion of the mesenchymal stem cells and the collection of the supernatant in step S3 during the anaerobic culture of the cells is respectively controlled:
comparative example 1 was prepared by the same procedure as in example 1.
Comparative example 2 differs from example 1 in that: selecting P3-P8 generation cells in the step S3 to inoculate the cells into a multilayer cell culture bottle coated with a Laminin-521 solution, culturing the cells under the gas concentration of 21% O2, 5% CO2 and 74% N2 for 8 hours after inoculation, then gradually reducing the oxygen concentration to 2%, continuing for 16 hours, recovering the supernatant, storing the supernatant at 4 ℃, replacing the paracrine special culture medium for continuing to culture for 24 hours, collecting the supernatant every 24 hours, replacing the fresh paracrine special culture medium once, and collecting the supernatant for 5 times in total.
Comparative example 3 differs from example 1 in that: selecting P3-P8 generation cells from the step S3, inoculating the cells into a multilayer cell culture bottle coated with a Laminin-521 solution, culturing the cells under the gas concentration of 21% O2, 5% CO2 and 74% N2 for 8 hours after inoculation, then gradually reducing the oxygen concentration to 15% and continuing culturing for 8 hours, then gradually reducing the oxygen concentration to 10% and continuing culturing for 8 hours, recovering the supernatant and storing the supernatant at the temperature of 4 ℃, replacing a special paracrine culture medium and continuing culturing for 8 hours after the oxygen concentration is reduced to 5%, then reducing the oxygen concentration to 2% and continuing culturing for 16 hours, collecting the supernatant every 24 hours, replacing a fresh special paracrine culture medium, and collecting the supernatant for 5 times in total.
Comparative example 4 differs from example 1 in that: the oxygen concentration is controlled to be 21% during the anaerobic culture in step S3.
And the cell state and the content of the cytokine in the supernatant collected in comparative examples 1 to 3 were measured, and the experimental results are shown in table 3.
TABLE 3 Effect of oxygen concentration on secretion of cytokines
As is clear from the experimental results in Table 3, the cell amplification speed in comparative example 1 was the fastest, apoptotic cells were few, and the concentration of the cell activity factor in the recovered supernatant was high, which is the most suitable hypoxic culture method.
Example 4
Recovery rate of cell active factors in freeze-dried powder under different freeze-drying formulas
In this example, based on the preparation process in example 1, the concentrated stock solution of stem cell active factors and the adjuvants are mixed according to the following 3 formulas, and the recovery rates of the relevant cell active factors in the lyophilized powder under different formulas are compared for measurement, and the measurement results are shown in table 4.
Formula 1: the freeze-dried auxiliary materials comprise mannitol with the final concentration of 40-50 wt%, oligopeptide-1 with the final concentration of 12-16 wt%, acetyl hexapeptide-8 with the final concentration of 12-16 wt%, octyl glycol with the final concentration of 12-16 wt%, sodium hyaluronate with the final concentration of 0.5-1 wt%, citric acid with the final concentration of 0.04-0.045 wt% and sodium citrate with the final concentration of 0.7-0.8 wt%.
Adding freeze-drying auxiliary materials to adjust the concentration of the stem cell active factor protein to 3 mug/mL.
And (2) formula: the freeze-drying auxiliary materials in the formula are the same as those in the formula 1, and the freeze-drying auxiliary materials are added to adjust the concentration of the stem cell active factor protein to 6 mug/mL.
And (3) formula: the freeze-drying auxiliary materials in the formula are the same as those in the formula 1, and the freeze-drying auxiliary materials are added to adjust the concentration of the stem cell active factor protein to 12 mug/mL.
As shown in Table 4, when the concentration of the stem cell active factor concentrated stock solution is adjusted to 6 mug/mL by adding the freeze-drying auxiliary material in the freeze-drying formula, the protein loss rate is the lowest, and the recovery rate of the effective protein factor is the highest.
