阅读说明：本技术 一种脂肪组织的冻存方法 (Cryopreservation method of adipose tissues ) 是由 龙婕 姜南 陈娟 孟宪星 董琪 于 2019-11-28 设计创作，主要内容包括：一种脂肪组织的冻存方法,步骤一、使用多孔注水针头在取材部位皮下均匀注射适量肿胀麻醉液,麻醉满意后,用吸脂针头接注射器,直接利用注射器管形成的负压将脂肪组织抽吸到注射器内,得到脂肪组织混合物,备用；步骤二、将抽取的脂肪组织混合物加适量生理盐水,上下倒置充分混匀后,静置,颗粒脂肪浮于上层,弃下层液体,将上层清洗去除大部分血液成分至颜色黄亮；然后置于控温离心机中离心,取颗粒脂肪组织,备用；步骤三、配置海藻糖溶液和油脂的混合液作为冻存液；步骤四、将颗粒脂肪组织与冻存液按1:1体积比混合,放入4℃冰箱2h后移入–20℃冰箱,4 h后移入–80℃深低温冰箱冷冻,12 h后移入液氮中冻存。本发明的冻存方法可保持冻存脂肪组织的活力。(A method for freezing adipose tissues comprises the steps of firstly, injecting a proper amount of swelling anesthetic liquid under the skin of a material-taking part by using a porous water injection needle, connecting a liposuction needle to a syringe after anesthesia is satisfied, and directly sucking the adipose tissues into the syringe by using negative pressure formed by the syringe tube to obtain an adipose tissue mixture for later use; step two, adding a proper amount of normal saline into the extracted adipose tissue mixture, inverting the mixture up and down, fully and uniformly mixing the mixture, standing the mixture until granular fat floats on the upper layer, discarding the liquid on the lower layer, and cleaning the upper layer to remove most blood components until the color is yellow and bright; centrifuging in a temperature-controlled centrifuge, and collecting granular adipose tissues for later use; step three, preparing a mixed solution of trehalose solution and grease as a frozen stock solution; and step four, mixing the granular adipose tissues with the freezing solution according to the volume ratio of 1:1, putting the mixture into a refrigerator at 4 ℃ for 2 hours, transferring the mixture into a refrigerator at 20 ℃ below zero, transferring the mixture into a deep low temperature refrigerator at 80 ℃ below zero for 4 hours for freezing, and transferring the mixture into liquid nitrogen for freezing after 12 hours. The cryopreservation method can keep the activity of the cryopreserved adipose tissues.)

1. A cryopreservation method of adipose tissues is characterized by comprising the following steps:

firstly, extracting adipose tissues: injecting proper amount of swelling anesthetic liquid under the skin of the material-taking part by using a porous water injection needle, connecting a liposuction needle with an injector after anesthesia is satisfied, and directly sucking adipose tissues into the injector by using negative pressure formed by the injector to obtain an adipose tissue mixture for later use;

step two, purifying adipose tissues: adding appropriate amount of normal saline into the extracted adipose tissue mixture, inverting, mixing, standing, floating granular fat on the upper layer, discarding the lower layer liquid, and cleaning the upper layer to remove most blood components to yellow and bright color; then placing the mixture in a temperature-controlled centrifuge, centrifuging the mixture for 2 to 4 min at 800-;

step three, preparing a freezing solution: preparing a 0.35mol/L mixed solution of a trehalose solution and oil as a frozen stock solution, wherein the volume ratio of the trehalose solution to the oil is 1: (1-3);

step four, freezing and storing granular adipose tissues: mixing the granular adipose tissues and the freezing solution according to the volume ratio of 1:1, placing the mixture into a refrigerator with the temperature of 4 ℃ for 2 hours, then transferring the mixture into a refrigerator with the temperature of 20 ℃ below zero, transferring the mixture into a deep low temperature refrigerator with the temperature of 80 ℃ below zero for 4 hours, freezing the mixture, and transferring the mixture into liquid nitrogen for freezing and storing the mixture after 12 hours.

2. The method of claim 1, wherein the freezing of adipose tissue comprises: in the first step, the injector is a 20mL injector.

3. The method of claim 1, wherein the freezing of adipose tissue comprises: in the second step, the mixture after centrifugal separation by the heat-preservation centrifugal machine is divided into an upper layer, a middle layer and a lower layer, the upper layer is lipid droplets overflowing from ruptured fat cells, the middle layer is granular adipose tissues, the lower layer is water and a small amount of fibrous tissues, the upper layer and the lower layer are discarded, and the granular adipose tissues in the middle layer are reserved.

