阅读说明：本技术 用于注射填充的含有自体血清和高浓度脂肪干细胞的复合脂肪胶及其制备方法 (Composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling and preparation method thereof ) 是由 陈晓波 韩洪起 韩颢 冯春玲 吴学涛 于 2021-10-15 设计创作，主要内容包括：本发明公开了一种用于注射填充的含有自体血清和高浓度脂肪干细胞的复合脂肪胶及其制备方法。本申请通过从自身外周血中分离提取血清、从自体脂肪组织中分离脂肪胶以及分离脂肪干细胞并进行体外扩增培养,再按比例将血清、脂肪干细胞和脂肪胶进行混合,从而得到复合脂肪胶。本申请方法避免了传统脂肪干细胞培养方法中非人源血清导致的外源性污染,同时凭借血清中的活性成分可为脂肪干细胞提供适宜生长的微环境,有利于其分化和发挥强大组织修复功能,使得复合脂肪胶具有相比玻尿酸、传统脂肪、脂肪胶填充具有更明显的抗衰效果,填充疗效更加持久。(The invention discloses a composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling and a preparation method thereof. According to the application, serum is separated and extracted from peripheral blood of a patient, fat gel is separated from autologous adipose tissues, adipose stem cells are separated, in-vitro amplification culture is carried out, and then the serum, the adipose stem cells and the fat gel are mixed in proportion, so that the composite fat gel is obtained. The method avoids exogenous pollution caused by non-human serum in the traditional adipose-derived stem cell culture method, and meanwhile, the active ingredients in the serum can provide a microenvironment suitable for growth of the adipose-derived stem cells, so that differentiation and strong tissue repair functions are facilitated, the composite adipose-derived gelatin has a more obvious anti-aging effect compared with hyaluronic acid, traditional fat and adipose-derived gelatin filling, and the filling curative effect is more durable.)

1. A preparation method of composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling is characterized by comprising the following steps: the method comprises the following steps:

s1, obtaining autologous serum

a. Mixing the peripheral blood with lotion, and standing for 10-15 min;

b. adding diluted blood into the separating medium containing Ficoll lymphocyte, and centrifuging for 20-30min at 400-600 g;

c. after centrifugation, taking the upper plasma for complement inactivation at 55-56 ℃ for 30-35min, centrifuging 2000g-3000g for 5-8min, and taking the upper serum;

s2 separating adipose tissue

Mixing adipose tissue in the adipose tissue collection bag with the adipose tissue cleaning solution, standing for 3-5min, and removing the lower layer cleaning solution to obtain adipose tissue;

s3, adipose-derived stem cell isolation culture

a. Mixing the adipose tissues separated in the step S2 with the digestive juice, and performing oscillatory digestion for 40-60min at 37 ℃ and an oscillation frequency of 100-150 rpm;

b. after digestion, adding a basic culture medium, uniformly mixing, and centrifuging for 5-10min at 500g of 300-;

c. after centrifugation, sucking and removing the upper floating tissue and supernatant, adding culture medium for basic suspension, filtering by using a 100um filter screen, and inoculating the filtered cell suspension into a culture medium for culture;

s4, preparing composite fatty glue:

fat gum separation

a. Putting the adipose tissues separated in the step S2 into a screw injector;

b. connecting the screw injector containing adipose tissues and the other injector with a three-way pipe;

c. pushing the injector push rod containing adipose tissues to make all adipose tissues enter the other injector through the three-way pipe, then pushing all tissues back to the original injector, and repeating the process for 10-15 times;

d. centrifuging the obtained adipose tissue at 400-600 g for 5-10min, and removing oil drop layer to obtain fat gel;

cell digestion

When the fusion degree of the adipose-derived stem cells cultured in the step S3c reaches 80% -95%, performing cell cultureDigesting and recovering to obtain 5 × 10⁶-1×10⁷Washing and centrifuging the individual cells;

III mixing

And (4) taking the autologous serum prepared in the step S1c to resuspend the cell sediment, filtering the cell sediment by using a 100-micron filter screen, adding the fat gel prepared in the step S4I, and uniformly mixing to obtain the composite fat gel.