In the invention, fat is obtained by adopting a liposuction method with a swelling method, and the obtained fat is filtered through a 500-micron filtering hole, so that liquid and grease in the fat can be effectively removed, and the content of SVF (stromal vascular cell population) in the obtained fat raw material is improved; fat is digested by mixed collagenase and then is pre-incubated by a low-concentration Trypsin-EDTA (Trypsin-EDTA) solution, so that adipose tissue blocks can be more effectively digested, the recovery rate of SVF is increased, and the single enzyme digestion can be reduced to damage cells by short-time incubation of the low-concentration Trypsin-EDTA solution and the mixed collagenase digestion with proper concentration, and the activity of the recovered cells is improved; the cell culture bottle is coated by adopting a Laminin-521 solution, so that the anchorage rate and the proliferation rate of cells are increased, and the growth of the cells is facilitated; the multilayer cell culture bottle is adopted to replace a conventional single-layer cell culture bottle, so that the utilization rate of the incubator is improved, the large-scale production is realized, the production cost is reduced, and the uniformity of a finally prepared product is ensured; in the process of cell culture, a serum substitute of human-derived components is adopted to be matched with a basic culture medium, and adhesion factors, growth factors and the like are added, so that adverse factors caused by serum can be reduced, cell proliferation is promoted, and the cell culture conditions are more stable; the oxygen content is adjusted at different stages of cell hypoxia culture, so that the rapid amplification of cells is realized, the apoptosis resistance of the cells is obviously enhanced, the paracrine performance of the cells is effectively improved, the content of the cell active factors in the collected supernatant is high, the stability is good, the paracrine special culture medium is matched to induce the cells to secrete more cell active factors while maintaining the growth state of the cells, and the yield of the final product and the content of the cell active factors in the product are improved; the collected supernatant is pressurized and concentrated, and the effective cell activity factor protein component can be retained to the maximum extent while the redundant components such as high salt, high sugar and the like in the liquid are removed; the freeze-drying auxiliary material is added in the freeze-drying process, and can be matched with the cell active factors to promote the regeneration of subcutaneous blood vessels, secrete protein, promote the healing of deep cell tissues of damaged skin and fade scars and acne marks.
The freeze-dried powder of the multi-cell active factors prepared by the preparation method is safe and reliable to use, has no adverse reaction, high yield, contains a large amount of cell active factors, has good activity and easy preservation, and can remarkably promote the healing of deep cell tissues of damaged skin and fade scars and acne marks.
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 (7)
1. A method for preparing adipose-derived stem cell multi-cell active factor freeze-dried powder is characterized by comprising the following steps:
s1: collecting human adipose tissues;
s2: primary isolation, culture and passage of adipose-derived stem cells;
s3: amplifying mesenchymal stem cells in a large scale and collecting supernatant;
s4: preparing a concentrated stock solution of the stem cell active factor;
s5: and (5) preparing freeze-dried powder.
2. The method for preparing the lyophilized powder of adipose-derived stem cell multi-cellular active factor according to claim 1, wherein the specific process of collecting the adipose tissues of the human body in step S1 is as follows: carrying out physical examination on a donor, then obtaining fat by adopting a swelling liposuction method under the state of local anesthesia, and filtering grease and water from the obtained fat through a 500-micron filtering hole to obtain light yellow fat.
3. The method for preparing the lyophilized powder of the adipose-derived stem cell multi-cellular activity factor according to claim 1, wherein the specific processes of primary isolation, culture and passage of the adipose-derived stem cells in step S2 are as follows:
(1) washing the fat obtained in the step S1 for multiple times by PBS (phosphate buffer solution) until the washing liquid is clear and transparent, the fat is in clean light yellow, then putting the obtained fat 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 ℃, and then adding a Trypsin inhibitor to uniformly mix and terminate 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 to obtain primary adipose-derived stem cells;
(2) adding the obtained primary adipose-derived stem cells into a serum-free culture medium for resuspension, then inoculating the primary adipose-derived stem cells into a cell culture bottle coated with a Laminin-521 solution, culturing the primary adipose-derived stem cells in a 5% CO2 culture box at 37 ℃ for 2 days, removing the culture solution, replacing a fresh serum-free culture medium, continuously culturing the cell culture bottle in a 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 the cells by using 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.
