Adipose cell composition for injection and filling and preparation method thereof

Abstract

The invention belongs to the technical field of medical cosmetology and plastic surgery and tissue engineering, and particularly relates to an adipocyte composition for injection and filling and a preparation method of the adipocyte composition. The invention provides an adipose cell composition for injection filling, which comprises adipose-derived stem cells and adipose microparticles, the volume ratio of the adipose-derived stem cells to the adipose microparticles is 1: (2-5), the density of the adipose-derived stem cells is 2 * 10 < 6 > / ml, and the particle size of the adipose microparticles is 0.8 mm. The fat cell composition disclosed by the invention is used for filling facial soft tissues, so that the survival rate of postoperative fat is increased, the purpose of treating facial soft tissue depression is achieved, the facial contour is effectively improved, and certain effects on reducing frontal lines and improving skin texture are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of medical cosmetic surgery and tissue engineering, and in particular relates to a fat cell composition for injection filling and a preparation method thereof. Background Art

[0002] In recent years, fat microparticle transplantation has played an important role in correcting facial soft tissue depressions. However, the low survival rate of fat after simple autologous fat microparticle transplantation is the main reason why patients are dissatisfied with the clinical effect. There is currently no standard fat transplantation technology. Therefore, how to improve the survival rate of fat microparticles after transplantation has always been a research hotspot.

[0003] Autologous adipose stem cells have a certain effect on improving the survival rate of fat cells, because adipose stem cells can secrete vascular endothelial growth factor, promote blood vessel regeneration, provide blood supply for transplanted fat, and replenish the number of cells reduced due to transplanted fat cell necrosis. Therefore, the existing technology uses autologous adipose stem cells in combination with fat transplantation to increase the survival rate of fat. At present, the homing rate of adipose stem cells after transplantation is still unknown, and there are large individual differences in the long-term survival and stability of transplanted autologous fat in the transplant recipient area. Summary of the invention

[0004] The object of the present invention is to provide a fat cell composition for injection filling.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned adipocyte composition.

[0006] According to a specific embodiment of the present invention, a fat cell composition for injection filling is provided, wherein the composition comprises adipose stem cells and fat particles, wherein the volume ratio of the adipose stem cells to the fat particles is 1:2-5, and the density of the adipose stem cells is 2×10 6 / ml, and the particle size of fat particles is 0.8mm.

[0007] Preferably, the volume ratio of adipose stem cells to fat particles is 1:3.

[0008] According to the fat cell composition for injection filling according to a specific embodiment of the present invention, the fat particles are prepared by the following method:

[0009] (1) Centrifuge the adipose tissue obtained by liposuction to remove the underlying fluid;

[0010] (2) adding physiological saline, centrifuging, retaining the middle layer, and obtaining purified fat;

[0011] (3) placing the fat obtained in step (2) in a homogenizer for homogenization;

[0012] (4) Processing the fat homogenized in step (3) into fat particles with a particle size of 0.8 mm.

[0013] According to a specific embodiment of the present invention, in the fat cell composition for injection filling, in step (1), the adipose tissue obtained by liposuction is centrifuged at 1000 r / min for 3-10 min.

[0014] According to the fat cell composition for injection filling according to a specific embodiment of the present invention, in step (2), after adding physiological saline, centrifugation is carried out at 1000r / min for 3-10min.

[0015] According to the fat cell composition for injection filling according to a specific embodiment of the present invention, the adipose stem cells are prepared by the following method:

[0016] (1) Purified adipose tissue is obtained, digested, centrifuged, and undigested adipose tissue is removed;

[0017] (2) Wash the adipose tissue obtained in step (1) and add (1-5) × 10 4 / cm 2 Inoculate the culture plate and culture in an incubator;

[0018] (3) The cells from the first inoculation are regarded as generation 0, denoted as P0. After the cells are 80-90% confluent, they are digested and subcultured at a ratio of 1:3. After 12-15 days, generation P3 is obtained, i.e., adipose stem cells.

