CN111449055A - Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method - Google Patents
Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention relates to an adipose tissue cryopreservation solution, a preparation method and an adipose tissue storage method, wherein the adipose tissue cryopreservation solution is prepared from the following raw materials in percentage by weight: 2-4% of trehalose, 8-15% of human serum albumin nanoparticles, 3-5% of phosphate buffer solution with the pH value of 6.5-7.2 and propylene glycol for complementing to 100%, wherein the human serum albumin nanoparticles are prepared by the following steps: dissolving 3-5 parts by weight of egg yolk lecithin in 40-50 parts by weight of absolute ethyl alcohol to obtain an emulsion; mixing 10-15 parts by weight of human serum albumin and 20-30 parts by weight of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution, adjusting pH to 4-4.5, stirring to solidify to obtain human serum albumin nanoparticles; the preserved adipose tissues have good biological activity and transplantation survival rate.
Description
Technical Field
The invention relates to the technical field of adipose tissue cryopreservation, in particular to an adipose tissue cryopreservation liquid, a preparation method and an adipose tissue storage method.
Background
Autologous fat transplantation filling is a common soft tissue filling method in clinic at present. The adipose tissues have the advantages of rich sources, simple operation, no rejection reaction and the like. Currently, fat transplantation has been widely used in the field of plastic cosmetology, such as: facial deformity, depressed scars, wrinkles, post-traumatic defects, depressed eyelids, depressed skin after liposuction, and the like are common techniques in the field of beauty or repair.
Although the survival rate of fat transplantation is obviously improved along with the deep research and the improvement of clinical technology, the largest problem at present is the necrotic absorption of fat tissue, most scholars think that the volume of the fat tissue after transplantation can only be maintained at about 50 percent, and the two points are that one, a small amount of repeated and multiple injections are needed, and the cell viability is kept to the maximum degree during transplantation.
Therefore, an appropriate adipose tissue in vitro storage technology is sought to achieve the purposes of one-time liposuction and multiple use, avoid repeated material taking, reduce the pain of patients and operators, and has important significance for clinical application.
In recent years, some researches on in vitro freezing storage and re-transplantation of fat have been carried out, and the observation of frozen adipose tissues shows that the cells can maintain the intact morphology and have no obvious difference from normal cells. After the frozen fat rewarming transplantation, a certain volume can be maintained, and the histology proves that the transplant has complete fat cells. However, it has been found that although transplantation or injection can be achieved only by freezing adipose tissues at a low temperature and then rewarming the tissues, the adipose tissues have a problem of volume reduction due to the absorption of substances, since substantially inactive cell substances are injected. In order to overcome the problem of inactivation of freezing damage of cells at low temperature, at present, an adipose tissue cryopreservation solution for performing cryoprotection on adipose tissues has appeared, so that the problem of cell inactivation can be improved.
Patent application publication No. CN 106719602a discloses a cryopreservation solution and a method for applying the same to cryopreservation of adipose tissues, the cryopreservation solution comprises: basal medium, NEAA, DMSO, propylene glycol and 1, 2-propylene glycol; the cryopreservation method comprises mixing the cryopreservation solution with adipose tissue, and soaking in liquid nitrogen. The prior art utilizes the freezing solution to freeze and store the adipose tissues, can protect the activity of the adipose tissues, and the cells in the adipose tissues still keep better biological activity after re-melting.
However, the above prior art has the following drawbacks: firstly, the frozen stock solution takes a basic culture solution as a matrix, and the components of the frozen stock solution are uncertain, so that certain immunogenicity may exist, and adverse reactions of later clinical patients are caused; secondly, about 20% of dimethyl sulfoxide is added into the frozen stock solution, so that the activity of adipose tissues and the survival rate after transplantation can be well preserved, but the dimethyl sulfoxide has cytotoxicity, and the toxic influence is greater after the addition amount exceeds 15%, so that the transplantation risk is improved; thirdly, due to the adoption of the vitrification cryopreservation technology of the one-step method, the fat tissue can be protected from freezing only by matching with the high-concentration cryopreservation liquid, but the high-concentration cryopreservation liquid can bring toxic damage to cells and devitrification ice crystal damage, thereby improving the transplantation risk.
Disclosure of Invention
The first purpose of the invention is to provide an adipose tissue freezing medium, and adipose tissues preserved by the adipose tissue freezing medium have good biological activity and transplantation survival rate.
