CN112155008A - Cryopreservation and recovery method of adipose tissues and adipose tissue cryopreservation recovery kit - Google Patents
Cryopreservation and recovery method of adipose tissues and adipose tissue cryopreservation recovery kit Download PDFInfo
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Abstract
The invention discloses a cryopreservation method of adipose tissues, which comprises the following steps: (1) pretreatment of adipose tissue; (2) adding a reagent for freezing and storing adipose tissues at a first stage, and centrifuging and washing; adding the adipose tissue freezing and storing two-stage reagent, and carrying out programmed cooling. The invention also discloses a recovery method of the cryopreserved adipose tissues, which comprises the following steps: and taking out the frozen adipose tissues from the liquid nitrogen, thawing, adding adipose tissue recovery liquid, and centrifugally washing to finish recovery. The invention also discloses an adipose tissue cryopreservation resuscitation kit, which comprises the adipose tissue cryopreservation first-stage reagent, the adipose tissue cryopreservation second-stage reagent and the adipose tissue resuscitation solution. The adipose tissues cryopreserved by the cryopreservation method can keep cell activity for a long time, the adipose stem cells extracted after recovery can express various stem cell markers, can be differentiated into adipose cells, bone cells and cartilage cells, have good stability, and greatly improve the utilization rate of the adipose tissues.
Description
Technical Field
The invention relates to a cryopreservation and recovery method of adipose tissues and an adipose tissue cryopreservation recovery kit, belonging to the field of cell biology.
Background
The autologous fat transplantation technology is widely applied in the field of plastic surgery, and as an ideal transplantation filler, fat has the advantages of abundant sources, convenient material acquisition, low cost, no immune rejection phenomenon and the like, but the problem of high absorption rate exists after fat transplantation, so that satisfactory postoperative effect cannot be achieved by only one-time filling operation. Although many clinicians and researchers have studied and improved the processes of obtaining adipose tissues, washing and purifying methods, injection techniques, etc., in recent years, the fat retention rate after transplantation is still not satisfactory and remains at a level of about 50%. When in clinical use, the expected effect can be achieved only by multiple times of liposuction, material drawing, injection and filling. Repeated surgical liposuction not only reduces the working efficiency of doctors, but also brings pain to patients and improves the incidence rate of complications. Obtaining a large quantity of fat at one time for cryopreservation for multiple treatments has great advantages in saving time and energy costs and reducing clinical complications.
Adipose-derived mesenchymal stem cells (ADSCs) gradually become important contents of stem cell storage and research hotspots of tissue engineering and regenerative medicine in recent years, have great application potential, and a great deal of research proves that: the ADSCs can proliferate and differentiate to form cells lacking damage, and the differentiation result proves that the ADSCs can be induced to differentiate into various cells, including fat cells, bone cells, cartilage cells, liver cells, nerve cells and the like.
Research proves that the survival rate of adipose tissues can be effectively improved by using a mixed transplantation mode of adipose mesenchymal stem cells extracted from the adipose tissues and the adipose tissues, and a better shaping effect is achieved. In order to avoid fat extraction and material drawing in multiple operations and realize the purposes of one-time obtaining and multiple treatments, it is important to explore a method for preserving autologous adipose tissues in vitro for a long time.
The deep low temperature freezing storage is the most common method for storing human tissues and cells, and the frozen adipose tissues can be used for extracting adipose mesenchymal stem cells.
At present, a uniform standardized scheme is not formed in the low-temperature preservation method of adipose tissues, the defects that adipose tissues are necrosed, the number of extracted mesenchymal stem cells is insufficient, and the activity is not strong can be caused when the extracted adipose tissues are not processed and are simply subjected to cryopreservation, the biological activity of cryopreserved fat and the safety of clinical application are not proved all the time, and how to preserve a large amount of obtained adipose tissues and keep the cell activity is a practical problem which needs to be solved urgently at present.
Disclosure of Invention
Aiming at the prior art, the invention provides an adipose tissue cryopreservation method and an adipose tissue resuscitation method, provides an adipose tissue cryopreservation solution and an adipose tissue resuscitation solution, and provides an adipose tissue cryopreservation and resuscitation kit. The invention establishes perfect sample collection specifications, establishes a standard adipose tissue cryopreservation process, establishes a cell recovery culture process at the same time, forms a set of complete adipose tissue cryopreservation recovery kit, can preserve adipose tissues treated by the kit for a long time after ultralow temperature freezing, and can keep stem cell related characteristics of adipose mesenchymal stem cells extracted after adipose tissue recovery.