[0019] The present invention also provides a method for preparing a fat cell composition for injection filling, comprising the following steps:

[0020] Take fat particles with a particle size of 0.8 mm;

[0021] Obtaining adipose stem cells;

[0022] After suspending adipose-derived stem cells in saline, they were added to fat particles. The volume ratio of adipose-derived stem cells to fat particles was 1:2-5, and the density of adipose-derived stem cells was 2×10 6 Pieces / ml.

[0023] According to the method for preparing the fat cell composition for injection filling in a specific embodiment of the present invention, the adipose stem cells are prepared by the following method:

[0024] (1) Purified adipose tissue is obtained, digested, centrifuged, and undigested adipose tissue is removed;

[0025] (2) washing the adipose tissue obtained in step (1), seeding the adipose tissue in a culture plate at a concentration of (1-5)×104 / cm2, and culturing the adipose tissue in an incubator;

[0026] (3) The cells from the first inoculation are regarded as generation 0, denoted as P0. After the cells are 80-90% confluent, they are digested and subcultured at a ratio of 1:3. After 12-15 days, generation P3 is obtained, i.e., adipose stem cells.

[0027] According to the method for preparing the fat cell composition for injection filling in a specific embodiment of the present invention, the fat particles are prepared by the following method:

[0028] (1) Centrifuge the adipose tissue obtained by liposuction to remove the underlying fluid;

[0029] (2) adding physiological saline, centrifuging, retaining the middle layer, and obtaining purified fat;

[0030] (3) placing the fat obtained in step (2) in a homogenizer for homogenization;

[0031] (4) Processing the fat homogenized in step (3) into fat particles with a particle size of 0.8 mm.

[0032] According to a specific embodiment of the present invention, a method for preparing a fat cell composition for injection filling is provided.

[0033] In step (1), the adipose tissue obtained by liposuction is centrifuged at 1000 r / min for 3-10 min;

[0034] In step (2), after adding physiological saline, centrifuge at 1000 r / min for 3-10 min.

[0035] Beneficial effects of the present invention:

[0036] The fat cell composition of the present invention is used to fill facial soft tissue, thereby improving the survival rate of postoperative fat, achieving the purpose of treating facial soft tissue depression, effectively improving facial contours, and having a certain effect on reducing forehead wrinkles and improving skin texture.

[0037] The present invention homogenizes the purified fat and selects granular fat with a particle size of 0.8 mm. Due to its small size and good homogenization, the fat particles can be more evenly distributed in the recipient area after being filled, further increasing the contact area with the recipient area tissue, which is beneficial to the early survival of the fat tissue and buys more time for vascular reconstruction. Therefore, the effective filling amount contained in the fat particles is greater than that of ordinary fat particle tissue.

[0038] The present invention selects adipose stem cells and fat particles to mix, which can promote the regeneration of tissue in the recipient area and repair tissue defects in the recipient area. The adipose stem cells and fat particles are mixed to increase the amount of stem cells in the autologous fat particles and improve the ratio of adipose stem cells to effective ingredients in the fat particles, thereby improving the survival rate of autologous fat. After transplantation, the active substances such as cytokines and adipose stem cells in the fat particles are mainly used to regulate immunity, induce differentiation, and promote regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0040] Figure 1 Shows the morphology of primary adipose-derived stem cells after 10 days of culture.

[0041] Figure 2 Shows the morphology of P3 adipose stem cells.

[0042] Figure 3 The expression of surface molecules of adipose stem cells was detected by flow cytometry.

[0043] Figure 4 The HE staining results are shown, where the left picture shows the staining of fat particle sections, and the right picture shows the staining of adipose stem cells and fat particle mixed preparation sections.

[0044] Figure 5 The following are photos of typical cases. DETAILED DESCRIPTION

[0045] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0046] Source of fat particles: provided by plastic surgery hospitals, donors are in good health, including: general physical examination, routine examination, ALT, TP, HIV, HBV, HCV, CMV, EBV, HTLV testing, and no genetic diseases or underlying diseases.