The second purpose of the invention is to provide a preparation method of the adipose tissue frozen stock solution.
The third purpose of the invention is to provide an adipose tissue storage method, which adopts a two-step method for cooling and can realize rapid cooling and freezing storage under the protection of freezing storage liquid with lower concentration by matching with the freezing storage liquid.
The first purpose of the invention is realized by the following technical scheme:
an adipose tissue cryopreservation liquid is prepared from the following raw materials in percentage by weight: 2-4% of trehalose, 8-15% of human serum albumin nanoparticles, 3-5% of phosphate buffer solution with the pH value of 6.5-7.2 and propylene glycol for complementing to 100%, wherein the human serum albumin nanoparticles are prepared by the following steps: dissolving 3-5 parts by weight of egg yolk lecithin in 40-50 parts by weight of absolute ethyl alcohol to obtain an emulsion; mixing 10-15 parts by weight of human serum albumin and 20-30 parts by weight of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution, adjusting pH to 4-4.5, and stirring at 20000-.
By adopting the technical scheme, trehalose (trehalose), a non-reducing disaccharide widely existing in nature, has no toxic or side effect and very stable chemical properties, has the general characteristics of oligosaccharide and unique biological characteristics, and can protect the tissues and the functions and the activities of macromolecules of organisms under severe environment; 1) stabilizing the structure of biofilms and proteins; 2) protecting the organism from thermal damage; 3) protecting an organism from damage by oxidative stress; oxygen free radicals can damage amino acids in cellular proteins, causing damage to cells, and high concentrations of trehalose in cells can prevent such damage, suggesting that trehalose has the role of an oxygen free radical scavenger; 4) trehalose protects mammalian cells from hypoxia. The human serum albumin nano particles are obtained by compounding the impermeable protective agent human serum albumin and the permeable protective agent dimethyl sulfoxide, and after the nano particles are added into the adipose tissue frozen stock solution, the effective content of the dimethyl sulfoxide is far lower than the mixing amount in the prior art, so that the cytotoxicity of the frozen stock solution is reduced. Trehalose is dispersed among human serum albumin nanoparticles in an amorphous cluster form, plays a role in supporting and protecting, and can overcome the problem that the nanoparticles are easy to agglomerate, so that the trehalose and the human serum albumin nanoparticles are uniformly dispersed in frozen stock solution, and the fat tissue is more favorably protected from freezing.
The raw materials of the adipose tissue freezing solution have no cytotoxicity or have cytotoxicity lower than that of the prior art, and are safer and more reliable; compared with a basic culture solution, the components are determined and immunogenicity does not exist, so that the possibility of adverse reaction of a patient is reduced; the preserved adipose tissues have good activity and survival rate of transplantation.
Preferably, in the preparation of the human serum albumin nanoparticles, after the solidification is completed, the human serum albumin nanoparticles are washed by phosphate buffer with pH of 6.5-7.0.
By adopting the technical scheme, under the normal condition, the pH value of the surface skin of a human body is 5.0-7.0, and the prepared human serum albumin nano particles are subjected to acid-base regulation in advance by cleaning with a phosphate buffer solution, so that the human serum albumin nano particles are more favorably matched with the acid-base of adipose tissues after being applied to a frozen stock solution.
Preferably, in the preparation of the human serum albumin nano-particles, the adding speed is controlled to be 5g/min in the process of adding the emulsion into the protein liquid.
By adopting the technical scheme, the emulsion is added at a slow speed, so that the emulsion and the protein liquid are fully mixed in advance, and the emulsification effect is ensured.
The second purpose of the invention is realized by the following technical scheme:
a method for preparing the adipose tissue freezing medium comprises the following steps: weighing trehalose, human serum albumin nanoparticles and propylene glycol, and uniformly mixing; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue.
By adopting the technical scheme, the raw materials are mixed and uniformly stirred, and the buffer solution is added to adjust the pH of the frozen stock solution, so that the pH environment similar to the internal pH environment of a human body is kept in the storage process of adipose tissues, and the stable storage of the adipose tissues is facilitated.
Preferably, the ambient temperature in the preparation process is 18-24 ℃, and the humidity is 50% -60%.
The third purpose of the invention is realized by the following technical scheme:
a method for storing adipose tissue, comprising the following steps:
(1) preprocessing adipose tissues;
(2) mixing the adipose tissue cryopreservation solution of any one of claims 1 to 3 with adipose tissue according to the weight ratio of (2-3) to 1 to obtain material A;
(3) cooling the material A to-35 ℃ at the speed of 1-2 ℃/min, and standing; transferring into liquid nitrogen for freezing preservation.
