阅读说明：本技术 从吸脂废液中制备的脂肪组织精华、其制备方法和应用 (Adipose tissue essence prepared from liposuction waste liquid, and preparation method and application thereof ) 是由 田卫东 于湄 伍月 刘磊 于 2021-09-30 设计创作，主要内容包括：本发明公开了从吸脂废液中制备的脂肪组织精华、其制备方法和应用,属于生物医学技术领域,解决现有技术中脂肪组织提取物有效成分含量低、无法满足临床需求的问题。本发明提供的从吸脂废液中制备脂肪组织精华的方法包括以下步骤：取吸脂手术产生的手术废弃液,去除油脂和脂肪颗粒,得到液体部分；将液体过滤,收集滤液；将滤液差速离心,收集上清液；将上清液超滤浓缩,即得。本发明以临床抽脂后的脂肪废液为原料,变废为宝,且来源广泛,便于临床推广。本发明制得的精华无活细胞,细胞外囊泡和脂肪因子成液体状态,可注射性好。实验表明,本发明的精华具有良好的促进包括毛发再生的组织再生的作用。(The invention discloses adipose tissue essence prepared from liposuction waste liquid, a preparation method and application thereof, belongs to the technical field of biomedicine, and solves the problems that in the prior art, an adipose tissue extract is low in effective component content and cannot meet clinical requirements. The method for preparing the adipose tissue essence from the liposuction waste liquid comprises the following steps: taking operation waste liquid generated in liposuction operation, and removing grease and fat particles to obtain a liquid part; filtering the liquid, and collecting filtrate; centrifuging the filtrate at a differential speed, and collecting supernatant; and (4) performing ultrafiltration concentration on the supernatant to obtain the product. The invention takes the fat waste liquid after clinical liposuction as the raw material, changes waste into valuable, has wide sources and is convenient for clinical popularization. The essence prepared by the invention has no living cells, and the extracellular vesicles and the fat factors are in a liquid state and have good injectability. Experiments show that the essence of the invention has good effect of promoting the regeneration of tissues including hair regeneration.)

1. A method for preparing adipose tissue essence from liposuction waste liquid is characterized by comprising the following steps:

s1, taking operation waste liquid generated by liposuction, and removing grease and fat particles to obtain a liquid part;

s2, filtering the liquid obtained in the step S1, and collecting filtrate;

s3, performing differential centrifugation on the filtrate obtained in the step S2, and collecting supernatant;

and S4, carrying out ultrafiltration concentration on the supernatant to obtain the adipose tissue essence.

2. The method of claim 1, wherein the surgical waste generated by liposuction includes residual fat, cells, interstitial fluid, blood, and anesthetic agents.

3. The method according to claim 1 or 2, wherein the filtration in S2 is filtration using a cell filtration sieve.

4. The method according to claim 3, wherein the cell filter sieve has a mesh size of 10 to 100 μm, preferably 30 μm.

5. The method according to claim 1 or 2, wherein the differential centrifugation in S3 comprises at least three times of centrifugation at different centrifugation speeds, and after each centrifugation, the supernatant is collected at 200-400 g/min, 1500-2500 g/min and 10000-20000 g/min respectively from low to high.

6. The method according to claim 5, wherein the centrifugal speed of the differential centrifugation is 300g/min, 2000g/min, 15000-20000 g/min from low to high respectively.

7. The method according to claim 1 or 2, wherein the concentration factor is 5 to 15; preferably 10 times.

8. The method of claim 1, further comprising the step of sterilizing: the supernatant obtained in S3 was subjected to filtration sterilization, followed by ultrafiltration and concentration.

9. An adipose tissue essence prepared by the method of any one of claims 1 to 9.

10. Use of the adipose tissue essence prepared by the method of any one of claims 1 to 9 for preparing a product for promoting tissue regeneration, preferably for preparing a product for promoting hair regeneration.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing adipose tissue essence from liposuction waste liquid, the adipose tissue essence prepared by the method and application.

Background

In recent years, with the increase of social competition and the deterioration of the environment, the incidence of alopecia has increased and the trend toward youth has become remarkable. Alopecia not only affects the appearance, but also may cause psychological problems. In the prior art, methods for treating alopecia include methods using minoxidil, finasteride, Platelet Rich Plasma (PRP), Low Level Laser Therapy (LLLT), stem cell therapy, hair follicle transplantation, and the like. Unfortunately, all of the prior art treatments have limitations. According to previous studies, minoxidil causes a high rate of adverse reactions, such as burning or itching of the application site, allergic contact dermatitis, cardiovascular diseases, and the like. Finasteride is associated with decreased libido, gynecomastia and psychological complications. Low level laser treatment may lead to adverse reactions such as dry skin, itching and scalp sensitivity. PRP does not achieve consistent dosing standards in terms of accurate concentration, dose and depth of injection, and often causes pain and erythema. Stem cell therapy has limited its clinical use due to factors such as its safety, effectiveness, and feasibility. Hair follicle transplantation is very expensive and the source of hair follicle donors is limited. Therefore, it is very necessary to search for a new treatment for alopecia.