2. The preparation method of the composite fat gel containing the autologous serum and the high-concentration adipose stem cells for injection filling according to claim 1, wherein the preparation method comprises the following steps: in step S1a, the washing solution is sodium chloride injection containing 25IU/ml heparin sodium.

3. The preparation method of the composite fat gel containing the autologous serum and the high-concentration adipose stem cells for injection filling according to claim 1, wherein the preparation method comprises the following steps: in step S2, the adipose tissue cleaning solution is sodium chloride injection containing gentamicin 10-50 IU/ml.

4. The preparation method of the composite fat gel containing the autologous serum and the high-concentration adipose stem cells for injection filling according to claim 1, wherein the preparation method comprises the following steps: in step S3c, the culture medium is a basal medium containing 10-20 v% of autologous serum.

5. The preparation method of the composite fat gel containing the autologous serum and the high-concentration adipose stem cells for injection filling according to claim 1, wherein the preparation method comprises the following steps: in step S4 III, the volume ratio of the autologous serum to the fat gel is 1 (2-8).

6. The composite fat gel prepared by the preparation method for the composite fat gel containing the autologous serum and the high-concentration adipose-derived stem cells for injection filling according to any one of claims 1 to 5.

Technical Field

The invention relates to the technical field of cell biology, in particular to a composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling and a preparation method thereof.

Background

The injection filling is a micro-plastic medical beauty treatment method for injecting a specific filling agent (or an injection) to a target position by a percutaneous injection method to achieve the effects of rejuvenation and beautifying, has the characteristics of micro-wound (non-wound), small pain, safety, high efficiency, customization according to the dosage requirement of a specific part and the like, and the main parts currently used for injection filling comprise the face, the chest, the hip and the like, wherein the injection filling of the face can play the roles of plastic contour, wrinkle removal, firmness, smoothness, three-dimensional and plump five sense organs and the like; the injection filling of the chest and the hip mainly aims to solve the problem of sagging, increase plasticity and repair local depressions caused by congenital or after surgical plastic surgery. At present, main fillers for injection filling comprise botulinum toxins, collagen, hyaluronic acid (sodium hyaluronate), autologous fat gel and the like, wherein the botulinum toxins, the collagen, the hyaluronic acid and the like have the defects of short retention time, easy absorption and consumption by surrounding tissues and the like, so regular continuous injection is needed for keeping the effect; although the problems can be solved by the autologous fat and the fat gel, the quantity of the viable fat cells is unstable due to the lack of peptide substances and individual difference, the ideal effect is difficult to achieve, and multiple times of filling are needed, so that certain defects exist in the technology.

Disclosure of Invention

The invention provides a compound fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling and a preparation method thereof in order to solve the technical problems.

In a first aspect, the invention provides a preparation method of a composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling, which is realized by adopting the following technical scheme.

A preparation method of composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling comprises the following steps:

s1, obtaining autologous serum

a. Mixing the peripheral blood with lotion, and standing for 10-15 min;

b. adding diluted blood into the separating medium containing Ficoll lymphocyte, and centrifuging for 20-30min at 400-600 g;

c. after centrifugation, taking the upper plasma for complement inactivation at 55-56 ℃ for 30-35min, centrifuging 2000g-3000g for 5-8min, and taking the upper serum;

s2 separating adipose tissue

Mixing adipose tissue in the adipose tissue collection bag with the adipose tissue cleaning solution, standing for 3-5min, and removing the lower layer cleaning solution to obtain adipose tissue;

s3, adipose-derived stem cell isolation culture

a. Mixing the adipose tissues separated in the step S2 with the digestive juice, and performing oscillatory digestion for 40-60min at 37 ℃ and an oscillation frequency of 100-150 rpm;

b. after digestion, adding a basic culture medium, uniformly mixing, and centrifuging for 5-10min at 500g of 300-;

c. after centrifugation, sucking and removing the upper floating tissue and supernatant, adding culture medium for basic suspension, filtering by using a 100um filter screen, and inoculating the filtered cell suspension into a culture medium for culture;

s4, preparing the composite fatty glue

Fat gum separation

a. Putting the adipose tissues separated in the step S2 into a screw injector;

