Protective agent for fat tissue freezing
阅读说明：本技术 脂肪组织冻存保护剂 (Protective agent for fat tissue freezing ) 是由 李青峰 周双白 张佩祺 于 2019-12-26 设计创作，主要内容包括：本发明提供一种甘油用于制备脂肪组织冻存保护剂的用途及一种脂肪组织冻存保护剂。以脂肪组织冻存保护剂总量为基准计,所述脂肪组织冻存保护剂包含：甘油体积分数为60％-80％；海藻糖0.090-0.200g/ml；溶剂为PBS缓冲液。本发明的脂肪组织冻存保护剂,无DMSO及其他含生物毒性的保护剂,对细胞及组织均无毒性,洗脱不彻底的情况下仍不会引起临床使用时的安全问题；无异种抗原,成本较低,甘油及海藻糖均为临床获批药物成分且成本较低的产品,且本发明不含有诸如人血白蛋白类成本较高的产品,故临床使用及推广的可能性较高；保护能力强,脂肪组织复苏后活性高,在移植后形成的炎性细胞少。(The invention provides application of glycerol in preparation of an adipose tissue cryopreservation protective agent and the adipose tissue cryopreservation protective agent. Based on the total amount of the fat tissue freezing protective agent, the fat tissue freezing protective agent comprises: the volume fraction of the glycerol is 60-80 percent; trehalose 0.090-0.200 g/ml; the solvent was PBS buffer. The fat tissue cryopreservation protective agent disclosed by the invention is free of DMSO (dimethyl sulfoxide) and other protective agents containing biotoxicity, has no toxicity to cells and tissues, and does not cause the safety problem in clinical use under the condition of incomplete elution; the invention has the advantages that the vaccine has no foreign antigen, the cost is lower, the glycerol and the trehalose are all clinical approved medicinal components and are lower in cost, and the vaccine does not contain products such as human serum albumin with higher cost, so the possibility of clinical use and popularization is higher; strong protective capability, high activity after the recovery of adipose tissues and less inflammatory cells formed after transplantation.)
1. Use of glycerol for the preparation of an adipose tissue cryopreservation protectant.
2. The use according to claim 1, wherein glycerol is an active ingredient of the adipose tissue cryopreservation agent.
3. The use of claim 1, wherein the adipose tissue cryopreservation agent does not comprise dimethyl sulfoxide, animal serum, or human serum albumin.
4. The use of claim 1, wherein the adipose tissue cryopreservation agent further comprises trehalose and PBS buffer.
5. The use according to claim 4, wherein the fat tissue cryopreservation agent comprises the following components in percentage by weight based on the total amount of the fat tissue cryopreservation agent:
the volume fraction of the glycerol is 60-80 percent;
trehalose 0.090-0.200 g/ml;
the solvent was PBS buffer.
6. The use of claim 1, wherein the adipose tissue cryopreservation agent enables stable storage of adipose tissue at-196 ℃ for at least 6 months.
7. An adipose tissue cryopreservation protective agent, which is characterized by comprising the following components in percentage by weight based on the total amount of the adipose tissue cryopreservation protective agent:
the volume fraction of the glycerol is 60-80 percent;
trehalose 0.090-0.200 g/ml;
the solvent was PBS buffer.
8. The adipose tissue cryopreservation agent of claim 7 wherein the adipose tissue cryopreservation agent does not comprise dimethyl sulfoxide, animal serum or human serum albumin.
9. The fat tissue cryopreservation agent of claim 7 wherein the fat tissue cryopreservation agent enables stable storage of fat tissue at-196 ℃ for at least 6 months.
10. A preparation method of a fat freezing protective agent is characterized by comprising the following steps:
1) adding trehalose into a PBS buffer solution to prepare a trehalose solution;
2) the fat freezing protective agent is prepared by using glycerol as a solute and a trehalose solution as a solvent.
11. The method of claim 10, further comprising one or more of the following features:
a. in the step 1), the concentration of the trehalose in the trehalose solution is 0.25-0.50 mol/L;
b. in the step 2), the fat freezing protective agent contains 60-80% of glycerol by volume fraction.