[0047] Example 1 Preparation of uniform fat particles

[0048] 1.1 Purification of fat particles

[0049] (1) Dispense 100 ml of granular adipose tissue obtained by liposuction into 2 to 4 50 ml centrifuge tubes and centrifuge at low speed (1000 rpm for 5 min). Use a pipette to discard the turbid liquid at the bottom (anesthetic drugs and blood cells).

[0050] (2) Add physiological saline and centrifuge (1000r / min for 5min) 1-3 times until the lower layer is clear. At this time, the fat tissue is divided into three layers, among which the upper layer is transparent liquid fat, the middle layer is light yellow fat particle tissue, and the lower layer is clear physiological saline.

[0051] (3) Use a Bass pipette to remove the upper layer of fat and the blood clots and cord fibers contained in the adipose tissue, remove the lower layer of saline, retain the plump fat particles in the middle layer, and obtain fat with higher purity for later use.

[0052] 1.2 Uniform fat particles

[0053] (1) Install a sterilized double-head fat homogenizer (3 sets) with 1.2 mm, 1.0 mm, and 0.8 mm pore diameter blades. Install a 50 ml sterile threaded syringe at one end of the double-head fat homogenizer.

[0054] (2) Take a portion of the purified middle layer fat and place it in a 50 ml disposable sterile threaded syringe.

[0055] (3) Install a sterile threaded syringe filled with plump fat on the fat homogenizer. In the first step, use a 1.2 mm aperture blade to push the syringe back and forth 2-3 times to remove the fascia.

[0056] (4) Replace the fat homogenizer and use a 1.0 mm aperture blade to push the syringe back and forth 2-3 times to remove the fascia again.

[0057] (5) Replace the fat homogenizer and use a 0.8 mm aperture blade to push the syringe back and forth 2-3 times to obtain uniform fat particles, which are placed in a centrifuge tube (stored at -196°C) for later use.

[0058] Example 2 Cultivation of adipose stem cells

[0059] 1. Take a portion of the purified fat particles prepared in Example 1 with a high purity: digest with 0.075% type I collagenase (Sigma, USA) at 37°C in a constant temperature shaker for 60 min, centrifuge at 1500 r / min for 10 min, and remove the undigested fat tissue on the upper layer.

[0060] 2. After the precipitate was resuspended in physiological saline, it was filtered through a 200-mesh sieve and centrifuged at 1500r / min for 10min. It was washed twice and resuspended in serum-free culture medium (Tianjin Haoyang).4 / cm 2 Inoculate to 10cm 2 The culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0061] 3. The cells inoculated for the first time were taken as the 0th generation, recorded as P0. After the cells were 80-90% confluent, they were digested with 0.25% trypsin and cultured at a ratio of 1:3. After 12-15 days, P3 adipose stem cells were obtained and frozen at -196℃ for later use.

[0062] Example 3: Mixing of adipose stem cells and fat particles

[0063] 1. The uniform fat particles (-196°C) prepared in Example 1 were rewarmed to 37°C, centrifuged at low speed (1200 r / min for 6 min) to remove the freezing solution, and the centrifugation was repeated for washing.

[0064] 2. The P3 adipose-derived stem cells prepared in Example 2 were revived in a 37° C. water bath, and centrifuged at a low speed (1200 rpm for 6 min) to remove the freezing solution, and the centrifugation was repeated for washing.

[0065] 3. Suspend P3 adipose-derived stem cells in physiological saline and add them to uniform fat particles to obtain a mixed preparation of adipose-derived stem cells and fat particles.

[0066] In the mixed preparation, adipose stem cells: fat particles = 1:3 (each 1 ml of the mixed preparation contains 2×10 6 P3 cells).

[0067] Microscopic morphology of adipose-derived stem cells

[0068] An inverted microscope was used to observe the growth of stem cells extracted from liposuctioned fat, and the growth, purification and proliferation of adipose stem cells adherently grown in culture bottles were continuously observed and photographed.