By adopting the technical scheme, the fat tissue frozen stock solution is cooled by adopting a two-step method, and is matched with the fat tissue frozen stock solution, the fat tissue frozen stock solution is firstly cooled to-35 ℃ by adopting a slow cooling mode, and in the process, the extracellular solution of the fat tissue generates ice to ensure that intracellular water is seeped outwards and the concentration of the intracellular solution is gradually increased; then directly transferring the cell into liquid nitrogen (-196 ℃) to realize vitrification of the solution in the cell at a very high speed so as to enable the cell to reach a relatively stable vitrification state. Due to the wrapping effect of the trehalose and the human serum albumin nano particles in the frozen stock solution, the adipose tissues can be well protected when being transferred into liquid nitrogen after being slowly cooled, the chemical damage of the adipose tissues can be reduced, and the good activity of the adipose tissues after being rewarming can be kept.
Preferably, in the step (1), the adipose tissue pretreatment operation is specifically: and (3) centrifugally cleaning the lipoaspirate with normal saline, standing for layering, and discarding a liquid layer to obtain the adipose tissue.
Preferably, the adipose tissues obtained preliminarily are crushed to obtain adipose tissues with a particle size of not more than 1.5 mm.
By adopting the technical scheme, the adipose tissues are granules or blocks, the frozen stock solution is difficult to completely permeate into the tissue blocks when the adipose tissues are mixed with the frozen stock solution, so that cells or tissues in the central part of the adipose tissues are difficult to be well protected, and the activity of the cells after freezing is greatly influenced. The invention breaks the adipose tissues to disperse the adipose tissues into a plurality of smaller particles, which is more beneficial for the frozen stock solution to permeate into the tissues and improves the cell activity after the frozen stock.
Preferably, in the step (3), the standing time is 8-12 min.
Preferably, the rewarming operation of the adipose tissues is specifically as follows: stirring and thawing in water bath at 37 ℃; repeatedly washing with physiological saline.
In conclusion, the invention has the following beneficial effects:
(1) the fat tissue freezing medium is not added with a basic culture solution, has determined components, does not have immunogenicity, and has higher safety;
(2) the adipose tissue frozen stock solution is used for storing adipose tissues, the average survival rate of the adipose tissues after rewarming is more than 97 percent, and the highest survival rate can reach 99.91 percent and is close to the survival rate of fresh adipose tissues; the expression of the cell marker is consistent with the expression of fresh adipose tissue; the absorbance value after thawing for 15 days is still as high as 0.37, and is basically the same as the survival capability of fresh adipose tissues;
(3) by adopting the adipose tissue cryopreservation liquid and matching with the two-step method cooling program, the adipose tissue can be well protected when being transferred into liquid nitrogen after being slowly cooled, and the chemical injury of the adipose tissue can be reduced, so that the adipose tissue after being rewarming can keep good activity.
Detailed Description
The present invention will be further described with reference to the following specific examples.
The following raw materials in the invention are all commercial products, and specifically comprise: the trehalose is selected from Japanese forest essence, and the content of effective substances is 99%; the egg yolk lecithin is selected from Shandong Lusen Biotech limited, and the content of effective substances is 99%; the human serum albumin is selected from Yika biotechnology limited Shanghai, code A0121, and the purity is more than or equal to 99 percent; selecting anhydrous ethanol with chromatographic grade; the propylene glycol is selected from chromatographic grade.
Preparation example 1
The human serum albumin nano particle is prepared by the following steps:
dissolving 3g of egg yolk lecithin in 40g of absolute ethyl alcohol to obtain an emulsion; mixing 10g of human serum albumin and 20g of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution at a speed of 5g/min, continuously stirring in the adding process, and adding hydrochloric acid to adjust the pH to 4 after the adding is finished; homogenizing at high speed with a high speed homogenizer at a stirring speed of 20000r/min for 50min to solidify to obtain human serum albumin nanoparticles; washing and filtering the human serum albumin nano particles twice by using phosphate buffer solution with the pH value of 6.5-7.0 to finally obtain the human serum albumin nano particles.