12. A method for cryopreserving adipose tissues, which is characterized by comprising the following steps:
1) mixing the fat cryopreservation agent of claim 7 with adipose tissue in a volume ratio of 1:1 to obtain a mixture 1;
2) placing the mixture 1 in a programmed cooling box, and carrying out programmed cooling at-80 ℃;
3) placing the mixture 1 obtained in the step 2) in liquid nitrogen for freezing storage to obtain frozen adipose tissues.
13. A method for rewarming cryopreserved adipose tissues is characterized by comprising the following steps:
a. removing the cryopreserved adipose tissue obtained in step 3) of claim 11 from the liquid nitrogen, and heating in a water bath;
b. eluting the adipose tissues obtained in the step a by using a PBS buffer solution, and centrifuging;
c. and (4) taking the centrifuged upper-layer substance, eluting with a PBS buffer solution, and centrifuging to obtain the upper-layer substance, namely the rewarming adipose tissue.
Technical Field
The invention relates to the field of medical treatment, in particular to an adipose tissue cryopreservation protective agent.
Background
With the continuous development of fat transplantation technology, autologous fat transplantation technology has been widely used clinically, and is a better solution to the problems of scar filling, facial atrophy, tissue reconstruction, wrinkles, breast filling and the like. However, fat transplantation has the problem of high reabsorption at present, and most cases still need multiple times of liposuction and immediate fat filling after operation so as to accurately achieve the repairing effect. Because the liposuction operation has serious local injury, great pain is caused to a patient, and the patient needs to be locally pressed for at least 1 month after the liposuction operation every time. Liposuction surgery also has a higher risk of infection. Therefore, if the fat extracted once can be effectively stored for a long time, and fat filling treatment is performed if necessary, the frequency of fat extraction in the operation can be reduced, the pain of a patient in the operation can be obviously relieved, the recovery time after the operation can be reduced, the economic burden and the operation risk of the operation can be reduced, and the treatment efficiency can be improved.
Cryopreservation is a method by which cells and tissues can be preserved for long periods of time. During freezing, a freezing protective agent is usually added to protect cells from being damaged by water molecule crystallization after low temperature.
However, the cryopreservation protection effect of the adipose tissues is controversial at present, and the problems of reduced tissue activity after cryopreservation, lower tissue retention rate after re-transplantation and the like exist; the existing tissue freezing protective agents contain biological toxic substances or xenoantigens, and the biological safety problem can be caused after the transplantation. No effective cryopreservation protective agent aiming at long-term cryopreservation of adipose tissues and being safe and non-toxic is found at present. Adipose tissue has higher cryoprotectant requirements than a simple cell suspension or other homogeneous tissue.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide an adipose tissue cryopreservation agent, which is used for solving the problems of high toxicity and poor cryopreservation effect of the cryopreservation agent in the prior art.
To achieve the above and other related objects, the present invention provides, in a first aspect, the use of glycerol for the preparation of an adipose tissue cryopreservation agent.
In a second aspect, the present invention provides an adipose tissue cryopreservation protective agent, comprising, based on the total amount of the adipose tissue cryopreservation protective agent:
the volume fraction of the glycerol is 60-80 percent;
trehalose 0.090-0.200 g/ml;
the solvent was PBS buffer.
The third aspect of the invention provides a preparation method of a fat freezing and preserving protective agent, which is characterized by comprising the following steps:
1) adding trehalose into a PBS buffer solution to prepare a trehalose solution;
2) the fat freezing protective agent is prepared by using glycerol as a solute and a trehalose solution as a solvent.
In a fourth aspect, the present invention provides a method for cryopreservation of adipose tissues, comprising the following steps:
1) mixing the fat freezing protective agent and adipose tissues in a volume ratio of 1:1 to obtain a mixture 1;
2) placing the mixture 1 in a programmed cooling box, and carrying out programmed cooling at-80 ℃;
3) placing the mixture 1 obtained in the step 2) in liquid nitrogen for freezing storage to obtain frozen adipose tissues.
