CN112779214B - Preparation method of clinical SVF - Google Patents
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Abstract
The invention provides a preparation method of clinical SVF, which comprises the following steps: (1) Rinsing human body fat with normal saline, removing other medicinal components of fat in operation, standing or centrifuging to obtain rinsed human body fat; (2) Placing the rinsed human body fat into a container, and adding a collagenase solution and normal saline into the container; (3) Placing the container in a shaker and digesting for 20-45 minutes at a temperature of 37 ℃, wherein the shaking frequency of the shaker is 175-400rpm per minute; (4) Centrifuging the container cultured by the shaking table in the step (3) for 5-15 minutes at 1500-2000rpm, removing the upper yellow grease layer in a sterile environment, and collecting the bottom material; and (5) centrifugally washing the filter residue by using the washing liquid. The SVF prepared by the method has better total cell number and cell activity.
Description
Technical Field
The invention relates to the field of SVF preparation, in particular to a preparation method of clinical SVF.
The present application claims the priority of applying collagenase for injection to SVF preparation and the method of SVF preparation, filed 3/6/2020/2020101526382.
Background
Adipose tissue has been considered an endocrine organ for its role in energy regulation, inflammatory response and immune response. In addition, it is one of the sources of multifunctional cells having a multipotentiality, which are present in the vascular stromal portion of adipose tissue. The vascular stromal fraction (SVF) in adipose tissue is obtained by collagenase digestion of adipose tissue, filtration, and centrifugation to remove mature adipocytes. Because SVF is easily extracted from Adipose tissue and contains abundant, very plastic Adipose-derived mesenchymal stem cells (ASC), it is necessary to investigate the extraction and preparation of SVF. SVF represents a heterogeneous group of cells that surround adipocytes in adipose tissue and functions include: 1) involvement in angiogenesis in vivo 2) repository of pluripotent stem cells, important in the reconstruction of fat, bone and cartilage 3) endocrine function: has the functions of energy regulation, inflammatory reaction and immune response.
The cellular components of SVF mainly include the following:
(1) Endothelial group cells: CD45-/CD31+/CD34+/CD133+
(2) Endothelial cells: CD31+/CD34-/CD133-
(3) Pericyte: CD146+/CD45-/CD31-
(4) SA-ASC (adipose precursor cells): CD34+/PDGFR alpha +/CD45-/CD31-
(5) Hematopoietic stem cells: CD45+/CD31-/CD146-/CD34-/CD206+/CD14-
(6)ADSC：CD45-/CD31-/CD44+/CD90+/CD73+/CD36+/CD106-
(7) Monocytes: CD14+/CD16+
(8) I macrophage cell: CD14+/CD45+/CD206-/CD86+
II macrophage cell: CD14+/CD45+/CD206+/CD163+/CD68+
The stem cell therapy has huge potential application space in a plurality of clinical fields, has a large number of relevant basic and clinical experimental researches at home and abroad, and has relevant clinical application development in the fields of plastic surgery, cardiology, vascular surgery, burn department, digestive department, hematology, rheumatism immunity department and the like at present. However, the following disadvantages exist for the prior art for the extraction and preparation of SVF: collagenase used in laboratories belongs to a biological agent which cannot be applied to human bodies. SVF prepared from collagenase for injection which can be used in human body has the problems of cell damage and weak proliferation ability.
Disclosure of Invention
The invention provides a preparation method of clinical SVF, which solves the problem of weak proliferation capacity of clinical SVF in the prior art.