[0069] After 24 hours of primary cell culture, the medium was completely replaced once, and the non-adherent cells were discarded. Microscopic observation showed that the cells were attached to the wall of the culture flask and were round, oval, and a small number of short spindle-shaped. The fat droplets were significantly reduced. After 72 hours, the medium was replaced again. Microscopic observation showed that the cells were spindle-shaped and the fat droplets basically disappeared. Figure 1 ).

[0070] As the number of medium changes increases, the cell proliferation rate accelerates, the cell morphology gradually becomes uniform, and the arrangement tends to be directional and swirl-shaped. After about 10-14 days, when the cells cover about 80% of the bottom of the bottle, the cells can be subcultured. The subcultured cells are uniformly distributed in a long spindle shape ( Figure 2 There was no obvious change in cell morphology between passages 3 and 8.

[0071] Cell surface molecular marker detection:

[0072] 1. Digest the third generation adipose-derived stem cells with trypsin and prepare a single cell suspension.

[0073] 2. Dispense the cells into 1.5 ml EP tubes, 100 ul per tube, and adjust the cell density to 10 6 / ml.

[0074] 3. Add 1ul each of anti-human CD34-PE, CD45-PE, CD105-PE, and CD90-PE monoclonal antibodies in sequence.

[0075] 4. Incubate at 4°C in dark for 30 minutes.

[0076] 5. After washing with PBS twice, resuspend the cells in 500ul PBS and detect by flow cytometry.

[0077] like Figure 3 As shown, adipose stem cells highly expressed CD90 and CD105, with positive rates of 100.00% and 100.00% respectively, and lowly expressed CD34 and CD45, with negative rates of 0.00%, 0.03%, and 0.82+0.15% respectively.

[0078] HE staining

[0079] One portion of the mixed preparation of adipose stem cells and adipose microparticles (experimental group) and one portion of adipose microparticles (control group) were taken out, fixed with 10% formaldehyde, embedded in paraffin, stained with hematoxylin and eosin (HE), and subjected to histological analysis.

[0080] The results are as follows Figure 4 As shown, the control group: most of it is hyperplastic fibrous connective tissue, with visible fibrous vesicles and fewer blood vessels; the experimental group: the cross-section shows rich blood vessels, and the fat stem cells are full and closely arranged. The white arrow represents the fat cells, the red arrow represents the vascular tissue, and the black arrow represents the fibrous tissue. Example 4 Application of the mixed preparation of fat stem cells and fat particles

[0081] 1.1 Fifteen patients with facial depression who visited the Department of Surgery of a Plastic Surgery Hospital were selected as the research subjects.

[0082] Inclusion criteria:

[0083] ① All met the diagnostic criteria for facial depression; ②) Aged 20 to 40 years old; ③ The patients voluntarily participated in this study and signed the informed consent form for surgery.

[0084] Exclusion criteria:

[0085] ① Patients with serious heart, liver, and other diseases; ② Patients with blurred consciousness; ③ Patients with mental and intellectual disabilities and language expression disorders.

[0086] Among the research subjects, there were 3 males and 12 females, aged 20 to 40 years old. Among them, there were 2 cases of forehead depression, 4 cases of temporal depression, and 9 cases of cheek depression.

[0087] 1.2 The surgical method uses a mixture of autologous adipose stem cells and fat particles for transplantation and filling, and autologous fat is extracted from the inner side of the thigh root.

[0088] 1.3 Observation indicators

[0089] The effect was judged 6 months after the first injection, using preoperative and postoperative photos as objective criteria, combined with the patient's subjective opinion: the three-level criteria of excellent, good, and poor were set:

[0090] Excellent: The sunken area is corrected, meeting the preoperative design requirements, and the patient is satisfied;

[0091] Good: The sunken area was partially corrected, close to the preoperative design requirements, and the patient was basically satisfied;

[0092] Poor: The sunken area is not significantly corrected and the patient is not satisfied.

[0093] Satisfaction = (satisfied + basically satisfied) / total number of cases x 100%.