The fifth aspect of the present invention provides a method for rewarming a cryopreserved adipose tissue, comprising the steps of:
a. taking out the frozen adipose tissues from liquid nitrogen, and heating in a water bath;
b. eluting the adipose tissues obtained in the step a by using PBS buffer solution, and centrifuging;
c. and (4) taking the centrifuged upper-layer substance, eluting by using PBS buffer solution, and centrifuging to obtain the upper-layer substance, namely the rewarming adipose tissue.
As described above, the fat tissue cryopreservation protective agent of the present invention has the following beneficial effects:
the fat tissue cryopreservation protective agent disclosed by the invention is free of DMSO (dimethyl sulfoxide) and other protective agents containing biotoxicity, has no toxicity to cells and tissues, and does not cause the safety problem in clinical use under the condition of incomplete elution; the preparation has no xenoantigen, and can cause hypersensitivity after fat transplantation under the condition that fetal calf serum is required to be used in the traditional protective agent, while the biological agent is not used in the preparation; the cost is low, the glycerol and the trehalose are all clinical approved medicinal components and are low-cost products, and the invention does not contain products with high cost such as human serum albumin, so the possibility of clinical use and popularization is high; strong protective capability, high activity after the recovery of adipose tissues and less inflammatory cells formed after transplantation.
Drawings
FIG. 1 shows a comparison of adipocytes and adipose tissue. The left image is the under-lens picture of the cells and the right image is adipose tissue (HE staining). It can be seen that adipose tissue has a large mass and a large number of cells, and there are many types of cells and its inherent tissue structure. The cell suspension only has single cell, the cells are dispersed, no obvious block exists, and the cryopreservation protective agent is easy to wrap each cell.
FIG. 2 shows a dorsal fat transplantation image of a nude mouse. A: the formula of the patent is preserved and then fat transplantation is carried out: the tissue block can be seen to be surrounded by the surrounding blood vessels, the graft is relatively uniform, and no obvious fat liquification necrosis area is seen.
B: fat transplantation after FBS + DMSO preservation: the tissue block blood vessel can be wrapped, but the upper and lower levels can be seen by comparing the liquefied necrotic area AB, and when the formula of the patent is applied, the transplanted tissue block after the adipose tissue is frozen is better in general form.
FIG. 3 is a graph showing the results of the G3PDH experiment for each sample, which is a quantitative measure of tissue mass activity.
Figure 4 shows the results of fat transplantation retention after cryopreservation at-196 ℃.
FIG. 5 shows the results of observation of section HE staining (in the figure,. about.. about.fibrosis tissue,. DELTA.inflammatory infiltration,. about..
Detailed Description
The traditional cell cryopreservation protective agent is mostly used for single type of cells, and has poor preservation effect on the composite tissue of adipose tissue. The freezing protection is effective for freezing protection of single cells and has poor protection effect on the three-dimensional structure of the tissue when the composite tissue is preserved. The cell suspension is frozen at low temperature after being treated by the freezing protective agent, the activity is usually 95 percent or more after recovery, and the activity of the tissue is usually only about 30 percent after being treated by the traditional freezing protective agent.
Adipose tissue is a complex tissue, the cell types of which are various, including adipocytes, adipose precursor cells, adipose stem cells, fibroblasts, vascular endothelial cells and the like (fig. 1), and the adipose tissue has a large mass, and the conventional cryopreservation protective agent is difficult to effectively infiltrate into the tissue. Adipose tissues are mainly composed of fat cells, the organelle components in the fat cells are few, most of the organelle components are fat drops rich in triglyceride, the fat drops are greatly different from other cells and tissues, and the conventional cryopreservation protective agent commonly used for other tissues has difficulty in effectively protecting special structural components of the adipose tissues. Secondly, the protective agent penetrating into the interior of the adipose tissue is often not removed sufficiently in elution, but in the traditional freezing protective agent taking Fetal Bovine Serum (FBS) + dimethyl sulfoxide (DMSO) as a formula, the protective agent contains heterogeneous antigens and the dimethyl sulfoxide is toxic to cells, and the residual protective agent can cause adverse effects such as toxicity and allergy to patients in clinical use (according to the Sigma product number Vetec-V900090 specification). Effective cryopreservation of adipose tissues not only requires good morphology immediately after being taken out, but also more importantly ensures the biological activity of cells in the tissues so as to ensure the survival rate and biological function after the tissues are transplanted again and reduce the tissue damage caused by local inflammatory reaction. Therefore, no cryopreservation protective agent which can be used for effectively and non-toxically cryopreserving adipose tissues for a long time, is low in cost and is suitable for mass popularization has been disclosed so far.