b. connecting the screw injector containing adipose tissues and the other injector with a three-way pipe;

c. pushing the injector push rod containing adipose tissues to make all adipose tissues enter the other injector through the three-way pipe, then pushing all tissues back to the original injector, and repeating the process for 10-15 times;

d. centrifuging the obtained adipose tissue at 400-600 g for 5-10min, and removing oil drop layer to obtain fat gel;

cell digestion

When the fusion degree of the adipose-derived stem cells cultured in the step S3c reaches 80% -95%, digesting and recovering the cells, and taking 5 × 10⁶-1×10⁷Washing and centrifuging the individual cells;

III mixing

And (4) taking the autologous serum prepared in the step S1c to resuspend the cell sediment, filtering the cell sediment by using a 100-micron filter screen, adding the fat gel prepared in the step S4I, and uniformly mixing to obtain the composite fat gel.

Further, in step S1a, the washing solution is sodium chloride injection containing 25IU/ml heparin sodium.

Further, in step S2, the adipose tissue-washing solution is sodium chloride injection containing gentamicin 10-50 IU/ml.

Further, in step S3c, the culture medium is a basal medium containing 10-20 v% of autologous serum.

Further, in step S4 III, the volume ratio of the autologous serum to the fat gel is 1 (2-8).

In a second aspect, the invention provides a composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling, which is realized by adopting the following technical scheme.

The composite fat gel for injection filling and prepared by the preparation method of the composite fat gel containing the autologous serum and the high-concentration adipose-derived stem cells.

The present application has the following advantageous effects.

The autologous serum contained in the composite fat gel contains a large amount of active ingredients such as growth factors, and the factors can promote the proliferation and activation of cells and the proliferation of collagen and elastic fibers; autologous adipose-derived stem cells directly participate in tissue reconstruction through self proliferation and differentiation, and stimulate the proliferation and differentiation of peripheral tissue cells and synthesize a large amount of nascent collagen, elastic fibers and hyaluronic acid under the action of active ingredients secreted by the autologous adipose-derived stem cells, so that the transplantation success rate of the adipose-derived glue at the filling part is assisted. The two components are self-derived and have no immunogenicity, so that the prepared composite fat gel has longer maintenance time and higher stability after being filled, and has better filling effect compared with the traditional fat and fat gel filling.

Drawings

FIG. 1 is an under-microscope image of adipose-derived stem cells cultured in the present application;

fig. 2 is a diagram of a finished product of the composite fat gel prepared by the method.

Detailed Description

The invention is further described below with reference to the figures and examples.

The invention provides a preparation method of a composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling, which comprises the following steps:

(1) obtaining autologous serum:

taking 15ml-30ml of autologous peripheral blood to a centrifuge tube, adding washing liquor (sodium chloride injection containing 25IU/ml heparin sodium) to 30-60ml, uniformly mixing, and standing for 10-15 min.

Secondly, slowly adding the diluted blood into a centrifuge tube containing 15-20ml of Ficoll lymphocyte separation liquid along the inner wall of the centrifuge tube, and centrifuging for 20-30min under the centrifugal force of 400-600 g.

③ after the centrifugation is finished, taking the upper layer of blood plasma to a new centrifugal tube, inactivating complement at 55-56 ℃ for 30-35min, centrifuging for 5-8min under the centrifugal force of 2000g-3000g, and taking the upper layer of blood serum to a new centrifugal tube for refrigerating and storing at 4 ℃.

(2) Fat tissue separation and cryopreservation:

pouring 80-120ml of adipose tissues in the fat collection bag into a 250ml centrifuge cup, pouring an adipose tissue cleaning solution (sodium chloride injection containing 10-50IU/ml of gentamicin) into the centrifuge tube to a total volume of 150-. And (3) subpackaging 30-50% of adipose tissues into 50ml centrifuge tubes, and freezing and storing at-80 ℃.

