Physical preparation method of purified adipose-derived stem cell gel with high shape plasticity and adipose-derived stem cell gel

Abstract

The invention discloses a physical preparation method of purified adipose-derived stem cell gel with high shape plasticity and the adipose-derived stem cell gel. A physical method is adopted in the whole preparation process, exogenous pollution is avoided to a great extent, and the safety and effectiveness of a purified product are guaranteed. According to the method, fat gum prepared from autologous fat tissue is purified, grease contained in the fat gum and liquid components left when autologous fat is obtained are further removed, the inflammatory reaction after fat filling injection is relieved, and pSVF-gel has better appearance plasticity and can improve the filling effect after injection. According to the method, known SVF cell components and extracellular matrixes of SVF-gel are reserved, and the treatment effect of the further concentrated SVF cells on the wound surface is further improved compared with traditional fat and fat gum.

Description

Technical Field

[0001] The present invention belongs to the technical field of fat transplantation beauty and wound repair, and particularly relates to a physical preparation method of purified adipose tissue stem cell gel with high shape plasticity and the adipose stem cell gel. Background Art

[0002] The fat obtained by vacuum-sucking autologous fat and then centrifuging and emulsifying it twice is divided into three layers. The gel product in the middle layer is adipose tissue stem cell gel (SVF-gel), which is a transplant material currently commonly used in cosmetic fillings.

[0003] SVF-gel is a concentrated fat product obtained by physical methods. It is rich in stromal vasculature fraction (SVF) and extracellular matrix (ECM), and has the advantages of mild inflammatory response, few adverse reactions, and high retention rate. It is widely used in medical cosmetology and the treatment of various diseases. SVF-gel is believed to have the potential to promote tissue repair and regeneration. Studies have shown that the cells and extracellular matrix components in SVF-gel can release growth factors, extracellular vesicles (such as exosomes), and other bioactive molecules, thereby promoting wound healing, angiogenesis, and tissue regeneration. It can also improve the function and structure of diseased tissues by promoting tissue repair and anti-inflammatory effects.

[0004] However, there is still some oil in SVF-gel. Experimental studies have found that oil has a certain inhibitory effect on wound healing, and SVF-gel still retains a certain liquid component, which affects the plasticity of fat transplantation. Therefore, further removing the oil in SVF-gel and further concentrating the fat gel can effectively enhance the therapeutic effect on the wound, and at the same time can significantly improve the plasticity of facial filling shape and postoperative retention rate in the plastic surgery field. Summary of the Invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a physical preparation method and adipose stem cell gel with high plasticity, further improving the healing effect on wounds, improving the filling effect of fat transplantation and the subsequent retention rate, and expanding the application prospects of pSVF-gel in the fields of wounds and beauty.

[0006] The present invention provides a preparation method of pSVF-gel with high shape plasticity, comprising the following steps.

[0007] 1) The original fat tissue of the patient's waist, abdomen or thigh area is obtained by suction and centrifuged to remove the lower layer of blood and swelling fluid. The upper layer of low-density fat is transferred to the syringe, and the middle layer of high-density fat is reserved.

[0008] 2) The upper layer of low-density fat transferred to the syringe in step 1) is connected to another syringe via a Luer connector for push injection, and this is repeated 6 to 10 times until the low-density fat tissue is fully mechanically emulsified and becomes chyloidal.

[0009] 3) The chylomicronized fat obtained in step 2) is centrifuged and separated into three layers: an upper layer of fat, a lower layer of blood and swelling fluid, and a gel-like substance in the middle layer, which is adipose tissue stem cell gel (SVF-gel).

[0010] 4) Transfer an appropriate amount of the upper layer of fat from step 3) to the remaining high-density fat from step 1) and repeat steps 2) and 3) to obtain another portion of SVF-gel.

[0011] 5) Place the SVF-gel obtained in steps 3) and 4) into a sieve, stir the SVF-gel in the sieve at a constant speed, and place gauze on the bottom of the sieve and wipe the bottom of the sieve at a constant speed to absorb the oil and liquid components exuded from the SVF-gel to obtain pSVF-gel.