Glycerol is a permeable cryoprotectant, plays a role in cryopreservation by inhibiting ice crystal formation, preventing permeability damage, protecting cell membranes and intracellular proteins and other mechanisms at low temperature, and is currently studied in cryopreservation of complex tissues such as skin, cartilage, testis and the like. In the aspect of adipose tissue preservation, the similarity between glycerol and triglyceride abundantly existing in fat cells can better preserve the activity of fat cells and the integrity of fat droplets, and the cytology has verified that the fat cells are infiltrated into glycerol solution to effectively inhibit the outflow of triglyceride in the fat cells; the permeability of the composite tissue is strong, and the composite tissue can better permeate and elute fat tissues with large lumps; has no tissue toxicity and lower cost, and is beneficial to clinical large-scale use. Therefore, the cryopreservation protective agent taking the glycerol as the main active ingredient can effectively protect the tissue activity of fat under the cryopreservation condition and reduce the apoptosis; in the recovery process, the toxic and side effects of the protective agent residue do not exist, and the tissue morphological structure after transplantation is better. In the invention, trehalose with a certain concentration is added at the same time to be used as an impermeable cryopreservation protective agent to strengthen the protection effect on tissues.
The invention discloses a cell cryopreservation protective agent which takes trehalose as a main component, wherein the main protection target is cell suspension rather than adipose tissue, the invention takes the single-purity trehalose as a contrast for verification, and the invention finds that the tissue morphology after transplantation is obviously superior to that of a group only containing trehalose after glycerol is added, and the tissue after trehalose protection is transplanted to have obvious inflammatory cell infiltration, possibly due to immune reaction caused by necrosis of fat cells due to poor protection effect after cryopreservation. The same experiment shows that after the treatment of the formula disclosed by the invention, the inflammatory infiltration after the adipose tissue transplantation is obviously less, and further provides a better evidence for the adipose tissue cryopreservation protective agent taking glycerol as a main component.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides application of glycerol in preparing an adipose tissue cryopreservation protective agent.
In the fat tissue freezing protective agent, glycerol is used as a permeability protective agent.
Furthermore, glycerol is an effective component of the fat tissue freezing preservation protective agent.
Further, the adipose tissue cryopreservation protective agent does not comprise dimethyl sulfoxide, animal serum or human serum albumin.
In one embodiment, the fat tissue cryopreservation protective agent further comprises trehalose and a PBS buffer.
In the fat tissue freezing protective agent, trehalose is used as an auxiliary non-permeability protective agent. More comprehensively reduces the formation of ice crystals and the damage to cell structures during the freezing process.
In one embodiment, the fat tissue cryopreservation agent comprises the following components in percentage by weight based on the total amount of the fat tissue cryopreservation agent:
the volume fraction of the glycerol is 60-80 percent;
trehalose 0.090-0.200 g/ml;
the solvent was PBS buffer.
In one embodiment, the trehalose is present in an amount of from 0.25 to 0.50 mol/L. Optionally, 0.25-0.3mol/L, 0.3-0.35mol/L, 0.35-0.4mol/L, 0.4-0.45mol/L, 0.45-0.5 mol/L.
Optionally, in the fat tissue cryopreservation protective agent, the volume fractions of glycerol are 60% -65%, 65% -70%, 70% -75% and 75% -80%.
Optionally, in the fat tissue freezing and preserving protective agent, the mass concentration of trehalose is 0.009-0.015g/ml, 0.015-0.020g/ml, 0.020-0.030g/ml, 0.030-0.040g/ml, 0.050-0.060g/ml, 0.060-0.070g/ml, 0.070-0.080g/ml, 0.080-0.090g/ml, 0.090-0.100g/ml, 0.010-0.120g/ml, 0.120-0.140g/ml, 0.140-0.160g/ml, 0.160-0.180g/ml and 0.180-0.200 g/ml.