The invention is realized by the following technical scheme:
a cryopreservation method of adipose tissues comprises the following steps:
(1) pretreatment of adipose tissue: centrifuging the collected fat specimen to remove the upper layer of grease, the lower layer of water layer, reserving the middle layer of adipose tissue, and washing the middle layer of adipose tissue with normal saline for 1-3 times to obtain washed adipose tissue;
(2) adding an adipose tissue cryopreservation first-stage reagent into the adipose tissue, and centrifuging and washing; centrifuging, removing washing solution, adding the adipose tissue cryopreservation two-stage reagent into the rest adipose tissue, mixing, pre-cooling at 4 deg.C for 15min, and transferring to a program cooling instrument for program cooling; after the programmed cooling is finished, transferring the frozen fat cells to a liquid nitrogen storage tank at the temperature of-196 ℃ for storage;
the program cooling procedure is as follows:
keeping the temperature at 4 ℃ for 5min, and cooling to 0 ℃ at 5 ℃/min;
keeping at 0 deg.C for 5min, and cooling to-15 deg.C at 2 deg.C/min;
keeping at-15 deg.C for 5min, and cooling to-65 deg.C at 1 deg.C/min;
keeping the temperature at minus 65 ℃ for 5min, and cooling to minus 80 ℃ at the temperature of 0.5 ℃/min.
The adipose tissue cryopreservation first-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide (DMSO) and 5-8% of trehalose.
The adipose tissue cryopreservation two-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide (DMSO), 3-8% of trehalose, 2-4% of propylene glycol, 0.5-1% of glutathione and 0.08-0.12% of sodium pyruvate.
In the step (1), the fat specimen is subcutaneous fat collected from thighs and abdomen under umbilicus of a person to be collected (with unlimited nature, preferably below 35 years old), the fat tissue content of the parts is rich, and the fat particles are fine, uniform and pure; during collection, a person to be collected is fixed on an operating table in a prone position, the person is disinfected and paved singly in a conventional way, subcutaneous fat is extracted by using a liposuction device such as a liposuction needle and the like according to requirements, the volume of a collected fat sample is 100-200 ml, and the sample is a single sample and cannot be a mixed sample; immediately after collection, the cells were placed in sterile collection bags (to avoid contamination due to prolonged exposure to the external environment) and stored at 4 ℃ for use.
Furthermore, the detected result of the collected person is qualified by HBsAg, Anti-HCV-IgG, Anti-HIVI/II-IgG, Anti-CMV-IgM, TP, Elisa, EBV and the like.
Further, the specific collection method of the fat specimen comprises the following steps: preparing 100ml of swelling solution ringer's solution, 30ml of 2% lidocaine injection and 1ml of epinephrine injection by using a conventional disinfection drape after marking according to preoperative design; after the swelling anesthesia of the pre-liposuction supply area of the patient is successful, a multi-side-hole liposuction needle with a side hole of 1mm in diameter and a needle tube of 3mm in diameter is connected with a 20ml syringe for liposuction; before liposuction, the injector is firstly pumped back by 10ml (at the moment, the negative pressure value in the injector is about-60 kpa); the fat cell obtained by low negative pressure liposuction is more in quantity and good in activity, and the obtained fat suction material is placed in a sterile syringe and is quickly transferred to a laboratory in a low-temperature environment.
Further, the specific operation mode of the step (1) is as follows: taking the collected fat sample, subpackaging and transferring the sample into a 50ml centrifuge tube, and centrifuging (800g, 8 min); sucking off the upper layer of grease, transferring the middle layer of adipose tissue into a new centrifuge tube, adding physiological saline with the same volume, centrifuging (800g for 8min), and discarding the supernatant; the saline washing of the adipose tissue was repeated twice.
Further, the step (1) is carried out in a biological safety cabinet, laminar flow is opened before subpackaging and transferring, an ultraviolet lamp of the biological safety cabinet is opened, experimental consumables are irradiated for 30min at the same time, and then follow-up operation is carried out.
Further, in the step (2), the volume ratio of the adipose tissues to the adipose tissue cryopreservation one-stage reagent is 1: 1.
Further, in the step (2), the volume ratio of the adipose tissues to the adipose tissue cryopreservation two-stage reagent is 1: 1.