[0012] Furthermore, the emulsified fat in step 2) can be injected into two syringes at equal volumes, while the syringe pistons are pulled back to observe the degree of fat emulsification.

[0013] Furthermore, in step 4), the amount of oil added is 30%-33% of the volume of the high-density fat.

[0014] Furthermore, the sieve in step 5) is not less than 400 mesh.

[0015] Furthermore, in step 5), every 10 mL of SVF-gel was purified to obtain pSVF-gel with a volume of about 2.5 mL.

[0016] Furthermore, in step 5), when the volume of the SVF-gel under constant stirring does not change significantly, pSVF-ge is obtained. At this time, there is little or no exuded oil and liquid components on the gauze.

[0017] Furthermore, the pSVF-gel finally obtained by the above method has lower fluidity than SVF-gel.

[0018] The present invention also provides pSVF-gel prepared by the above method, which is a component of facial filling material and wound repair material.

[0019] Furthermore, the above-mentioned pSVF-gel is a component of the fat gel filling area of ​​the nose base or glabellar lines.

[0020] The present invention has the following beneficial effects:

[0021] 1) The purification process of the present invention does not use any exogenous chemical reagents and is a physical preparation method, so there is no possibility of chemical contamination;

[0022] 2) The present invention is a product obtained by processing and purifying autologous adipose tissue, is non-immunogenic, and does not pose any ethical issues;

[0023] 3) The present invention has simple operation steps, does not require the use of expensive tools, and has a low time cost;

[0024] 4) The operation process of the present invention has universal applicability. The autologous fat tissue is widely available and easy to obtain, making it suitable for all types of people who need facial augmentation.

[0025] 5) The present invention can use a lipofilling needle suitable for SVF-gel injection for precise facial filling, with better plasticity and longer retention time;

[0026] 6) The present invention further removes mature fat cells, oil and liquid components in SVF-gel, which greatly improves the wound treatment effect and facial filling effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in multiple embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 A flow chart of the fat purification process provided by the present invention;

[0029] Figure 2 Comparison of the appearance and concentration of SVF-gel and pSVF-gel prepared in the present invention;

[0030] Figure 3 Schematic diagram of live-dead staining of pSVF-gel cells provided by the present invention;

[0031] Figure 4 Schematic diagram of the intervention effects of SVF-gel and pSVF-gel provided by the present invention on HUVEC cell migration;

[0032] Figure 5This is a wound image of the SVF-gel and pSVF-gel intervention experiment on rat wound healing provided by the present invention;

[0033] Figure 6 Schematic diagram of the lipidomics comparison results of SVF-gel and pSVF-gel provided by the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0035] Example 1

[0036] This embodiment provides a physical preparation method for purified adipose stem cell gel with high plasticity, comprising the following steps.

[0037] Step 1): Aspirate and obtain the original fat tissue from the patient's waist, abdomen, or thigh area. Centrifuge the collected original fat tissue to remove the blood and swelling fluid in the lower layer. Transfer the upper low-density fat into a syringe, and reserve the middle high-density fat for later use.

[0038] Specifically, fat was aspirated from the patient's thigh or abdomen using a 4 mm diameter multi-hole liposuction needle at -0.75 atm. The harvested fat was transferred to a 60 mL sterile needle-free syringe and centrifuged at 1200 g / min for 3 min (e.g., Figure 1 As shown), remove the blood and swelling fluid in the lower layer to obtain Coleman fat. According to the amount of fat, the upper layer of Coleman fat with more fat is marked as low density, and the lower layer of Coleman fat with less fat is marked as high density (as shown). Figure 1 shown).

[0039] Step 2): The upper layer of low-density fat transferred to the syringe in step 1) is connected to another syringe via a Luer connector for push injection, and this is repeated 6 to 10 times until the low-density fat tissue is fully mechanically emulsified and becomes chyloidal.