The fat tissue freezing protective agent can ensure that the fat tissue can be stably stored for at least 6 months at the temperature of-196 ℃.
The fat tissue cryopreservation protective agent provided by the invention comprises the following components in percentage by weight based on the total amount of the fat tissue cryopreservation protective agent:
the volume fraction of the glycerol is 60-80 percent;
trehalose 0.090-0.200 g/ml;
the solvent was PBS buffer.
In one embodiment, the trehalose is present in an amount of from 0.25 to 0.50 mol/L.
Optionally, in the fat tissue cryopreservation protective agent, the volume fractions of glycerol are 60% -65%, 65% -70%, 70% -75% and 75% -80%.
Optionally, in the fat tissue freezing and preserving protective agent, the mass concentration of trehalose is 0.009-0.015g/ml, 0.015-0.020g/ml, 0.020-0.030g/ml, 0.030-0.040g/ml, 0.050-0.060g/ml, 0.060-0.070g/ml, 0.070-0.080g/ml, 0.080-0.090g/ml, 0.090-0.100g/ml, 0.010-0.120g/ml, 0.120-0.140g/ml, 0.140-0.160g/ml, 0.160-0.180g/ml and 0.180-0.200 g/ml.
The solutes of the PBS buffer were: 200mmol/L Na2HPO4，35mmol/LKH2PO42.74mol/L NaCl, 53 mmol/LKCl. The solvent is water. pH7.2-7.6.
Further, the adipose tissue cryopreservation protective agent does not comprise dimethyl sulfoxide, animal serum or human serum albumin.
The preparation method of the fat freezing protective agent provided by the invention comprises the following steps:
1) adding trehalose into a PBS buffer solution to prepare a trehalose solution;
2) the fat freezing protective agent is prepared by using glycerol as a solute and a trehalose solution as a solvent.
In one embodiment, in step 1), said concentration of trehalose in said trehalose solution is from 0.25mol/L to 0.50 mol/L. Optionally, 0.25-0.3mol/L, 0.3-0.35mol/L, 0.35-0.4mol/L, 0.4-0.45mol/L, 0.45-0.5 mol/L.
In one embodiment, in step 2), the fat cryopreservation agent comprises glycerol in a volume fraction of 60% to 80%. Optionally, the glycerol has a volume fraction of 60% -65%, 65% -70%, 70% -75%, 75% -80%.
Further, the step 1) also comprises the step of carrying out high-pressure steam sterilization on the obtained trehalose solution.
Further, step 2) is performed under aseptic conditions.
The glycerol is medical sterile glycerol.
The method for freezing and storing the adipose tissues, provided by the invention, comprises the following steps of:
1) mixing the fat freezing protective agent and adipose tissues in a volume ratio of 1:1 to obtain a mixture 1;
2) placing the mixture 1 in a programmed cooling box, and carrying out programmed cooling at-80 ℃;
3) placing the mixture 1 obtained in the step 2) in liquid nitrogen for freezing storage to obtain frozen adipose tissues.
Further, the step 1) also comprises sealing the mixture 1.
In step 2), the programmed cooling time is at least 12 hours.
The invention provides a method for rewarming frozen adipose tissues, which comprises the following steps:
a. taking out the frozen adipose tissues from liquid nitrogen, and heating in a water bath;
b. eluting the adipose tissues obtained in the step a by using PBS buffer solution, and centrifuging;
c. and (4) taking the centrifuged upper-layer substance, eluting by using PBS buffer solution, and centrifuging to obtain the upper-layer substance, namely the rewarming fat tissue.
Further, in step a, the temperature of the water bath heating is 37 ℃.
Further, in step a, the heating time of the water bath is 2 minutes.
In one embodiment, in step b, the elution time is 3 minutes.
In one embodiment, in step b, centrifugation conditions are 500rpm for 3 minutes.
In one embodiment, in step c, the elution time is 3 minutes.
In one embodiment, in step c, centrifugation conditions are 500rpm for 3 minutes.
The method for freezing and preserving the adipose tissues or the method for rewarming the frozen adipose tissues do not relate to the purposes of diagnosis and treatment. Can be for health care purposes or for basic research purposes.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.