Further, the specific operation mode of the step (2) is as follows: adding equal volume of adipose tissue freezing first stage reagent into the adipose tissue, centrifuging (1500rpm, 5min), and washing; centrifuging, removing washing liquid, stirring the rest adipose tissue with glass rod, subpackaging into 5ml freezing tubes, adding adipose tissue freezing two-stage reagent 2.5ml per tube, mixing in vortex oscillator, transferring into 4 deg.C refrigerator, pre-cooling for 15min, and transferring into program cooling instrument for program cooling.
A method for resuscitating cryopreserved adipose tissue comprising: taking out the fat tissue frozen by the freezing method from the liquid nitrogen, and melting; adding adipose tissue recovery liquid into the melted liquid, and centrifuging and washing; after centrifugation, the washing liquid is removed, recovery is completed, and the fat sediment can be subjected to primary culture.
The adipose tissue resuscitation solution consists of the following components in percentage by weight: 75-85% of DMEM medium, 15-25% of dextran, 1-2% of L-glutamine and 1-2% of D-glucose.
Furthermore, the recovery of adipose tissues is carried out in a biological safety cabinet, laminar flow is opened before operation, an ultraviolet lamp of the biological safety cabinet is opened, experimental consumables are irradiated for 30min at the same time, and then follow-up operation is carried out.
Further, the resuscitation method specifically comprises the following steps: taking out the fat tissue from the liquid nitrogen, and melting in a water bath at 37 deg.C (preheated to 37 deg.C in advance); adding adipose tissue resuscitation agent into the thawed (after about 1 min), and centrifuging and washing (1500rpm, 5 min); and removing the washing liquid after centrifugation to obtain the fat sediment.
Further, the method for performing primary culture comprises: adding equal volume of digestive juice and equal volume of type I collagenase solution (concentration is 0.2%) into the fat sediment, mixing well, sealing, placing into a 37 ℃ shaking table, and digesting for 1 hour at 150 r/min; after digestion, centrifugation (800g, 10 min), removal of upper fat, resuspension of the cell pellet in DMEM/F12 medium (DMEM medium mixed with F12 medium at a volume ratio of 1: 1), centrifugation (800g, 10 min); discarding supernatant, resuspending the cell pellet with DMEM/F12 medium, filtering with 100 μm sieve to obtain adipocytes, centrifuging (800g, 10 min), discarding supernatant, resuspending adipocytes with complete medium, inoculating into culture flask to ensure the concentration of inoculated cells to be 2.5 × 104cells/ml, placed at 37 ℃ in CO2Culturing in an incubator, changing the cell culture solution after 24 hours, and removing non-adherent cells; the solution was changed every three days later. The cell growth density reaches about 90% after the culture is about the fifth day, and the cell can be used for subculture.
An adipose tissue cryopreservation resuscitation kit comprises the adipose tissue cryopreservation first-stage reagent, the adipose tissue cryopreservation second-stage reagent and an adipose tissue resuscitation solution. Further, the specification is as follows: freezing and storing the adipose tissues in one stage, wherein the volume of the reagent is 500 ml/bottle; freezing and storing the adipose tissues in a first bottle of 200 ml/bottle of the two-stage reagent; one bottle of adipose tissue recovery liquid, 200 ml/bottle; during practical application, the freezing kit is taken according to experimental needs, and theoretically one set of the freezing kit can be used for freezing 500ml of adipose tissues.
The technical scheme of the invention is summarized to form a freezing and thawing kit for preserving stem cells in a manner of freezing adipose tissues, which mainly comprises: the adipose tissue frozen stock solution is used for freezing and storing adipose tissues, the frozen and stored adipose tissues are used for reviving, the mesenchymal stem cells can be prepared and extracted, the cultured cells have high amplification efficiency and normal cell morphology, and the flow type identification result accords with the relevant characteristics of the adipose mesenchymal stem cells. And after the adipose tissues are frozen and restored, observing the cell extraction efficiency of the adipose mesenchymal stem cells extracted after the frozen and restored adipose mesenchymal stem cells, the proliferation and differentiation capacity of the extracted mesenchymal stem cells and the expression of stem cell markers. The obtained adipose-derived stem cells grow in an adherent manner, the adipose-derived mesenchymal stem cells can be stably subcultured and proliferated within 5 generations, the doubling time between the generations is about 3 days, adipose-derived stem cell markers such as CD29, CD44 and CD105 are expressed, and CD34, CD45 and HLA-DR are not expressed.