[0040] Specifically, the low-density Coleman fat after centrifugation was transferred to two 10 mL sterile syringes of equal volume, which were connected via 2.4 mm Luer connectors. The low-density Coleman fat was repeatedly injected between the two syringes using the pistons of the syringes at a rate of 10 mL / sec for 2 min to make the fat into a chyme state, thereby obtaining nano-fat (such as Figure 1 shown).

[0041] In this step, the emulsified fat can be injected into two syringes at equal volumes while pulling the syringe pistons backward to observe the degree of fat emulsification.

[0042] Step 3): The chylomicronized fat obtained in step 2) is centrifuged and separated into three layers: an upper layer of fat, a lower layer of blood and tumescent fluid, and a gel-like substance in the middle layer, which is adipose tissue stem cell gel (SVF-gel).

[0043] Specifically, the nano fat is centrifuged for a second time at 1600g / min for 3 minutes. After centrifugation, it is divided into three layers: the upper layer of fat and the lower layer of liquid. The gel product in the middle layer is SVF-gel.

[0044] Step 4): Pour an appropriate amount of the upper layer of the nanofat from the second centrifugation into the remaining unprocessed high-density Coleman fat. Repeat steps 2) and 3) to obtain another portion of SVF-gel. In this step, the amount of fat added should be 30%-33% of the volume of the high-density fat.

[0045] Step 5): Transfer the prepared SVF-gel to a sieve, stir the SVF-gel in the sieve at a constant speed, and place a gauze pad on the bottom of the sieve. Wipe the bottom of the sieve at a constant speed to absorb the oil and liquid components that seeped out of the SVF-gel to obtain pSVF-gel (such as Figure 1 (as shown). In this step, the mesh size should be no smaller than 400 mesh. For every 10 mL of SVF-gel, approximately 2.5 mL of pSVF-gel is purified. When the volume of the SVF-gel remains unchanged during constant stirring, pSVF-gel is obtained, and little or no exuded oil or liquid remains on the gauze.

[0046] Example 2

[0047] Step 1): Aspirate and obtain the original fat tissue from the patient's waist, abdomen, or thigh area. Centrifuge the collected original fat tissue to remove the blood and swelling fluid in the lower layer. Transfer the upper low-density fat into a syringe, and reserve the middle high-density fat for later use.

[0048] Specifically, a 3.5 mm diameter multi-side hole liposuction needle was used to aspirate fat from the patient's thigh or abdomen at an air pressure of -0.75 atm. The harvested fat was transferred to a 60 ml sterile needle-free syringe and centrifuged at 1000 g / min for 3 minutes to remove the blood and tumescent fluid in the lower layer to obtain Coleman's fat. Based on the amount of fat, the upper layer of Coleman's fat with more fat was marked as low density, and the lower layer of Coleman's fat with less fat was marked as high density (e.g., Figure 1 shown);

[0049] Step 2): The upper layer of low-density fat transferred to the syringe in step 1) is connected to another syringe via a Luer connector for push injection, and this is repeated 6 to 10 times until the low-density fat tissue is fully mechanically emulsified and becomes chyloidal.

[0050] Specifically, the low-density Coleman fat after centrifugation was transferred to two 10 mL sterile syringes of equal volume, which were connected via a 2.4 mm Luer connector. The low-density Coleman fat was repeatedly injected between the two syringes using the pistons of the syringes at a rate of 20 mL / sec for 1 min to turn the fat into a chyme, thereby obtaining nano-fat (e.g., Figure 1 shown);

[0051] Step 3): The chylomicronized fat obtained in step 2) is centrifuged and separated into three layers: an upper layer of fat, a lower layer of blood and tumescent fluid, and a gel-like substance in the middle layer, which is adipose tissue stem cell gel (SVF-gel).