(3) And (3) carrying out isolated culture on the adipose-derived stem cells:

taking the remaining 50-70% of adipose tissues in (2) for adipose stem cell isolation:

firstly, absorbing adipose tissues according to a tube of 15-20ml, adding the adipose tissues into a centrifuge tube containing 2-4ml of digestive juice (basic culture medium contains 1-2% of I-type collagenase), reversing and mixing for 3-5 times, inserting the mixture into a steel wire mesh of a gas bath constant temperature oscillator preheated to 37 ℃ in advance at an angle of 45 degrees, adjusting the oscillation frequency to be 100 plus 150rpm, and performing oscillation digestion for 40-60 min.

② after the digestion, the centrifuge tube is taken out for observation, the tissue blocks are obviously reduced and become small, and the state of semitransparent connective tissue is presented. 10-15ml of the basal medium was added, mixed by inversion 3-5 times, and then centrifuged at 500g of 300-500g for 5-10 min.

Thirdly, absorbing and removing the upper floating tissue and the supernatant after the centrifugation is finished, adding 30-40ml of culture medium (the culture medium is a basic culture medium containing 10-20 v% of autologous serum) into each tube for re-suspension, filtering by using a 100um filter screen, averagely inoculating the filtered cell suspension into 2T-75 cell culture bottles, and putting CO into the culture bottles₂Culturing in an incubator.

(4) Preparing a composite fatty gum:

when the growth state of the cells in the T-75 cell culture bottle is good and the fusion degree reaches 80-95%, the preparation of the composite fat gel is carried out.

a. Taking the fat tissue frozen in the step (2), thawing, rewarming and then separating fat gel:

sucking the adipose tissues into a 10ml screw-type syringe according to 8-10 ml/branch.

And secondly, taking 1 medical three-way pipe, rotating a knob to enable the medical three-way pipe to be in a state that all through openings are communicated, and connecting the injector filled with the adipose tissues and a new 10ml injector to the main through opening and the side through opening of the medical three-way pipe. The main port is opened to discharge air in the syringe, and the knob is rotated to connect the main port and the side port in a communicated state.

Thirdly, pushing the injector push rod containing the adipose tissues to ensure that all the adipose tissues enter a new 10ml injector through the three-way pipe, and then pushing all the tissues back to the original injector, wherein the process is repeated for 10-15 times.

The syringe containing the adipose tissue was removed and the adipose tissue was injected into a centrifuge tube.

And fourthly, continuously treating the adipose tissues of 2-6 tubes according to the third step, centrifuging for 5-10min under the centrifugal force of 400-600 g, removing an oil drop layer after the centrifugation is finished, and taking the fat gel in the centrifuge tube for subsequent preparation operation.

b. Cell digestion:

when the growth state of the cells in the T-75 cell culture bottle is good and the fusion degree reaches 80% -95%, digesting and recovering the cells, and taking 5 multiplied by 10⁶-1×10⁷The cells are centrifugally cleaned for 3-4 times (300-.

c. Mixing and filling:

adding 2-4ml of autologous serum to resuspend the cell precipitate, filtering by a 100um filter screen, adding 8-16ml of fat gel, and uniformly mixing. And filling 10-20ml of the uniformly mixed composite fatty gum into a 10ml prefilled syringe.

The implementation case is as follows:

the patient was Liezhi, age 43, apple muscle drop, and temple depression.

A preparation method of composite fat gel containing autologous serum and high-concentration adipose-derived stem cells for injection filling comprises the following steps:

(1) obtaining autologous serum:

taking 30ml of autologous peripheral blood to a centrifuge tube, supplementing washing liquor (sodium chloride injection containing 25IU/ml heparin sodium) to 60ml, uniformly mixing, and standing for 10 min.

② slowly adding the diluted blood into a centrifuge tube containing 15ml of Ficoll lymphocyte separating medium along the inner wall of the centrifuge tube, and centrifuging for 23min under the centrifugal force of 500 g.

③ after the centrifugation is finished, taking 23.5ml of upper layer plasma to a new centrifuge tube, inactivating complement at 56 ℃ for 30min, centrifuging for 5min under 3000g of centrifugal force, taking 21.5ml of upper layer serum to a new centrifuge tube, and refrigerating and storing at 4 ℃.