The technical scheme of the invention is realized as follows:
a method of preparing clinical-grade SVF, comprising:
(1) Rinsing human body fat with normal saline, removing other medicinal components of fat in operation, standing or centrifuging to obtain rinsed human body fat;
(2) Placing the rinsed human fat in a container, and adding a collagenase solution and normal saline into the container; the collagenase solution is as follows: adding 1-3ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase for injection; wherein the injectable collagenase is an agent for the treatment of herniated disc nucleus pulposus;
adding 0.05-0.15ml of collagenase solution into every 1ml of fat, and then adding physiological saline till the total volume is 2 times of the fat;
(3) Placing the container in a shaker and digesting for 20-45 minutes at a temperature of 37 ℃, wherein the shaking frequency of the shaker is 175-400rpm per minute;
(4) Centrifuging the container cultured by the shaking table in the step (3) for 5-15 minutes at 1500-2000rpm, removing the upper yellow grease layer in a sterile environment, and collecting the bottom substance; adding a washing solution into the bottom layer substance, repeatedly blowing, resuspending, filtering, removing the filtrate, and keeping the filter residue for later use;
the washing solution consists of amino acid, glucose and normal saline, and specifically, 800ul of amino acid and 10ml of 10% glucose are added into each 100ml of normal saline;
(5) And (3) centrifugally washing the filter residue by using the washing liquid, wherein the centrifugal force is 1200-1500g, each time lasts for 3-5 minutes, and the lower-layer cell mass is SVF.
In some embodiments, the amino acid is: alanyl glutamine, alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, L-alanine, L-alanyl-L-glutamine, L-arginine-HCL, L-aspartic acid-H 2 O, L-cystine 2HCL, L-glutamic acid, L-glutamine, glycine, L-histidine HCL H 2 And O or a combination of more than one of the combination of O.
In some embodiments, the collagenase solution is: adding 2ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase.
In some embodiments, 0.05-0.15ml of the collagenase solution is added per 1ml of fat.
In some embodiments, the step (5) centrifugal washing 1-3 times. More preferably, the washing is performed 2 times by centrifugation.
In some embodiments, the filtering of step (5) is performed using a 40-100 μm single cell filter.
Advantageous effects
(1) The SVF prepared by the method has better total cell number and cell activity.
(2) The method of the invention adds the washing solution in the washing process, thus improving the problem of cell adherence.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.
FIG. 1: under different treatment conditions, the invention prepares the comparison of the total cell number, the living cell number, the dead cell number and the cell activity of the SVF;
FIG. 2: under different treatment conditions, a comparison graph of the total number of the SVF cells is prepared;
FIG. 3: under different treatment conditions, a contrast chart of the cell activity of the SVF is prepared;
FIG. 4: the adherence condition of SVF obtained under different treatment conditions during culture;
FIG. 5: a comparison of the total number of cells of SVF was made under different treatment conditions of Table 1;
FIG. 6: a comparative plot of the cell viability of SVF was prepared for different treatment conditions in Table 1;
FIG. 7: growth curves of the SVF prepared in example 2 and SVF obtained by other methods.
FIG. 8: cell proliferation of SVF prepared in example 2.
FIG. 9: cell proliferation of SVF prepared by the conventional method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to solve the problem of weak proliferation ability of cells obtained by collagenase preparation with physiological saline alone, i.e., the problem of impaired cell function, a washing solution capable of maintaining cell function in place of a laboratory culture solution was obtained by the following experiment.
1. Different methods of preparation of SVF.
Collagenase and physiological saline
Preparing a collagenase solution: adding 3ml of normal saline into 900 units of collagenase for injection, and fully mixing to obtain the collagenase for injection; wherein the collagenase for injection is a preparation for treating herniated disc nucleus pulposus.
The method specifically comprises the following steps:
(1) Rinsing human body fat with normal saline for 3 times, centrifuging at 600rpm for 2min, removing upper free fat drop and lower swelling solution, and purifying fat in the middle layer.
(2) 20ml of purified fat was placed in a container, and the entire amount of the prepared collagenase solution and 17ml of physiological saline were added to the container.
(3) The vessel was placed in a shaker and digested at 37 ℃ for 45 minutes with shaking at a frequency of 175rpm on the shaker.
(4) Centrifuging the container cultured by the shaking table in the step (3) for 5 minutes at 1500rpm, removing the upper yellow grease layer in a sterile environment, and collecting the bottom material; resuspending the bottom layer material, filtering, removing the filtrate, and filtering the residue for use; the adopted filter screen is a 100 mu m single cell filter screen.
(5) Centrifuging and washing filter residues, wherein the centrifugal force is 1500g, and centrifuging for 3 minutes; and (4) centrifuging again for washing, wherein the centrifugal force is 1500g, and centrifuging for 3 minutes to obtain a lower layer cell mass which is SVF.