Preparation example 2
The human serum albumin nano particle is prepared by the following steps:
dissolving 3g of egg yolk lecithin in 40g of absolute ethyl alcohol to obtain an emulsion; mixing 10g of human serum albumin and 20g of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution at a speed of 5g/min, continuously stirring in the adding process, and adding hydrochloric acid to adjust the pH to 4 after the adding is finished; homogenizing at high speed with a high speed homogenizer at a stirring speed of 25000r/min for 45min until solidification to obtain human serum albumin nanoparticles; washing and filtering the human serum albumin nano particles twice by using phosphate buffer solution with the pH value of 6.5-7.0 to finally obtain the human serum albumin nano particles.
Preparation example 3
The human serum albumin nano particle is prepared by the following steps:
dissolving 3g of egg yolk lecithin in 40g of absolute ethyl alcohol to obtain an emulsion; mixing 10g of human serum albumin and 20g of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution at a speed of 5g/min, continuously stirring in the adding process, and adding hydrochloric acid to adjust the pH to 4 after the adding is finished; homogenizing at high speed with a high speed homogenizer at a stirring speed of 30000r/min for 40min to solidify to obtain human serum albumin nanoparticles; washing and filtering the human serum albumin nano particles twice by using phosphate buffer solution with the pH value of 6.5-7.0 to finally obtain the human serum albumin nano particles.
Preparation example 4
The human serum albumin nano particle is prepared by the following steps:
taking 4g of egg yolk lecithin, and adding 45g of absolute ethyl alcohol for dissolving to obtain an emulsion; mixing 13g of human serum albumin with 25g of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution at a speed of 5g/min, continuously stirring in the adding process, and adding hydrochloric acid to adjust the pH to 4.5 after the adding is finished; homogenizing at high speed with a high speed homogenizer at a stirring speed of 25000r/min for 45min until solidification to obtain human serum albumin nanoparticles; washing and filtering the human serum albumin nano particles twice by using phosphate buffer solution with the pH value of 6.5-7.0 to finally obtain the human serum albumin nano particles.
Preparation example 5
The human serum albumin nano particle is prepared by the following steps:
dissolving 5g of egg yolk lecithin in 50g of absolute ethyl alcohol to obtain an emulsion; mixing 15g of human serum albumin with 30g of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution at a speed of 5g/min, continuously stirring in the adding process, and adding hydrochloric acid to adjust the pH to 4.5 after the adding is finished; homogenizing at high speed with a high speed homogenizer at a stirring speed of 25000r/min for 45min until solidification to obtain human serum albumin nanoparticles; washing and filtering the human serum albumin nano particles twice by using phosphate buffer solution with the pH value of 6.5-7.0 to finally obtain the human serum albumin nano particles.
Example 1
An adipose tissue cryopreservation solution prepared by the following operations:
according to the mixing amount shown in table 1, trehalose, the human serum albumin nanoparticles of preparation example 1 and propylene glycol are respectively weighed, mixed and stirred uniformly; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue; wherein the ambient temperature in the preparation process is 18 ℃ and the humidity is 54%.
Example 2
An adipose tissue cryopreservation solution prepared by the following operations:
according to the mixing amount shown in table 1, trehalose, the human serum albumin nanoparticles of preparation example 2 and propylene glycol are respectively weighed, mixed and stirred uniformly; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue; wherein the ambient temperature in the preparation process is 21 deg.C, and the humidity is 50%.
Example 3
An adipose tissue cryopreservation solution prepared by the following operations:
according to the mixing amount shown in table 1, trehalose, the human serum albumin nanoparticles of preparation example 3 and propylene glycol are respectively weighed, mixed and stirred uniformly; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue; wherein the ambient temperature in the preparation process is 24 deg.C, and the humidity is 57%.
Example 4
An adipose tissue cryopreservation solution prepared by the following operations:
according to the mixing amount shown in table 1, trehalose, the human serum albumin nanoparticles of preparation example 4 and propylene glycol are respectively weighed, mixed and stirred uniformly; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue; wherein the ambient temperature in the preparation process is 20 deg.C, and the humidity is 60%.
Example 5
An adipose tissue cryopreservation solution prepared by the following operations:
according to the mixing amount shown in table 1, trehalose, the human serum albumin nanoparticles of preparation example 5 and propylene glycol are respectively weighed, mixed and stirred uniformly; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue; wherein the ambient temperature in the preparation process is 23 deg.C, and the humidity is 58%.