The cryopreservation method for adipose tissues can be used for cryopreservation of a large number of adipose tissues in batches, so that adipose tissue resources can be efficiently utilized, and the procedures of digestion, inoculation, culture and the like of the adipose tissues are not required during cryopreservation, so that a large amount of time and economic cost are saved. The frozen adipose tissues can be preserved for a long time in a large amount. The frozen adipose tissues can be revived and digested as required to obtain required cells, and experiments prove that the frozen adipose tissues can keep the cell activity for a long time, and the adipose stem cells extracted after revival can express various stem cell markers and can be differentiated to adipose, bone and cartilage cells, so that the method has very good stability and greatly improves the utilization rate of the adipose tissues.
The invention has the following beneficial effects:
the invention confirms an adipose tissue cryopreservation and recovery kit which can perform deep low temperature cryopreservation on fresh adipose tissues extracted from a human body to ensure the activity of the adipose tissues, fat mesenchymal stem cells can be stably extracted from the recovered frozen adipose tissues, the fat mesenchymal stem cells can efficiently express adipose stem cell markers such as CD29, CD44, CD105 and the like, and do not express CD34, CD45 and HLA-DR.
The cryopreservation method can be used for cryopreserving a large amount of adipose tissues in batches, the activity of the cryopreserved adipose tissues is kept for a long time, only the adipose tissues are cryopreserved, revived and extracted when the fat is used, and the human body fat is not required to be extracted for multiple times, so that the pain of a patient is relieved.
Before the adipose tissues are frozen, sample treatment is carried out to remove redundant tumescent liquid, thick connective tissues and blood mixtures in the adipose tissues, redundant transparent grease is removed by centrifugation, finally, the adipose tissue particles in the middle layer are kept for next-step experiments, and the utilization efficiency of the adipose tissues is improved.
Before the adipose tissues are frozen, the adipose tissues are rinsed by using an adipose tissue freezing first-stage reagent, redundant water in the adipose tissues is diluted and cleaned, the formation of ice crystals inside and outside cells during freezing is reduced, the damage to the cells during freezing is reduced, and then the adipose tissues are frozen and stored by using an adipose tissue freezing second-stage reagent, so that the cell survival rate is improved.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.
Drawings
FIG. 1: the invention relates to a freezing method and a resuscitating method of adipose tissue, and a process flow chart thereof.
FIG. 2: adipose-derived mesenchymal stem cells (100X) cultured for 7 days were inoculated by freeze-stored adipose resuscitation digestion.
FIG. 3: passage P02 adipose-derived mesenchymal stem cells (100 ×).
FIG. 4: schematic diagram of detection result of flow cytometry.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The various liquids used in the present invention are illustrated below:
complete culture medium of adipose-derived mesenchymal stem cells: the commercial adipose tissue-derived mesenchymal stem cell culture medium is purchased, and the adipose tissue-derived mesenchymal stem cell culture medium mentioned herein is purchased from Beijing Yingfeng Dingcheng bioengineering technology Co., Ltd, Catharan No. 16070007.
Adipose tissue digestive juice: consists of collagenase type I, pancreatin and DMEM/F12. Wherein the collagenase concentration is 0.1-0.3g/100ml, such as 0.120g/100ml, and the pancreatin concentration is 0.125-0.25%.
Fat stem cell digestive juice: consists of trypsin at a concentration of 0.25-0.3g/100ml, e.g. 0.27g/100 ml.
The adipose-derived mesenchymal stem cell culture medium is a common adipose-derived mesenchymal stem cell culture medium and can be purchased from commercial companies, and the adipose-derived mesenchymal stem cell culture medium mentioned in the text is purchased from Beijing Yifengdingding Chengcheng biotechnology Limited company with the product number of 16070007.
Collagenase, a reagent for hydrolyzing collagen components in connective tissues, is commercially available from commercial companies, and is type I collagenase, available from SIGMA, having a commercial designation of C0130, DMEM/F12, a basic medium commonly used in cell culture, and DMEM/F12, available from Gibco, having a commercial designation of: c11330500 BT.
PBS is a common reagent in cell culture, purchased from Hyclone, cat #: SH 30256.01.