(di) collagenase + culture solution
The collagenase (I) and physiological saline (DMEM) were replaced with collagenase (II) and the same procedure was repeated.
(III) adding a washing solution during centrifugation
The washing liquid comprises the following components: 800ul alanylglutamine and 10ml10% glucose were added to 100ml of physiological saline.
In the above procedure for preparing SVF using collagenase (i) and physiological saline, washing solution was added during the washing in step (5), and the rest of the procedure was the same as in step (i).
(IV) control group of collagenase in laboratory
(1) Adipose tissue is washed 2-3 times by liposuction with normal saline;
(2) Moving into a centrifugal tube at 600rpm for 2-3mins, and removing upper oil and lower swelling liquid;
(3) Prepare 0.2% collagenase NB4 etc (laboratory grade) solution: 0.2g collagenase dry powder +100ml low sugar DMEM (i.e. conventional cell culture solution laboratory preparation), sterile filter screen filtration;
(4) 1, adding collagenase preparation with the same amount as that of the mixed solution into digested fat cells, and treating the fat cells by a shaking table at 37 ℃ for 60mins by fully shaking at 150 rpm;
(5) Filtering with a filter screen with the aperture of 100 mu m after digestion, removing upper-layer grease, and centrifuging at 1500rpm for 5 mins;
(6) Dumping and discarding the supernatant, adding PBS to wash for 2 times at 1500rpm 5 mins;
(7) The supernatant was decanted and the cells pelleted as SVF.
The experimental collagenase NB4 and the low-sugar DMEM are not suitable for clinical human body application, and have long digestion and filtration time.
The SVF prepared by the above four methods is shown in FIG. 1-FIG. 3 for comparison of data.
As can be seen from fig. 1 to 3:
adding amino acid and glucose cell group during centrifugation, wherein the total number of cells is higher than three groups;
the cell activity of the amino acid + glucose cell group was significantly higher in the collagenase plus saline group (p = 0.0063) and the experimental collagenase control group (p = 0.0467) at centrifugation.
2. Comparison of cell adherence
The cell anchorage capacity of the amino acid + glucose cell group is significantly better than that of the other three groups during centrifugation, as shown in FIG. 4.
3. The optimal proportion and the addition time of the washing solution are determined, and the experimental data are shown in figures 5-6.
The data obtained by using different processing conditions in table 1 are very different.
TABLE 1 data under different treatment conditions
Based on the above experiments, the formulation of the selected washing solution was: adding 800ul amino acid and 10ml10% glucose into 100ml normal saline; the timing of addition was when centrifugation was performed after fat digestion. See example 1 for details.
The alanylglutamine is added during elution, and various proteasomes, lysosomes and autophagosomes released by the disrupted cells can convert the alanylglutamine into alanyl-glutamine.
The above experimental results show that the addition during elution is more effective than the addition during digestion, probably because digestion is when digestive enzymes are acting; the elution is not interfered by digestive enzyme, and the amino acid can play a better protection role.
Example 1
A method of preparing clinical-grade SVF, comprising:
preparing a collagenase solution: adding 3ml of normal saline into 900 units of collagenase for injection, and fully mixing to obtain the collagenase for injection; wherein the collagenase for injection is a preparation for treating herniated disc nucleus pulposus.
Preparing a washing solution: 800ul alanylglutamine and 10ml10% glucose were added to 100ml of physiological saline.
(1) Rinsing human body fat with normal saline for 3 times, centrifuging at 600rpm for 2min, removing upper free fat drop and lower swelling solution, and purifying fat in the middle layer.
(2) 20ml of purified fat was placed in a container, and the entire amount of the prepared collagenase solution and 17ml of physiological saline were added to the container.
(3) The vessel was placed in a shaker and digested at 37 ℃ for 45 minutes with shaking at a frequency of 175rpm on the shaker.