Examples 6 to 16
The procedures of preparation and operation, ambient temperature and humidity of the frozen adipose tissue stocks of examples 6 to 16 were the same as those of example 4, and the types of the respective raw materials of the frozen adipose tissue stocks were the same, except that the amounts of the respective raw materials of the frozen adipose tissue stocks were different, as shown in Table 1.
TABLE 1 blending amounts (unit: g) of respective raw materials in examples 1 to 16
Comparative example 1
Comparative example 1 is the frozen stock solution of example 3 of the patent application with application publication No. CN 106719602 a.
Comparative example 2
The frozen stock solution of comparative example 2 is different from that of example 1 in that it is prepared by mixing and stirring the following raw materials: trehalose 2%, human serum albumin 8%, phosphate buffer solution with pH 6.5-7.0 3%, propylene glycol make up to 100%.
Comparative example 3
The frozen stock solution of comparative example 3 is different from that of example 1 in that the raw material human serum albumin nanoparticles are prepared by directly adding the emulsion to the protein solution at one time, and the rest is the same as that of example 1.
Control group
The same amount of physiological saline as in example 1.
Application example 1
The adipose tissue frozen stock solution is applied to storage of adipose tissues, and specifically comprises the following operation steps:
(1) pretreatment of adipose tissues: centrifuging and cleaning lipoaspirate with normal saline at 800r/min, standing at 5 deg.C for 10min, layering, and discarding the liquid layer after the adipose tissue and swelling liquid are layered to obtain adipose tissue; cutting and crushing the primarily obtained adipose tissues by using a nano-adipose converter with the aperture of 2mm, and then performing secondary crushing by using a nano-adipose converter with the aperture of 1.5mm to finally obtain adipose tissues with the particle size of not more than 1.5 mm;
(2) mixing the adipose tissue frozen stock solution of example 1 with the adipose tissues according to the weight ratio of 2:1 to obtain a material A;
(3) quickly adding the material A into a freezing tube, placing the tube in a program-controlled rate freezer, cooling the material A to-35 ℃ at the speed of 1 ℃/min, and standing for 8 min; transferring into liquid nitrogen for freezing preservation.
Application example 2
The adipose tissue storage method of application example 2 is different from that of application example 1 in that:
(3) quickly adding the material A into a freezing tube, placing the tube in a program-controlled rate freezer, cooling the material A to-35 ℃ at the speed of 2 ℃/min, and standing for 12 min; transferring into liquid nitrogen for freezing and storing; the rest of the process was the same as in application example 1.
Application example 3
The adipose tissue storage method of application example 3 differs from application example 1 in that:
(3) quickly adding the material A into a freezing tube, placing the tube in a program-controlled rate freezer, cooling the material A to-35 ℃ at the speed of 2 ℃/min, and standing for 10 min; transferring into liquid nitrogen for freezing and storing; the rest of the process was the same as in application example 1.
Application example 4
The adipose tissue storage method of application example 4 differs from that of application example 3 in that:
(2) mixing the adipose tissue frozen stock solution of example 1 with the adipose tissues according to the weight ratio of 2.5:1 to obtain a material A; the rest of the process was the same as in application example 3.
Application example 5
The adipose tissue storage method of application example 5 differs from application example 3 in that:
(2) mixing the adipose tissue frozen stock solution of example 1 with the adipose tissues according to the weight ratio of 3:1 to obtain a material A; the rest of the process was the same as in application example 3.
Application examples 6 to 20
The adipose tissue storage methods of application examples 6 to 20 are different from application example 4 in that: respectively mixing the adipose tissue frozen stock solution of the embodiments 2-16 and adipose tissues according to the weight ratio of 2.5:1 in the step (2) to obtain a material A; the rest of the process was the same as in application example 4.
In the invention, the specific operations of rewarming and thawing the fat tissue after freezing storage are as follows: taking out the frozen tube, immediately placing the tube in a water bath at 37 ℃, and stirring and thawing; and in the thawing process, absorbing and removing the frozen stock solution in time, and repeatedly cleaning with normal saline for 3 times to obtain the final product.
Application of comparative examples 1 to 3
The adipose tissue storage method using comparative examples 1-3 was different from application example 4 in that: respectively mixing the adipose tissue frozen stock solution of the comparative examples 1-3 and the adipose tissue according to the weight ratio of 2.5:1 in the step (2) to obtain a material A; the rest of the process was the same as in application example 4.