4. The method for preparing the lyophilized powder of adipose-derived stem cell multi-cellular active factor according to claim 1, wherein the specific processes of the large-scale expansion of the mesenchymal stem cells and the collection of the supernatant in step S3 are as follows:
(1) washing and centrifuging a large amount of adipose-derived stem cells obtained in the step S2 for multiple times by PBS, then inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, carrying out subculture in a 5% CO2 incubator at 37 ℃, and digesting and passaging by 0.125% Trypsin-EDTA digestive juice when the cells grow and fuse to 80-90%;
(2) selecting P3-P8 generation cells, inoculating the cells into a multilayer cell culture bottle coated by a Laminin-521 solution, culturing the cells under the gas concentration of 21% O2, 5% CO2 and 74% N2 for 8 hours after inoculation, then gradually reducing the oxygen concentration to 10% and continuing culturing for 8 hours, gradually reducing the oxygen concentration to 5% again and continuing culturing for 8 hours, wherein the cell growth is fused to 90%, recovering the supernatant and storing the supernatant at 4 ℃, replacing a paracrine special culture medium and reducing the oxygen concentration to 2% and continuing culturing for 24 hours, collecting the supernatant every 24 hours, replacing a fresh paracrine special culture medium once, and collecting the supernatant for 5 times in total.
5. The method for preparing the lyophilized powder of the adipose-derived stem cell multi-cellular active factor according to claim 1, wherein the preparation process of the concentrated stock solution of the adipose-derived stem cell multi-cellular active factor in step S4 is as follows:
(1) filtering the supernatant collected in the step S3 by a 0.45-micrometer filter membrane in sequence to remove cell debris, filtering and sterilizing by a 0.22-micrometer filter membrane, and then carrying out sterile detection, mycoplasma detection, endotoxin detection, protein concentration detection and key cytokine concentration detection on the collected supernatant;
(2) and then, pressurizing and concentrating the qualified supernatant through a 100KDa filter membrane to obtain a stem cell active factor concentrated stock solution for later use.
6. The method for preparing the lyophilized powder of adipose-derived stem cell multi-cellular active factor according to claim 1, wherein the lyophilized powder prepared in step S5 is prepared by the following steps: adding freeze-drying auxiliary materials into the stem cell active factor concentrated stock solution obtained in the step S4, adjusting the protein concentration of the stem cell active factor to 5-10 mu g/mL, uniformly mixing, quickly subpackaging into penicillin bottles, carrying out freeze-drying treatment under certain conditions, and then capping and sealing to obtain the multi-cell active factor freeze-dried powder of the adipose-derived stem cells; the freeze-drying auxiliary materials comprise mannitol with the final concentration of 40-50 wt%, oligopeptide-1 with the final concentration of 12-16 wt%, acetyl hexapeptide-8 with the final concentration of 12-16 wt%, octyl glycol with the final concentration of 12-16 wt%, sodium hyaluronate with the final concentration of 0.5-1 wt%, citric acid with the final concentration of 0.04-0.045 wt% and sodium citrate with the final concentration of 0.7-0.8 wt%.
7. The method for preparing the lyophilized powder of the adipose-derived stem cell multi-cellular active factor according to claim 6, wherein the lyophilization process is specifically operated as follows: rapidly cooling to-50 deg.C, vacuumizing and maintaining for 1 hr; then, the temperature is raised to-40 ℃, and the temperature is kept for 1 hour after the temperature is stabilized; then heating to-30 ℃, and keeping for 2 hours after the temperature is stable; continuously heating to-25 ℃, and keeping for 2 hours after the temperature is stable; then heating to-20 ℃, and keeping for 2 hours after the temperature is stable; then heating to-15 ℃, and keeping for 2 hours after the temperature is stabilized; then heating to-10 ℃, and keeping for 2 hours after the temperature is stable; finally, the temperature is raised to 20 ℃, and the temperature is kept for 12 hours after the temperature is stabilized.
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|---|---|---|---|---|
| CN112602704A (en) * | 2020-12-28 | 2021-04-06 | 杜琳琳 | Mesenchymal stem cell factor freeze-drying protective agent and application thereof |
| CN113712996A (en) * | 2021-09-09 | 2021-11-30 | 江苏中衍生科细胞技术研究院有限公司 | Method for promoting hair regeneration by adipose-derived mesenchymal stem cell factor compound |
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| CN113712996A (en) * | 2021-09-09 | 2021-11-30 | 江苏中衍生科细胞技术研究院有限公司 | Method for promoting hair regeneration by adipose-derived mesenchymal stem cell factor compound |
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