(4) Centrifuging the container cultured by the shaking table in the step (3) for 5 minutes at 1500rpm, removing the upper yellow grease layer in a sterile environment, and collecting the bottom substance; adding 10ml of washing liquid into the bottom layer material, repeatedly blowing, resuspending, filtering, removing the filtrate, and keeping the filter residue for later use; the adopted filter screen is a 100 mu m single cell filter screen.
(5) Centrifuging and washing the filter residue by using 10ml of washing liquid, wherein the centrifugal force is 1500g, and centrifuging for 3 minutes; and adding 10ml of washing solution again for centrifugal washing, wherein the centrifugal force is 1500g, and centrifuging for 3 minutes to obtain the lower layer cell mass which is the SVF.
In the method, a washing solution is added in the washing process in the step (5) for washing, so that the total cell number, the viable cell number and the cell activity of the prepared SVF are superior to those of the prior laboratory culture method.
The existing laboratory method can not be applied to human bodies due to substances such as fetal calf serum and the like.
Therefore, the addition of the washing solution during the washing in step (5) as described above not only improves the relevant data of SVF, but also makes the SVF clinically useful.
Example 2
Preparing collagenase for injection into collagenase solution for digestion, which comprises the following steps: adding 2ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase.
The method for preparing SVF using the collagenase for injection comprises:
(1) Standing the sucked human body fat until the fat and the liquid are layered, removing the liquid, rinsing the fat with normal saline for 2 times, and removing the medical components such as lidocaine, epinephrine and the like in the operation.
(2) 10ml of the washed fat was taken in a 50ml sterile centrifuge tube and 1ml of collagenase solution was added followed by physiological saline until the total solution volume in the sterile centrifuge tube reached 20 ml.
(3) And (3) obliquely placing the sterile centrifuge tube obtained in the step (2) in a constant-temperature shaking table. The specific parameters can be selected from digestion at 37 deg.C for 20-120 min, and the oscillation frequency of constant temperature shaking table is 150-400rpm per minute. Digestion was carried out for 30 minutes in this example, with an oscillation frequency of 200rpm.
(4) Placing the sterile centrifuge tube obtained in the step (3) into a centrifuge for centrifugation for 10 minutes, wherein the centrifugal force is 1500g, sucking out supernatant in a sterile environment, adding physiological saline into a lower layer for resuspension, filtering through a single cell filter screen with the size of 70 mu m, removing filtrate, and keeping filter residues for later use;
(5) And (5) centrifugally washing the filter residue obtained in the step (4) for 1-3 times, wherein the centrifugal force is 500g, and each time lasts for 3-5 minutes, and the precipitated cell mass is SVF. The prepared SVF has collagenase content of less than 0.001U/ml.
Example 3
Collagenase for injection (Shanghai Qiaoyuan) was used in the preparation of SVF.
Preparing collagenase for injection into a collagenase solution for digestion, which comprises the following steps: adding 2ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase.
A method for producing SVF using the collagenase for injection, comprising:
(1) Standing the sucked human body fat until the fat and the liquid are layered, removing the liquid, rinsing the fat with normal saline for 2 times, and removing the medical components such as lidocaine, epinephrine and the like in the operation.
(2) 10ml of the washed fat was taken in a 50ml sterile centrifuge tube and 3ml of collagenase solution was added followed by physiological saline until the total solution volume in the sterile centrifuge tube reached 20 ml.
(3) And (3) obliquely placing the sterile centrifuge tube obtained in the step (2) in a constant-temperature shaking table. The specific parameters can be selected from digestion at 37 deg.C for 20 min, and oscillation frequency of constant temperature shaker at 400rpm.
(4) Placing the sterile centrifuge tube obtained in the step (3) into a centrifuge for centrifugation for 15 minutes, wherein the centrifugal force is 500g, sucking out supernatant in a sterile environment, adding normal saline into a lower layer for resuspension, filtering through a 40-micron single-cell filter screen, removing filtrate, and keeping filter residues for later use;
(5) And (5) centrifugally washing the filter residue in the step (4) for 2 times, wherein the centrifugal force is 300g, and each time lasts for 3-5 minutes, and the settled cell mass is SVF. The prepared SVF has collagenase content of less than 0.001U/ml.