Application comparative example 4
The adipose tissue storage method using comparative example 4 was different from application example 4 in that: (1) pretreatment of adipose tissues: centrifuging and cleaning lipoaspirate with normal saline, standing at 5 deg.C for 10min, layering, discarding the liquid layer after the adipose tissue and swelling liquid are layered to obtain adipose tissue without crushing adipose tissue; the rest of the process was the same as in application example 4.
Comparative application example 5
The adipose tissue storage method using comparative example 5 was different from application example 4 in that: (3) quickly adding the material A into a freezing storage tube, placing the tube in a program-controlled rate freezer, and cooling the material A to-80 ℃ at the speed of 2 ℃/min for freezing storage; the rest of the process was the same as in application example 4.
Comparative application example 6
The adipose tissue storage method using comparative example 6 was different from application example 4 in that: (3) quickly adding the material A into a freezing tube, and directly placing the material A into liquid nitrogen for freezing and storing; the rest of the process was the same as in application example 4.
Blank control group
The fat storage method of application example 4 was used, and the frozen stock solution of application example 4 was replaced with physiological saline solution of the control group, and the rest was the same as in application example 4.
Performance detection
Preparation of fresh adipose tissue: centrifuging and cleaning lipoaspirate with normal saline at 800r/min, standing at 5 deg.C for 10min, layering, and discarding the liquid layer after the adipose tissue and swelling liquid are layered to obtain adipose tissue; cutting and crushing the primarily obtained adipose tissues by using a nano fat converter with the aperture of 2mm, and then performing secondary crushing by using a nano fat converter with the aperture of 1.5mm to finally obtain fresh adipose tissues with the particle size of not more than 1.5 mm.
The method for thawing the adipose tissues frozen and stored for 3 months respectively comprises the following steps of thawing the adipose tissues in application examples 1-20, application comparative examples 1-6 and a blank control group, and detecting the adipose tissues and the fresh adipose tissues after the thawing by the rewarming method respectively as follows:
trypan blue staining for cell viability: the specific operation requirements are referred to the 'viability influence of frozen fat particle tissues under different protective agents', the gangen sensitivity, the xiehong torch, the 2014 Master graduation paper of southern university, page 10; the detection results are shown in Table 2;
biological activity detection of cells, namely, cleaning each group of adipose tissues for 3 times after digestion to prepare cell suspension, adding an antibody, and detecting and analyzing the expression conditions of CD73, CD90, CD105, CD11b, CD19, CD34, CD45 and H L A-DR by a flow cytometer, wherein the detection results are detailed in Table 3;
MTT assay to determine the viability of cells in vitro: the specific operation requirements are referred to the vitality influence of frozen fat particle tissues under different protective agents, the treatise on the seal, the Xiehong torch, the university of south China 2014 Master, pages 9-10; the results are shown in Table 4.
TABLE 2 cell viability of different adipose tissues (unit/%)
TABLE 3 biological Activity of different adipose tissues (Unit/%)
TABLE 4 viability of different adipose tissues (absorbance A)
The detection results in table 2 show that after the adipose tissues are frozen for 3 months by using the freezing solution and the storage method of the invention, the cell survival rate of the adipose tissues is more than 97% on average, and can reach 99.91% at most, which is close to the cell survival rate of fresh adipose tissues and is far higher than that of the prior art (application comparative example 1); while the survival rate of cells in adipose tissues stored in physiological saline was very low. The detection result of the comparative example 2 shows that the human serum albumin nano particles in the frozen stock solution have great influence on the frozen stock solution; after the replacement of the normal human serum albumin, the survival rate of the adipose tissues is reduced from 96.11% to 63.54% after rewarming. According to the detection result of the comparative example 3, in the preparation of the human serum albumin nano-particle, the emulsion is added once, so that the freezing effect of the frozen stock solution is greatly influenced, and the survival rate of adipose tissues after rewarming is reduced to 70.71% from 96.11%. According to the detection result of the comparative example 4, when the adipose tissues are pretreated, the freezing effect can be greatly influenced without crushing the adipose tissues, and the survival rate of the adipose tissues after rewarming is reduced from 96.11% to 66.80%. By applying the detection results of the comparative examples 5 to 6, the cooling procedure in the adipose tissue storage method has a great influence on the cryopreservation effect; the two-step cooling is adjusted into one-step slow cooling and one-step vitrification cooling, and the survival rate of adipose tissues after rewarming is greatly reduced.