Dextran is a blood volume expander, has the functions of increasing the plasma colloid osmotic pressure, increasing the plasma volume and maintaining the blood pressure, is a substitute of the plasma, and is usually used for replacing the plasma in vitro.
DMSO is a permeability protectant that lowers the cell freezing point, reduces ice crystal formation, mitigates free radical damage to cells, alters the permeability of biological membranes to electrolytes, drugs, poisons and metabolites, and as used herein DMSO is purchased from WAK, cat #: WAK-DMSO-70.
DMEM is a basic medium commonly used in laboratories, and is manufactured by Hyclone under the trade name SH 30022.01.
DMEM/F12 is a basic medium commonly used in laboratories, and DMEM/F12 used herein is available from Hyclone, cat #: SH30023.01B are provided.
L-glutamine is a common reagent in cell culture, glutamic acid generated after decomposition is a necessary nutrient for cell growth, and the L-glutamine is purchased from Gibco company, and the cargo number is as follows: SH 30034.01.
The various consumables used in the present invention are described below: both 10ml pipettes and 50ml centrifuge tubes were purchased from Corning, 10ml pipettes having a product number of 4488 and 50ml centrifuge tubes having a product number of 430828.
Example 1 cryopreservation and Resuscitation of adipose tissue
The cryopreservation method of the adipose tissues comprises the following steps:
(1) pretreatment of adipose tissue:
firstly, the collection requirement of a fat sample is as follows: detecting the virus of the collected person: the detection results of (HBsAg), (Anti-HCV-IgG), (Anti-HIVI/II-IgG), (Anti-CMV-IgM), (TP, Elisa) and (EBV) are qualified;
a sample collection method: preparing 100ml of swelling solution ringer's solution, 30ml of 2% lidocaine injection and 1ml of epinephrine injection by using a conventional disinfection drape after marking according to preoperative design; after the swelling anesthesia of the pre-liposuction supply area of the patient is successful, a multi-side-hole liposuction needle with a side hole of 1mm in diameter and a needle tube of 3mm in diameter is connected with a 20ml syringe for liposuction; before liposuction, the injector is firstly pumped back by 10ml (at the moment, the negative pressure value in the injector is about-60 kpa); the low negative pressure liposuction is higher than the negative pressure liposuction, the obtained fat cells are more in quantity and good in activity, and the obtained fat suction substance is placed in a sterile injector and is quickly transferred to a laboratory in a low-temperature environment;
processing adipose tissues: storing the fat specimen extracted by the liposuction operation at a low temperature (4 ℃ in an ice box), and transporting to a laboratory within 22 h;
opening laminar flow, opening an ultraviolet lamp of the biological safety cabinet, and simultaneously irradiating experimental consumables for 30 min; transferring the adipose tissues into a 50ml centrifuge tube, and centrifuging at 800g for 8 min; absorbing the upper layer of grease from the centrifuged adipose tissue, transferring the middle layer of adipose tissue to a new centrifugal tube, adding the same volume of normal saline, centrifuging (800g for 8min), and discarding the supernatant; the saline washing of the adipose tissue was repeated twice.
(2) Adding equal volume of adipose tissue freezing first stage reagent into the adipose tissue, centrifuging (1500rpm, 5min), and washing; centrifuging, removing washing liquid, stirring the rest adipose tissues with glass rod, subpackaging into 5ml freezing tubes, adding adipose tissue freezing two-stage reagent 2.5ml per tube, mixing in vortex oscillator, transferring into 4 deg.C refrigerator, pre-cooling for 15min, and transferring into program cooling instrument for program cooling; after the programmed cooling is finished, transferring the frozen fat cells to a liquid nitrogen storage tank at the temperature of-196 ℃ for storage;
the program cooling procedure is as follows:
keeping the temperature at 4 ℃ for 5min, and cooling to 0 ℃ at 5 ℃/min;
keeping at 0 deg.C for 5min, and cooling to-15 deg.C at 2 deg.C/min;
keeping at-15 deg.C for 5min, and cooling to-65 deg.C at 1 deg.C/min;
keeping the temperature at minus 65 ℃ for 5min, and cooling to minus 80 ℃ at the temperature of 0.5 ℃/min.
The adipose tissue cryopreservation first-stage reagent consists of the following components in percentage by weight: 78% of DMEM medium, 15% of dimethyl sulfoxide (DMSO) and 7% of trehalose.