Example 4
Collagenase for injection (jogao source in shanghai) was used in the preparation of SVF.
Preparing collagenase for injection into a collagenase solution for digestion, which comprises the following steps: adding 2ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase.
A method for producing SVF using the collagenase for injection, comprising:
(1) Standing the sucked human body fat until the fat and the liquid are layered, removing the liquid, rinsing the fat with normal saline for 2 times, and removing the medicinal components such as lidocaine, adrenaline and the like in the operation.
(2) 10ml of the washed fat was taken in a 50ml sterile centrifuge tube and 0.5ml of collagenase solution was added followed by physiological saline until the total solution volume in the sterile centrifuge tube reached 20 ml.
(3) And (3) obliquely placing the sterile centrifuge tube obtained in the step (2) in a constant-temperature shaking table. The specific parameters can be selected from digestion at 37 deg.C for 20-120 min, and the oscillation frequency of constant temperature shaking table is 150-400rpm per minute. Digestion was carried out for 120 minutes in this example, with an oscillation frequency of 150rpm.
(4) Placing the sterile centrifuge tube obtained in the step (3) into a centrifuge for centrifugation for 5 minutes, wherein the centrifugal force is 2000g, removing an upper yellow grease layer in a sterile environment, adding normal saline into a lower layer for resuspension, filtering through a 100-micron single-cell filter screen, removing filtrate, and keeping filter residues for later use;
(5) And (4) centrifugally washing the filter residue in the step (4) for 1-3 times, wherein the centrifugal force is 1500g, each time lasts for 3-5 minutes, and the settled cell mass is SVF. The prepared SVF has collagenase content of less than 0.001U/ml.
Comparative example 1
The laboratory collagenase CollageneseeNB 4 (Sigma) was used in the preparation of SVF.
Preparing laboratory collagenase into a collagenase solution for digestion, and the specific process is as follows: adding 50ml of normal saline into 40mg of laboratory collagenase, and fully mixing to obtain the collagenase.
A method for preparing SVF using the laboratory collagenase, comprising:
(1) Standing the sucked human body fat until the fat and the liquid are layered, removing the liquid, rinsing the fat with normal saline for 2 times, and removing the medical components such as lidocaine, epinephrine and the like in the operation.
(2) 10ml of the washed fat was placed in a 50ml sterile centrifuge tube and 10ml of the prepared laboratory collagenase was added.
(3) And (3) obliquely placing the sterile centrifuge tube obtained in the step (2) in a constant-temperature shaking table. The specific parameters can be selected from digestion at 37 deg.C for 30 min, and oscillation frequency of constant temperature shaker 200rpm per minute. Digestion was carried out for 30 minutes in this example, with an oscillation frequency of 200rpm.
(4) Placing the sterile centrifuge tube obtained in the step (3) into a centrifuge for centrifugation for 10 minutes, wherein the centrifugal force is 1500g, removing an upper yellow grease layer in a sterile environment, adding physiological saline into a lower layer for heavy suspension, filtering through a 40-micron single-cell filter screen, removing filtrate, and keeping filter residues for later use;
(5) And (4) centrifugally washing the filter residue in the step (4) for 2 times, wherein the centrifugal force is 700g, each time lasts for 3-5 minutes, and the settled cell mass is SVF.
The SVF prepared by the method of example 2 of the present invention was compared to the SVF prepared by the prior art (comparative example 1) as follows:
1. detection of collagenase residues:
the usual collagenases in the literature have a digestion concentration of 0.075-0.2% of digested fat.
In the present invention, the digestion concentration of collagenase is 0.0015% -0.015%, which is lower than the digestion concentration of conventional collagenase, and the residual amount of collagenase in the obtained SVF is very low and can be ignored.
2. Comparison of collagenase amounts:
20ml fat, the method of the present invention uses collagenase for injection that is already on the market, while the conventional method uses 24mg collagenase (test agent), and the amount of SVF finally prepared is not significantly different, respectively: 2.64 +/-0.06 multiplied by 10 7 (n = 5) and 2.63 ± 0.04 × 10 7 (n＝5)p>0.05。
20ml of fat refers to:
(1) The fat used in 2 trials in example 2 was 10ml each.