[0052] The nano fat was centrifuged for a second time at 1800g / min for 3 minutes. After centrifugation, it was divided into three layers: the upper layer of fat and the lower layer of liquid. The gel product in the middle layer was SVF-gel.

[0053] Step 4): Pour an appropriate amount of the upper layer of the nanofat from the second centrifugation into the remaining unprocessed high-density Coleman fat. Repeat steps 2) and 3) to obtain another portion of SVF-gel. In this step, the amount of fat added should be 30%-33% of the volume of the high-density fat.

[0054] Specifically, an appropriate amount of upper layer fat after the second centrifugation of nanofat was poured into the remaining untreated high-density Coleman fat, and steps 2) and 3) were repeated to obtain another portion of SVF-gel.

[0055] Step 5): Transfer the prepared SVF-gel to a sieve, stir the SVF-gel in the sieve at a constant speed, and place a gauze pad on the bottom of the sieve. Wipe the bottom of the sieve at a constant speed to absorb the oil and liquid components that seeped out of the SVF-gel to obtain pSVF-gel (such as Figure 1 (as shown). In this step, the mesh size should be no smaller than 400 mesh. For every 10 mL of SVF-gel, approximately 2.5 mL of pSVF-gel is purified. When the volume of the SVF-gel remains unchanged during constant stirring, pSVF-gel is obtained, and little or no exuded oil or liquid remains on the gauze.

[0056] Example 3

[0057] Step 1): Aspirate and obtain the original fat tissue from the patient's waist, abdomen, or thigh area. Centrifuge the collected original fat tissue to remove the blood and swelling fluid in the lower layer. Transfer the upper low-density fat into a syringe, and reserve the middle high-density fat for later use.

[0058] Specifically, a 4mm-caliber multi-side hole liposuction needle was used to aspirate fat from the patient's thigh or abdomen at an air pressure of -0.75 atm. The harvested fat was transferred to a 60mL sterile needle-free syringe and centrifuged at 1100g / min for 3 minutes to remove the blood and tumescent fluid in the lower layer. Coleman's fat was obtained. Based on the amount of fat, the upper layer of Coleman's fat with more fat was labeled as low-density, and the lower layer of Coleman's fat with less fat was labeled as high-density.

[0059] Step 2): The upper layer of low-density fat transferred to the syringe in step 1) is connected to another syringe via a Luer connector for push injection, and this is repeated 6 to 10 times until the low-density fat tissue is fully mechanically emulsified and becomes chyloidal.

[0060] Specifically, the low-density Coleman fat after centrifugation was transferred to two 10 mL sterile syringes of equal volume, which were connected via a 2.4 mm Luer connector. The low-density Coleman fat was repeatedly injected between the two syringes using the pistons of the syringes at a rate of 10 mL / sec for 2 min to make the fat into a chyme state, thereby obtaining nano-fat (such as Figure 1 shown).

[0061] Step 3): The chylomicronized fat obtained in step 2) is centrifuged and separated into three layers: an upper layer of fat, a lower layer of blood and tumescent fluid, and a gel-like substance in the middle layer, which is adipose tissue stem cell gel (SVF-gel).

[0062] Specifically, the nano fat was centrifuged for a second time at 2000g / min for 3 minutes. After centrifugation, it was divided into three layers: the upper layer of fat and the lower layer of liquid. The gel product in the middle layer was SVF-gel.

[0063] Step 4): Pour an appropriate amount of the upper layer of the nanofat from the second centrifugation into the remaining unprocessed high-density Coleman fat. Repeat steps 2) and 3) to obtain another portion of SVF-gel. In this step, the amount of fat added should be 30%-33% of the volume of the high-density fat.

[0064] An appropriate amount of the upper layer of fat after the second centrifugation of the nanofat was poured into the remaining untreated high-density Coleman fat, and steps 2) and 3) were repeated to obtain another portion of SVF-gel.