When the substances generated in the liposuction operation are placed in the same container, the liposuction operation is mainly divided into an upper part and a lower part: the upper layer is an oily fat layer, and the lower layer is liposuction operation waste liquid containing fat, cells, tissue fluid, blood and anesthetic fluid. In the prior art, stem cells and extracellular matrix products can be prepared by using an upper fat layer substance, but the lower fat layer waste liquid of liposuction operation is always used as medical waste liquid treatment, and related reports are not found to be used for utilizing the waste liquid.

In addition, it has been found in clinical practice that the skin condition of the area to which autologous fat transplantation is applied is improved. And the effects of adipose tissue acellular derivatives, such as adipose stem cell culture media and adipose stem cell exosomes, on resisting skin aging, promoting wound healing and reducing scar formation are proved in experiments and clinics. However, the extraction and mass amplification culture of stem cells have high requirements on the operating environment, and the operation process is complicated, time-consuming and labor-consuming. Then, the effective components, such as exosome, are obtained from the culture medium, which is a complex process that is time-consuming and labor-consuming. If we can develop and utilize the large-volume liposuction waste liquid and solve the problem of rapidly and efficiently obtaining the effective components in the liquid, a new idea is provided for changing the liposuction waste into valuable and meeting the clinical treatment requirement.

Disclosure of Invention

One of the objectives of the present invention is to provide a method for preparing adipose tissue essence, which solves the problem in the prior art that the waste liquid from liposuction surgery is not effectively utilized.

The second object of the present invention is to provide an adipose tissue essence prepared by the above method.

The present invention also provides the use of the adipose tissue essence prepared by the above method.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides a preparation method of adipose tissue essence, which comprises the following steps:

s1, taking operation waste liquid generated by liposuction, and removing grease and fat particles to obtain a liquid part;

s2, filtering the liquid obtained in the step S1, and collecting filtrate;

s3, performing differential centrifugation on the filtrate obtained in the step S2, and collecting supernatant;

and S4, carrying out ultrafiltration concentration on the supernatant to obtain the adipose tissue essence.

In some embodiments of the invention, the surgical waste fluid from liposuction includes residual fat, cells, interstitial fluid, blood, and anesthetic agents.

In the operation process of the liposuction operation, after a swelling solution is firstly injected into a subcutaneous fat layer, tissues are dissociated (part of cells are broken in the process) through the movement of an ultrasonic or liposuction needle, and the fat cells are sucked out through the negative pressure effect of the liposuction needle. Blood vessels are in the subcutaneous fat layer, so that not only fat cells but also other blood are pumped in the process of liposuction. The material extracted by liposuction is divided into an upper layer and a lower layer after standing, the upper layer is oily fat, and the lower layer is the raw material of the invention: surgical waste generated by liposuction. Thus, the surgical waste fluid includes residual fat, cells, interstitial fluid, blood, and anesthetic.

In some embodiments of the invention, the filtration in S2 is filtration using a cell filtration sieve.

In some embodiments of the present invention, the mesh size of the cell filter screen is 10-100 μm, preferably 30 μm.

The invention removes the large fat tissue suspended substance after filtering the liquid obtained in S1.

In some embodiments of the present invention, the differential centrifugation in S3 includes at least three times of centrifugation at different centrifugation speeds, and after each centrifugation, the supernatant is collected, and the centrifugation speeds are respectively 200-400 g/min, 1500-2500 g/min, and 10000-20000 g/min from low to high.

In some embodiments of the invention, the first and second centrifugation times are both 5-20 min, and the third centrifugation time is 0.5-2 hours.

Preferably, the time for the first and second centrifugation is 10min and the time for the third centrifugation is 1 hour.

The filtrate obtained in S2 is centrifuged for three times, the first centrifugation removes cell components (10-30 μm) in the liquid, the second centrifugation removes cell debris components (1-10 μm), and the third centrifugation removes large vesicles (0.3-2 μm) in the liquid, such as apoptotic bodies.

In some embodiments of the invention, the concentration factor is 5 to 15; preferably 10 times.

In some embodiments of the present invention, the method further comprises the step of sterilizing: the supernatant obtained in S3 was subjected to filtration sterilization, followed by ultrafiltration and concentration.