(2) Fat tissue separation and cryopreservation:

pouring 110ml of adipose tissues in the adipose collection bag into a 250ml centrifuge cup, pouring an adipose tissue cleaning solution (sodium chloride injection containing 50IU/ml of gentamicin) into the centrifuge tube to the total volume of 200ml, standing for 5min after the mixture is reversed, and sucking away the lower layer cleaning solution to obtain 55ml of adipose tissues in total. 25ml of adipose tissues were dispensed into 250ml centrifuge tubes and stored frozen at-80 ℃.

(3) And (3) carrying out isolated culture on the adipose-derived stem cells:

the remaining 30ml of adipose tissues in (2) above were used for adipose stem cell isolation:

30ml of adipose tissues are averagely added into two centrifuge tubes containing 4ml of digestive juice (1% of I-type collagenase contained in a basic culture medium), inverted and mixed for 5 times, inserted into a steel wire mesh of a gas bath constant temperature oscillator preheated to 37 ℃ in advance at an angle of 45 degrees, the oscillation frequency is adjusted to be 120rpm, and the mixture is subjected to oscillation digestion for 45 min.

② after the digestion, the centrifuge tube is taken out for observation, the tissue blocks are obviously reduced and become small, and the state of semitransparent connective tissue is presented. 10ml of the basal medium was added to each tube, mixed 5 times by inversion, and centrifuged at 300g for 8 min.

Thirdly, absorbing and discarding the upper floating tissue and the supernatant after the centrifugation is finished, adding 15ml of culture medium (containing 3ml of autologous serum) into each tube for resuspension, filtering by using a 100um filter screen, inoculating the filtered cell suspension into 1T-75 cell culture bottle, inoculating two T-75 culture bottles together, and putting CO into the two T-75 culture bottles₂Culturing in an incubator.

The photograph under the lens of the cultured adipose-derived stem cells is shown in FIG. 1.

(4) Preparing a composite fatty gum:

after culturing for 4 days, the growth state of the cells in the T-75 cell culture bottle is good, the fusion degree reaches 90 percent, and the preparation of the composite fat gel is carried out.

a. Taking the fat tissue frozen in the step (2), thawing, rewarming and then separating fat gel:

sucking the adipose tissues into a 10ml screw-type syringe according to 8 ml/branch.

And secondly, taking 1 medical three-way pipe, rotating a knob to enable the medical three-way pipe to be in a state that all through openings are communicated, and connecting the injector filled with the adipose tissues and a new 10ml injector to the main through opening and the side through opening of the medical three-way pipe. The main port is opened to discharge air in the syringe, and the knob is rotated to connect the main port and the side port in a communicated state.

Thirdly, pushing the injector push rod containing the adipose tissues to ensure that all the adipose tissues enter a new 10ml injector through the three-way pipe, and then pushing all the tissues back to the original injector, wherein the process is repeated for 15 times.

The syringe containing the adipose tissue was removed and the adipose tissue was injected into a centrifuge tube.

And fourthly, continuously treating 3 tubes of adipose tissues according to the third-fourth method, centrifuging for 8min under the centrifugal force of 500g, removing an oil drop layer after the centrifugation is finished, and taking 24ml of fatty glue in a centrifuge tube for subsequent preparation operation.

b. Cell digestion:

digesting and recovering cells in 2T-75 culture bottles, counting, and taking 1 × 10⁷Individual cells were washed 3 times (300 g, 8 min) by centrifugation in compound electrolyte solution.

c. Mixing and filling:

adding 4ml of autoserum to resuspend the cell sediment, filtering by a 100um filter screen, adding 16ml of fat gel, and uniformly mixing. The 20ml of the mixed composite fat gel is filled into a 5ml prefilled syringe with 4 branches.

The finished product of the composite fat gum is shown in figure 1.

The patient receives one-time injection filling treatment, and 20ml of compound fat gel is injected into apple muscles on both sides and temple at one time. The swelling disappears in 3 days, the filling effect appears, the effect is evaluated in 14 days, the apple muscle sagging disappears, and the cheeks are smooth and elastic; the temple is full, and the whole stereoscopic impression of the face is enhanced compared with that before an operation.

The return visit is carried out after 180 days, the filling effect is kept good, and the filling is more natural and full.

The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.