[0094] 1.4 Results

[0095] 14 out of 15 patients were satisfied with the surgical results, and only 1 patient felt that the improvement was not obvious and was dissatisfied with the treatment effect. The surgical satisfaction rate was as high as 93.33%. The dissatisfied patient underwent secondary filling one year after the operation. After the operation and during the follow-up period, none of the 15 patients had complications such as fat liquefaction, infection, hematoma, nodules, etc. The postoperative effect was long-lasting and natural. Typical cases are shown in Figure 5 .

[0096] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A fat cell composition for injection filling, characterized in that: The composition is adipose stem cells and adipose microparticles, the volume ratio of adipose stem cells to adipose microparticles is 1:2-5, and the density of adipose stem cells is 2×10 6 / ml, and the particle size of fat particles is 0.8mm.

2. The fat cell composition for injection filling according to claim 1, characterized in that: The volume ratio of adipose stem cells to fat particles is 1:

3.

3. The fat cell composition for injection filling according to claim 1, characterized in that: The fat particles were prepared by the following method: (1) Centrifuging the micro-adipocytes obtained by liposuction to remove the underlying liquid; (2) adding physiological saline, centrifuging, retaining the middle layer, and obtaining purified fat; (3) placing the fat obtained in step (2) in a homogenizer for homogenization; (4) Processing the fat homogenized in step (3) into fat particles with a particle size of 0.8 mm.

4. The fat cell composition for injection filling according to claim 1, characterized in that: In step (1), the micro-fat tissue obtained by liposuction is centrifuged at 1000 r / min for 3-10 min.

5. The fat cell composition for injection filling according to claim 1, characterized in that: In step (2), after adding physiological saline, centrifuge at 1000 r / min for 3-10 min.

6. The fat cell composition for injection filling according to claim 1, characterized in that: Adipose-derived stem cells are prepared by the following method: (1) Purified adipose tissue is obtained, digested, centrifuged, and undigested adipose tissue is removed; (2) Wash the adipose tissue obtained in step (1) and add 1×10 4 -5×10 4 / cm 2 Inoculate the culture plate and culture in an incubator; (3) The cells from the first inoculation are regarded as generation 0, denoted as P0. After the cells are 80-90% confluent, they are digested and subcultured at a ratio of 1:

3. After 12-15 days, generation P3 is obtained, i.e., adipose stem cells.

7. The method for preparing the fat cell composition for injection filling according to claim 1, characterized in that: Take fat particles with a particle size of 0.8 mm; Obtaining adipose stem cells; After suspending adipose-derived stem cells in saline, they were added to fat particles. The volume ratio of adipose-derived stem cells to fat particles was 1:2-5, and the density of adipose-derived stem cells was 2×10 6 Pieces / ml.

8. The method for preparing the fat cell composition for injection filling according to claim 7, characterized in that: Adipose-derived stem cells are prepared by the following method: (1) Purified adipose tissue is obtained, digested, centrifuged, and undigested adipose tissue is removed; (2) Wash the adipose tissue obtained in step (1) and add 1×10 4 -5×10 4 / cm 2 Inoculate the culture plate and culture in an incubator; (3) The cells from the first inoculation are regarded as generation 0, denoted as P0. After the cells are 80-90% confluent, they are digested and subcultured at a ratio of 1:

3. After 12-15 days, generation P3 is obtained, i.e., adipose stem cells.

9. The method for preparing the fat cell composition for injection filling according to claim 7, characterized in that: The fat particles were prepared by the following method: (1) Centrifuge the adipose tissue obtained by liposuction to remove the underlying fluid; (2) adding physiological saline, centrifuging, retaining the middle layer, and obtaining purified fat; (3) placing the fat obtained in step (2) in a homogenizer for homogenization; (4) Processing the fat homogenized in step (3) into fat particles with a particle size of 0.8 mm.

10. The method for preparing the fat cell composition for injection filling according to claim 9, characterized in that: In step (1), the adipose tissue obtained by liposuction is centrifuged at 1000 r / min for 3-10 min; and / or, In step (2), after adding physiological saline, centrifuge at 1000 r / min for 3-10 min.

Images