The adipose tissue essence prepared by the preparation method provided by the invention.

The application of the adipose tissue essence prepared by the preparation method provided by the invention in preparing products for promoting tissue regeneration, preferably in preparing products for promoting hair regeneration.

The adipose tissue essence obtained by the method not only contains high-concentration extracellular vesicles, but also contains a large amount of protein components (such as fat factors), and has a better effect of promoting hair regeneration.

Compared with the prior art, the invention has the following beneficial effects:

the invention has scientific design and ingenious conception, and changes waste into valuable by taking the operation waste liquid generated by liposuction as the raw material.

The raw materials of the invention have wide sources and are convenient for clinical popularization.

The method is simple, is simple and convenient to operate, can quickly and efficiently obtain the extracellular vesicles and the proteins from the large-volume sample, and has the potential of large-scale production.

The method adopts pure physical operation, and the essence obtained is not added with any chemical reagent, so that the method has good safety and is convenient for clinical application.

The essence prepared by the method has no living cells, and does not need to further detect the activity of the cells. The fine powder is in a liquid state, and the injectability is good; and the concentrated essence has high content of effective components (extracellular vesicles and proteins), and the concentration of the anesthetic (lidocaine) is lower than the concentration of the injection for operation, so that the subsequent in vivo application can be safely carried out. Experiments show that the essence has good effects of promoting hair regeneration, blood vessel regeneration and tissue regeneration.

Drawings

FIG. 1 is a flow chart of the preparation of AT-XOs according to the present invention;

FIG. 2 is a micrograph of an adipose tissue mass.

FIG. 3A is a graph showing the comparison of the sizes of the extracellular vesicles of AT-XOs AT different concentrations;

FIG. 3B is a graph showing a comparison of the protein content of AT-XOs AT different concentration factors for the same volume;

FIG. 3C is a graph showing the comparison of the number of extracellular vesicle particles in AT-XOs AT different concentration ratios for the same volume;

FIG. 4 is a graph showing a comparison of the results of lidocaine detection in test example 1;

FIG. 5 is a graph showing the results of measurement of adipokines contained in AT-XOs prepared in example 1;

FIG. 6 is a schematic overview of groups of mice at different time points in Experimental example 1;

FIG. 7 is a typical graph showing the growth of hair on the back of hair-shedding regions of mice in each group on day 12 of test example 1;

FIG. 8 is a back skin inner surface view of a control group and an experimental group on day 15 of test example 1;

FIG. 9 is a histological analysis chart of back skin samples of a control group and an experimental group in test example 1; wherein, the A picture is the histological analysis picture of the back skin sample of the control group, and the C picture is the enlarged part of the frame line of the A picture; panel B is a histological analysis of a skin sample on the back of the experimental group, and panel D is an enlarged view of a portion of the outline of panel B.

Detailed Description

The above-described aspects of the present invention will be described in further detail with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All technical solutions realized based on the above contents of the present invention belong to the scope of the present invention.

Example 1

The present embodiment discloses a method for preparing adipose tissue essence, the flow of which is shown in fig. 1, and specifically comprises the following steps:

s1, taking operation waste liquid (a lower liquid part of a container shown in the attached drawing 1) generated by liposuction, and removing grease and fat particles to obtain a liquid part;

s2, filtering the liquid obtained in the step S1 through a 500-mesh cell filter screen (with the aperture of 30 mu m), and collecting filtrate;

s3, centrifuging the filtrate obtained in the step S2 at a differential speed, firstly centrifuging for 10min under the condition of a rotating speed of 300g/min, and collecting the centrifuged supernatant;

centrifuging the supernatant at 2000g/min for 10min, and collecting the centrifuged supernatant;

centrifuging the supernatant obtained after the second centrifugation for 1h at the rotating speed of 15000g/min, and collecting the centrifuged supernatant.

S4, filtering and sterilizing the supernatant by using a filter with the pore diameter of 0.22 mu m, concentrating the filtrate by 10X through a tangential flow ultrafiltration system, and installing a 500KD Millipore filter to obtain the reflux liquid which is adipose tissue essence (AT-XOs).

Example 2

The microstructure of the adipose tissue essence prepared in example 1 was observed, the adipose tissue essence was fixed with 4% paraformaldehyde for 10min, diluted appropriately, dropped on a sample-carrying copper mesh, excess liquid was removed with filter paper, dried, negatively stained with 1% uranyl acetate, and observed under a transmission electron microscope. See figure 2 for results.

As shown in FIG. 2, the adipose tissue seminal plasma mainly contains vesicles below 200nm (as indicated by the open arrows).