[0065] Step 5): Transfer the prepared SVF-gel to a sieve, stir the SVF-gel in the sieve at a constant speed, and place a gauze pad on the bottom of the sieve. Wipe the bottom of the sieve at a constant speed to absorb the oil and liquid components that seeped out of the SVF-gel to obtain pSVF-gel (such as Figure 1 (as shown). In this step, the mesh size should be no smaller than 400 mesh. For every 10 mL of SVF-gel, approximately 2.5 mL of pSVF-gel is purified. When the volume of the SVF-gel remains unchanged during constant stirring, pSVF-gel is obtained, and little or no exuded oil or liquid remains on the gauze.

[0066] The pSVF-gel finally obtained by the above method has lower fluidity than SVF-gel, is colloid-like, and has stronger cohesive force.

[0067] The physically purified pSVF-gel prepared above can further enhance wound healing, improve the filling effect of fat grafting, and improve the retention rate after transplantation. The pSVF-gel can be directly injected through a fine needle for cosmetic filling or mixed with hydrogel materials to directly cover the wound surface.

[0068] The pSVF-gel prepared by the method of the present invention is a component of facial filling materials and wound repair materials. Furthermore, it is a component of the fat gel filling area of ​​the nose base or glabellar lines.

[0069] The pSVF-gel prepared above was further studied and tested.

[0070] (1) Volume concentration ratio and gross comparison of pSVF-gel and SVF-gel (retention rate)

[0071] Experimental methods:

[0072] The experimental method was the same as in Example 1. SVF-gel was obtained by step 4). 5 mL of SVF-gel was used to obtain pSVF-gel by the method of step 5). The pSVF-gel was transferred to a syringe to obtain the corresponding volume concentration ratio. 1 mL of SVF-gel and pSVF-gel were transferred to 1 mL syringes respectively and injected simultaneously onto gauze to create the same pattern and observe the general appearance.

[0073] Place the two in a 1.5mL centrifuge tube and a culture dish to observe the general difference between the two. At the same time, inject 1mL of pSVF-gel and 1mL of SVF-gel from a 1mL syringe onto sterile gauze to observe the changes in properties after injection, as well as the changes in properties and the degree of retention of the two at 7 days, 15 days, 45 days, 90 days, and 180 days, and calculate the retention rate.

[0074] Experimental results and conclusions:

[0075] Test results such as Figure 2 As shown in Figure 2, the model shaped by pSVF-gel after physical purification has a clearer structure and cohesion. The above results indicate that pSVF-gel has better plasticity than SVF-gel.

[0076] (2) pSVF-gel live-dead staining

[0077] Experimental methods:

[0078] The experimental method was the same as in Example 1. The prepared pSVF-gel was stained according to the operating procedures of the live and dead cell staining kit, and the number of live and dead cells was observed under an inverted fluorescence microscope.

[0079] Experimental results and conclusions:

[0080] Test results such as Figure 3 As shown, pSVF-gel retained a large number of living cells after physical purification. The above results indicate that the cells retained by pSVF-gel in wound treatment can promote wound healing by secreting a variety of bioactive factors.

[0081] (3) pSVF-gel cell migration assay

[0082] Experimental methods:

[0083] Cultured human umbilical vein endothelial cells (HUVEC cells) were seeded in 24-well plates. The cell plating was observed under a microscope. After the wells were fully covered with cells, the cells were scratched. The cells were intervened with pSVF-gel and SVF-gel components. Pictures were taken at different time points to calculate the cell migration rate.

[0084] Experimental results and conclusions:

[0085] Test results such as Figure 4 As shown in the figure, under the same culture conditions, the migration rate of HUVEC cells treated with pSVF-gel was higher than that treated with SVF-gel. It can be seen that the pSVF-gel of the present invention can promote cell migration and shorten the repair time of wounds.

[0086] (4) Comparative experiment on pSVF-gel promoting wound healing

[0087] Experimental methods:

[0088] Full-thickness skin defects were created on the backs of SD rats after adaptive feeding. The pSVF-gel of the present invention was mixed with a light-cured hydrogel and then applied to the wound surface. A sterile dressing was then applied. In the control group, SVF-gel and light-cured hydrogel were mixed and injected at the same volume. Wound healing was observed 3, 7, and 14 days after surgery, and the corresponding wound areas were calculated.