4. The method for freezing adipose tissues according to claim 1, wherein superoxide dismutase with a concentration of 800IU/mL is added to the freezing solution in the third step.

5. The method for cryopreserving adipose tissues according to claim 1, wherein 5 to 25mmol/mL glutathione is further added to the cryopreserved solution of the third step.

6. The method for freezing adipose tissues according to claim 1, wherein 2-4mg/mL tocopherol is further added to the freezing solution of the third step.

7. The method for cryopreserving adipose tissues according to claim 1, wherein the preparation method of the cryopreserving solution comprises the following steps: mixing 0.35mol/L trehalose solution with oil, placing into a freezing tube, and adding superoxide dismutase (SOD) with the set concentration of 400-800IU/mL or/and 5-25mmol/mL Glutathione (GSH) or/and 2-4mg/mL tocopherol (VE).

8. The method for cryopreserving adipose tissues according to claim 1, wherein in the fourth step, the purified fat is filled into a special fat cryopreserving bag, the fat cryopreserving liquid is extracted and slowly added into the fat cryopreserving bag, after sufficient mixing, air is discharged, and the connecting pipe is sealed by a heat sealing machine; placing the freezing bag into a 4 ℃ vaccine box, transferring to a freezing chamber, placing in a 4 ℃ refrigerator for 2h, transferring to a-20 ℃ refrigerator, transferring to a-80 ℃ deep low temperature refrigerator for 4 h, freezing, and transferring to liquid nitrogen for freezing after 12 h.

Technical Field

The invention relates to the technical field of biological medical treatment, in particular to a cryopreservation method of adipose tissues.

Background

Autologous fat particles are an ideal soft tissue filling material; however, the low survival rate and high absorption rate of the transplanted fat particles are major problems that plague the wide clinical development and application of fat transplantation. Small amount, repeated and multiple injections are the ideal method for improving the survival rate of fat at present. However, repeated fat aspiration not only increases the pain of the patient, but also increases the medical risk and cost, so that more fat is extracted once for subsequent fractionated transplantation, the problems can be solved to a great extent, and the workload of medical staff can be reduced. "one-time liposuction and multiple injections" rely on the improvement of in vitro fat cryopreservation techniques. In previous studies, physiological saline or dimethyl sulfoxide (DMSO) was often used as a freezing base solution, but the cell activity of cells after storage in physiological saline was low; DMSO has certain toxicity, and cannot be used for clinical fat cell reinfusion.

At present, the existing frozen fat technology has certain damage to the activity of fat cells. Shochanni suggested that the main cause of the impairment of adipocyte viability was due to the freezing and rewarming processes, not to the length of the freezing time. After the human granular adipose tissues are subjected to processes of cooling, freezing and the like, the viability and the transplantation survival rate of fat cells in the tissues are lower than those of fresh human granular adipose tissues, wherein the main reasons comprise that: firstly, coarse dendritic ice crystals generated in cells directly destroy the cell structure when the temperature is reduced; on the other hand, the cells generate excessive peroxide by oxidative stress in the process of cooling, so that the cell activity is damaged, and the apoptosis of the cells is accelerated.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for freezing adipose tissues, which can eliminate the mechanical damage to cells caused by the formation of ice crystals, and is advantageous for maintaining the viability of the frozen adipose tissues by using a freezing solution containing an antioxidant for preservation.

The technical scheme provided by the invention for solving the problems is as follows: a cryopreservation method of adipose tissues comprises the following steps:

firstly, extracting adipose tissues: injecting proper amount of swelling anesthetic liquid under the skin of the material-taking part by using a porous water injection needle, connecting a liposuction needle with an injector after anesthesia is satisfied, and directly sucking adipose tissues into the injector by using negative pressure formed by the injector to obtain an adipose tissue mixture for later use;

step two, purifying adipose tissues: adding appropriate amount of normal saline into the extracted adipose tissue mixture, inverting, mixing, standing, floating granular fat on the upper layer, discarding the lower layer liquid, and cleaning the upper layer to remove most blood components to yellow and bright color; then placing the mixture in a temperature-controlled centrifuge, centrifuging the mixture for 2 to 4 min at 800-;

step three, preparing a freezing solution: preparing a 0.35mol/L mixed solution of a trehalose solution and oil as a frozen stock solution, wherein the volume ratio of the trehalose solution to the oil is 1: (1-3);

step four, freezing and storing granular adipose tissues: mixing the granular adipose tissues and the freezing solution according to the volume ratio of 1:1, placing the mixture into a refrigerator with the temperature of 4 ℃ for 2 hours, then transferring the mixture into a refrigerator with the temperature of 20 ℃ below zero, transferring the mixture into a deep low temperature refrigerator with the temperature of 80 ℃ below zero for 4 hours, freezing the mixture, and transferring the mixture into liquid nitrogen for freezing and storing the mixture after 12 hours.