The results of Table 3 show that the expression conditions of the cell markers of comparative example 1 are consistent, negative cell markers (hematopoietic cell markers CD34, CD45, CD11, H L A-DR and CD19) are not expressed, and positive cell markers (CD90, CD73 and CD105) are highly expressed and are consistent with the expression conditions of fresh adipose tissues, so that the adipose tissues obtained by the invention well maintain the original activity, and the obtained adipose stem cells are not different from the fresh adipose tissues.
The detection results in table 4 show that when the fat tissue is frozen by using the freezing solution and the storage method of the invention, the thawed fat tissue still has good survival ability, the absorbance value after 15 days is still as high as 0.37, and the survival ability of the thawed fat tissue is basically the same as that of the fresh fat tissue, which is far superior to that of the fat tissue only using physiological saline (blank control group) and the fat tissue stored in the prior art (application comparative example 1). The results of the application comparative examples 2-3 show that the human blood albumin nano-particles in the frozen stock solution and the preparation method thereof have great influence on the frozen stock solution, thereby influencing the survival capability of the fat stem cells after thawing and reducing the survival capability. The results of the application comparative examples 4 to 6 show that the control of the pretreatment and cooling procedures of the adipose tissues in the adipose tissue storage method of the present invention has a great influence on the survival ability of the thawed adipose-derived stem cells.
The above-mentioned embodiments are merely illustrative and not restrictive, and those skilled in the art can modify the embodiments without inventive contribution as required after reading this specification, but only fall within the scope of the claims of the present invention.
Claims (10)
1. The fat tissue freezing solution is characterized by being prepared from the following raw materials in percentage by weight: 2-4% of trehalose, 8-15% of human serum albumin nanoparticles, 3-5% of phosphate buffer solution with the pH value of 6.5-7.0, and the balance of propylene glycol to 100%, wherein the human serum albumin nanoparticles are prepared by the following steps: dissolving 3-5 parts by weight of egg yolk lecithin in 40-50 parts by weight of absolute ethyl alcohol to obtain an emulsion; mixing 10-15 parts by weight of human serum albumin and 20-30 parts by weight of dimethyl sulfoxide, and uniformly stirring to obtain protein liquid; adding the emulsion into the protein solution, adjusting pH to 4-4.5, and stirring at 20000-.
2. The adipose tissue cryopreservation liquid of claim 1, wherein: in the preparation of the human serum albumin nano particles, after solidification, the human serum albumin nano particles are washed by phosphate buffer solution with pH of 6.5-7.0.
3. The adipose tissue cryopreservation liquid of claim 1, wherein: in the preparation of the human serum albumin nano-particles, the adding speed is controlled to be 5g/min in the process of adding the emulsion into the protein liquid.
4. A method for preparing an adipose tissue frozen stock solution according to any of the claims 1 to 3, characterized in that it comprises the following operative steps: weighing trehalose, human serum albumin nanoparticles and propylene glycol, and uniformly mixing; adding phosphate buffer solution with pH of 6.5-7.0, and stirring to obtain frozen stock solution of adipose tissue.
5. The method for preparing an adipose tissue frozen stock solution according to claim 4, wherein: the environmental temperature in the preparation process is 18-24 ℃, and the humidity is 50% -60%.
6. An adipose tissue storage method, characterized in that it comprises the following operating steps:
(1) preprocessing adipose tissues;
(2) mixing the adipose tissue cryopreservation solution of any one of claims 1 to 3 with adipose tissue according to the weight ratio of (2-3) to 1 to obtain material A;
(3) cooling the material A to-35 ℃ at the speed of 1-2 ℃/min, and standing; transferring into liquid nitrogen for freezing preservation.
7. The method for storing adipose tissue according to claim 6, wherein: in the step (1), the adipose tissue pretreatment operation specifically comprises: and (3) centrifugally cleaning the lipoaspirate with normal saline, standing for layering, and discarding a liquid layer to obtain the adipose tissue.
8. The method for storing adipose tissue according to claim 7, wherein: and (3) crushing the preliminarily obtained adipose tissues to obtain the adipose tissues with the particle size of not more than 1.5 mm.
9. The method for storing adipose tissue according to claim 6, wherein: in the step (3), the standing time is 8-12 min.
10. The method for storing adipose tissue according to claim 6, wherein: the rewarming operation of the adipose tissues comprises the following specific steps: stirring and thawing in water bath at 37 ℃; repeatedly washing with physiological saline.
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