[0089] Experimental results and conclusions:

[0090] Test results such as Figure 5 As shown, the wound images taken show that the wound covered by pSVF-gel has a smaller wound area at each time point than the SVF-gel wound group at the same time, and has a higher healing rate. It can be seen that the pSVF-gel of the present invention has a better wound treatment effect.

[0091] (5) Lipidomics comparison between pSVF-gel and SVF-gel

[0092] Experimental methods:

[0093] Fat samples from 6 individuals were retained as pSVF-gel and SVF-gel samples for lipidomics analysis.

[0094] Experimental results and conclusions:

[0095] Test results such as Figure 6 As shown in the volcano plot of lipidomics detection, it can be seen that 19 metabolites were significantly upregulated and 31 metabolites were significantly downregulated in the control group (SVF-gel) and PS group (pSVF-gel).

[0096] The above-mentioned specific embodiments of the present invention are all preferred embodiments of the present invention, but the described embodiments are only part of the embodiments of the present invention. The implementation technology of the present invention is not limited to the above-mentioned embodiments. Other equivalent changes made based on the structure and principles of the technology that do not deviate from the basic characteristics and spirit of the technical method, as well as other embodiments used without creative innovation, all fall within the scope of protection of the present invention.

Claims

1. A physical preparation method for purified adipose stem cell gel with high plasticity, characterized in that: The following steps are involved: 1) Aspirate and obtain the original fat tissue from the patient's waist, abdomen, or thigh area. Centrifuge the collected original fat tissue to remove the blood and swelling fluid in the lower layer. Transfer the upper low-density fat into a syringe, and keep the middle high-density fat for later use. 2) Connect the upper layer of low-density fat transferred to the syringe in step 1) to another syringe via a Luer connector and inject it, repeating 6 to 10 times until the low-density fat tissue is fully mechanically emulsified and becomes chyloidal; 3) Centrifuging the chylomicronized fat obtained in step 2) to separate it into three layers: an upper layer of fat, a lower layer of blood and tumescent fluid, and a gel-like substance in the middle layer, which is adipose tissue stem cell gel (SVF-gel); 4) Transfer an appropriate amount of the upper layer of fat from step 3) to the remaining high-density fat from step 1) and repeat steps 2) and 3) to obtain another portion of SVF-gel; 5) Place the SVF-gel obtained in steps 3) and 4) into a sieve, stir the SVF-gel in the sieve at a constant speed, and place gauze on the bottom of the sieve and wipe the bottom of the sieve at a constant speed to absorb the oil and liquid components exuded from the SVF-gel to obtain pSVF-gel.

2. The method according to claim 1, characterized in that The emulsified fat in step 2) can be injected into two syringes at equal volumes while pulling the syringe pistons backward to observe the degree of fat emulsification.

3. The method according to claim 1, characterized in that In step 4), the amount of oil added is 30%-33% of the volume of the high-density fat.

4. The method according to claim 1, wherein The sieve in step 5) is not less than 400 mesh.

5. The method according to claim 1, wherein Step 5) For every 10 mL of SVF-gel, purify to obtain pSVF-gel with a volume of approximately 2.5 mL.

6. The method according to claim 1, characterized in that Step 5) When the volume of the SVF-gel under constant stirring does not change significantly, pSVF-gel is obtained. At this time, there is little or no exuded oil and liquid components on the gauze.

7. The method according to claim 1, wherein: The final pSVF-gel obtained had lower fluidity than SVF-gel.

8. pSVF-gel prepared by the method according to any one of claims 1 to 7, characterized in that It is a component of facial filling materials and wound repair materials.

9. The pSVF-gel according to claim 8, characterized in that It is a component of the fat gel filling area of ​​the nose base or glabellar lines.

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