Example 3

This example examines the concentration factor of the adipose tissue extract. The preparation method of the adipose tissue essence is the same as that of example 1 except that the concentration factor of step S4 is different. The supernatant after S3 centrifugation is respectively concentrated by 5 times, 10 times and 15 times, and the quantity of Extracellular Vesicles (EV) and the protein concentration of the concentrated adipose tissue essence are evaluated.

The results are shown in FIGS. 3A, 3B and 3C.

From the DLS data (fig. 3A), it can be seen that the concentrated adipose tissue essence by the tangential flow ultrafiltration system contains vesicles with a particle size of about 100nm, and the particle sizes of AT-XOs obtained by different concentration times are not significantly different.

BCA protein quantification (fig. 3B) results show that protein concentration in adipose tissue seminal plasma increases in equal proportion with increasing fold concentration.

The results of counting the extracellular vesicles in the adipose tissue refined waviness at different concentration ratios by Nanoparticle Tracking Analysis (NTA) (fig. 3C) show that the concentration of the extracellular vesicles in the adipose tissue refined waviness increases with increasing concentration ratio, but the concentration of the extracellular vesicles after 15-fold concentration is not significantly improved compared with the sample after 10-fold concentration compared with the proportion of the extracellular vesicles after 5-fold concentration and 10-fold concentration.

Therefore, the concentration efficiency of extracellular vesicles and proteins is evaluated in two aspects, 5-15 times of concentration can obtain essence, and 10 times of concentration is the best.

Test example 1 detection of residual Lidocaine in adipose tissue essence

The adipose tissue essence was prepared according to the method of example 1, and a 10-fold concentrated sample of S4 was taken to detect the content of lidocaine, an anesthetic, by high performance liquid chromatography-tandem mass spectrometry (LC-MS, agilent). The result is shown in figure 4, the concentration of lidocaine in the 10-fold concentrated adipose tissue sperm is 40 mug/ml, and the dosage of lidocaine calculated according to the clinical use of 1ml is far lower than the clinical use amount of 4.0-4.5 mg/kg for children.

Test example 2 measurement of the content of adipokines in AT-XOs

Adipose tissue extracts were prepared according to the method of example 1, and 3 independent samples of the extracts were analyzed using human adipokine solid phase antibody chip (QAH-ADI-3) according to the manual (Raybiotech, USA). Reference is made to fig. 5. The results show that: adipose tissue seminal plasma contains a variety of adipose factors. Studies have shown that various adipokines play important roles in tissue regeneration processes (fat regeneration, hair regeneration, and vascular regeneration, etc.), including: adipoectin, leptin, PAI-1, IL-6, IGF-1, PDGF-BB, VEGF, etc.

Test example 3 measurement of in vivo Hair-regenerating ability of AT-XOs

The adipose tissue essence prepared by the method of example 1 was used for the hair regeneration capacity test, which specifically comprises the following steps:

c57 mice, which were 7 weeks old, were randomly divided into 4 groups after back dehairing, i.e., PBS group, and AT-XOs-1, AT-XOs-2, and AT-XOs-3 groups from different donors, respectively. PBS and AT-XOs were injected intradermally into the back of mice AT 100. mu.l each, once every other day. Recordings were taken at four time points, day 2, day 10, day 12 and day 15, respectively. The results are shown in FIGS. 6-9.

FIG. 6 shows the general view of groups of mice at different time points.

From the in vivo results, it can be seen that the three adipose tissue extracts from different donor sources all promote hair regeneration, and especially the effect difference is most significant at day 12 after injection (fig. 7).

FIG. 8 shows an internal view of the dorsal skin after 15 days, no visible revascularization was seen in the control group, and no visible tissue regeneration was seen in the silicone sheet (shown by solid arrows); the adipose tissue panel was seen with a large number of new blood vessels, and the silicone sheet was filled with new tissue (indicated by the open arrows).

FIG. 9 shows hematoxylin-eosin (HE) staining of 15-day back skin samples of control and experimental groups, with the red solid frame showing a partial enlarged view of the control group, the red dotted frame showing a partial enlarged view of the experimental group, and the black dotted lines showing regenerated tissue thickness; black filled triangular arrows indicate regenerated blood vessels.

The results show that the adipose tissue essence prepared by the method has good effect of promoting tissue regeneration, particularly promoting hair regeneration.

The foregoing is merely a preferred embodiment of this invention, which is intended to be illustrative, not limiting; those skilled in the art will appreciate that many variations, modifications, and even equivalent variations are possible within the spirit and scope of the invention as defined in the appended claims.