In the present invention, preferably, in the first step, the syringe is a 20mL syringe.

In the present invention, preferably, in the second step, the mixture after centrifugation by the centrifuge is divided into three layers, namely, an upper layer, a middle layer and a lower layer, the upper layer is lipid droplets overflowing from ruptured adipocytes, the middle layer is granular adipose tissue, the lower layer is water and a small amount of fibrous tissue, the upper layer and the lower layer are discarded, and the granular adipose tissue in the middle layer is retained.

In the present invention, preferably, 400-800IU/mL superoxide dismutase is further added into the frozen stock solution in the third step.

In the present invention, preferably, 5 to 25mmol/mL glutathione is further added to the frozen stock solution of the third step.

In the present invention, preferably, 2-4mg/mL tocopherol is further added to the frozen stock solution of the third step.

In the present invention, preferably, the frozen stock solution is prepared by a method comprising: mixing 0.35mol/L trehalose solution with oil, placing into a freezing tube, and adding superoxide dismutase (SOD) with the set concentration of 400-800IU/mL or/and 5-25mmol/mL Glutathione (GSH) or/and 2-4mg/mL tocopherol (VE).

Preferably, in the fourth step, the purified fat is filled into a special fat cryopreservation bag, the fat cryopreservation liquid is extracted and slowly added into the fat cryopreservation bag, after the fat cryopreservation liquid and the fat cryopreservation liquid are fully mixed, air is discharged, and a connecting pipe is sealed by a heat sealing machine; placing the freezing bag into a 4 ℃ vaccine box, transferring to a freezing chamber, placing in a 4 ℃ refrigerator for 2h, transferring to a-20 ℃ refrigerator, transferring to a-80 ℃ deep low temperature refrigerator for 4 h, freezing, and transferring to liquid nitrogen for freezing after 12 h.

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

according to the method for freezing and preserving the adipose tissue, on one hand, trehalose solution is used as a novel adipose cell stabilizer, grease is a better solvent, the activity of frozen adipose cells can be well maintained after the trehalose solution and the fat are mixed, and the trehalose is an ideal fat freezing and preserving protective agent, is insoluble in fat, so that the added grease is used as an organic solvent and can be used as a carrier to carry trehalose into the adipose tissue cells, so that the adipose tissue cells are effectively cooled and protected, and mechanical damage to the cells caused by formation of ice crystals in the freezing and cooling process can be eliminated;

on the other hand, the addition of antioxidants can not only inhibit the production of active oxygen, but also directly scavenge active oxygen, or reduce the production of active oxygen by directly acting on antioxidant reaction-linking elements to increase the level of endogenous antioxidant substances or inhibit the expression of oxygenases. Therefore, the intervention of the antioxidant can effectively improve the activity of the granular adipose tissues of the cryopreserved human and avoid the excessive peroxide formation of cells due to oxidative stress in the cooling process. In summary, the cryopreservation solution described herein facilitates cryopreservation of human granular adipose tissue.

By the adipose tissue cryopreservation liquid, one-time liposuction and long-term use are really realized theoretically and technically; providing theoretical and technical basis for clinically applying frozen human granular adipose tissue transplantation filling to treat soft tissue defects caused by various reasons; meanwhile, the pain of liposuction surgery for the patient caused by repeated material taking is avoided, and the treatment cost of the patient is reduced; on the other hand, the workload and medical resources of medical staff can be reduced, in fat filling transplantation, the fat extraction time occupies more than half of the whole operation time, and doctors often adopt a syringe to form negative pressure manual liposuction in order to reduce the damage of fat cells as much as possible.

By freezing the adipose tissues, an in vitro fat depot can be established, and the patient can use autologous fat transplantation for life, and even the adipose tissues at the young age can be stored for anti-aging treatment when the coming soft tissues are aged and withered. The technology has wide clinical application prospect, is certainly well received by doctors and patients, and the obtained research data can provide relevant laboratory data and theoretical reference for the future establishment of fat banks by human beings and also can provide certain reference basis for the cryopreservation of other tissues or organs of the human beings.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.