(2) The fat used in 2 trials in comparative example 1 was 10ml each.
The amount of SVF produced at the end refers to:
(1) SVF prepared in 2 trials in example 2.
(2) SVF produced in 2 trials in comparative example 1.
3. The results of testing the cell activities of the SVFs prepared by different methods by the CCK8 method are shown in FIG. 7, wherein A in FIG. 7 is the SVF prepared by the method of example 2, and B is the SVF prepared by the prior method, therefore, the cell activities of the SVF prepared by the method of example 2 are not obviously different from those of the SVF prepared by the prior method.
4. When SVFP0 prepared by a different method was cultured for 7 days (40X), in which FIG. 8 is SVF prepared in example 2 and FIG. 9 is SVF prepared by a conventional method (comparative example 1), it was found by comparison that the proliferation activity of SVF prepared by the method of the present invention was not significantly different from that of SVF prepared by the conventional method.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (4)
1. A method for preparing clinical-grade SVF, comprising:
(1) Rinsing human body fat with normal saline, removing other medicinal components of fat in operation, standing or centrifuging to obtain rinsed human body fat;
(2) Placing the rinsed human fat in a container, and adding a collagenase solution and normal saline into the container; the collagenase solution is as follows: adding 1-3ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase for injection; wherein the injectable collagenase is an agent for the treatment of herniated disc nucleus pulposus;
adding 0.05-0.15ml of collagenase solution into every 1ml of fat, and then adding physiological saline until the total volume is 2 times of the volume of the fat;
(3) Placing the container in a shaker and digesting for 20-45 minutes at a temperature of 37 ℃, wherein the shaking frequency of the shaker is 175-400rpm per minute;
(4) Centrifuging the container cultured by the shaking table in the step (3) for 5-15 minutes, wherein the centrifugal force is 1500-2000g, removing the upper yellow grease layer in a sterile environment, and collecting the bottom material; adding a washing solution into the bottom layer substance, repeatedly blowing, resuspending, filtering, removing the filtrate, and keeping the filter residue for later use;
the washing liquid consists of alanyl-glutamine, glucose and normal saline, and specifically, 800ul alanyl-glutamine and 10ml10% glucose are added into each 100ml of normal saline;
(5) And (3) centrifugally washing the filter residue by using the washing liquid, wherein the centrifugal force is 1200-1500g, each time lasts for 3-5 minutes, and the lower-layer cell mass is SVF.
2. The method of claim 1, wherein the collagenase solution is: adding 2ml of normal saline into 600 units of collagenase for injection, and fully mixing to obtain the collagenase.
3. The method of preparing SVF according to claim 1, wherein said step (5) comprises 1-3 centrifugal washing steps.
4. The method of claim 1, wherein the filtration of step (4) is performed using a 40-100 μm single cell filter.
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| EP2792741A1 (en) * | 2013-04-18 | 2014-10-22 | Jaroslav Michalek | Method for isolation of adipose tissue-derived stromal vascular fraction cells |
| CN111321114A (en) * | 2020-03-06 | 2020-06-23 | 上海交通大学医学院附属第九人民医院 | Application of collagenase for injection to preparation of SVF (singular value Fall) and preparation method of SVF |
| CN111690597A (en) * | 2020-06-29 | 2020-09-22 | 海口健康岛生物科技有限公司 | Extraction reagent of human autologous fat vascular stroma component SVF and extraction, amplification and culture method |
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| EP2792741A1 (en) * | 2013-04-18 | 2014-10-22 | Jaroslav Michalek | Method for isolation of adipose tissue-derived stromal vascular fraction cells |
| CN111321114A (en) * | 2020-03-06 | 2020-06-23 | 上海交通大学医学院附属第九人民医院 | Application of collagenase for injection to preparation of SVF (singular value Fall) and preparation method of SVF |
| CN111690597A (en) * | 2020-06-29 | 2020-09-22 | 海口健康岛生物科技有限公司 | Extraction reagent of human autologous fat vascular stroma component SVF and extraction, amplification and culture method |
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