The adipose tissue cryopreservation two-stage reagent consists of the following components in percentage by weight: 78% of DMEM culture medium, 12% of dimethyl sulfoxide (DMSO), 6.1% of trehalose, 3% of propylene glycol, 0.8% of glutathione and 0.1% of sodium pyruvate.
The method for recovering the frozen adipose tissues comprises the following steps: opening laminar flow, and opening ultraviolet lamp of biological safety cabinet to irradiate for 30 min; taking out the fat tissue from the liquid nitrogen, and melting in a water bath at 37 deg.C (preheated to 37 deg.C in advance); adding adipose tissue resuscitation agent into the thawed (after about 1 min), and centrifuging and washing (1500rpm, 5 min); and (4) removing the washing liquid after centrifugation to obtain a fat precipitate, and performing primary culture on the fat precipitate.
The adipose tissue resuscitation solution consists of the following components in percentage by weight: 78% of DMEM medium, 19% of dextran, 1.5% of L-glutamine and 1.5% of D-glucose.
The method for performing primary culture comprises the following steps: adding equal volume of digestive juice and equal volume of type I collagenase solution (concentration is 0.2%) into the fat sediment, mixing well, sealing, placing into a 37 ℃ shaking table, and digesting for 1 hour at 150 r/min; after digestion, centrifugation (800g, 10 min), removal of upper fat, resuspension of the cell pellet in DMEM/F12 medium (DMEM medium mixed with F12 medium at a volume ratio of 1: 1), centrifugation (800g, 10 min); discarding supernatant, resuspending the cell pellet with DMEM/F12 medium, filtering with 100 μm sieve to obtain adipocytes, centrifuging (800g, 10 min), discarding supernatant, resuspending adipocytes with complete medium, inoculating into culture flask to ensure the concentration of inoculated cells to be 2.5 × 104cells/ml, placed at 37 ℃ in CO2Culturing in an incubator, changing the cell culture solution after 24 hours, and removing non-adherent cells; the solution was changed every three days later. Culturing until about the fifth day, wherein the cell growth density reaches about 90%, and using for subculture.
The utility model provides an adipose tissue's cryopreserving recovery kit, includes foretell adipose tissue cryopreserving first stage reagent, adipose tissue cryopreserving second stage reagent, adipose tissue resuscitation fluid, the specification is: freezing and storing the adipose tissues in one stage, wherein the volume of the reagent is 500 ml/bottle; freezing and storing the adipose tissues in a first bottle of 200 ml/bottle of the two-stage reagent; one bottle of adipose tissue recovery liquid, 200 ml/bottle; during practical application, the freezing kit is taken according to experimental needs, and theoretically one set of the freezing kit can be used for freezing 500ml of adipose tissues.
Experiment 1
The tissue cryopreservation method of example 1 was performed, and it was confirmed that adipose mesenchymal stem cells extracted from adipose tissues within 6 months of adipose tissue cryopreservation were in a fibrous morphology, uniform in size, densely arranged, and grown in a vortex shape, as shown in fig. 2 and 3.
Flow cytometry detection results show that cells generated by digestion after adipose tissue cryopreservation recovery, surface markers CD29 and CD 44; CD105 was positively expressed (> 99%), cell surface markers CD34, CD45, HLA-DR were negatively expressed (< 2%), as shown in figure 4.
Experiment 2
Tissue cryopreservation was performed as in example 1, with conventional cryopreservation as a control. The results are shown in tables 1 and 2.
The conventional cryopreservation method of the adipose tissues comprises the following steps: centrifuging adipose tissue, separating oil and fat components and oil-water mixture, directly freezing adipose tissue, and storing in refrigerator at-80 deg.C. From all detection results, the particles and biological activity of the adipose tissues are gradually reduced along with the prolongation of the cryopreservation time, and the change of the adipose tissue results observed under a microscope is not obvious in one week and four weeks at the early stage of cryopreservation.
The fat tissue is frozen by using the method of the invention, the fat is revived and extracted when the fat is frozen for 4 weeks, 8 weeks and 12 weeks, and the cell number is still more and the cell activity is higher after the fat is frozen for 12 weeks, which are obviously superior to the conventional freezing method.
TABLE 1 direct cryopreservation
| Freezing time | Freezing for 0 week | Freezing for 1 week | Freezing for 12 weeks |
| Extraction of cell number | 2.5±0.2*106 | 0.34±0.1*106 | 0.28±0.1*106 |
| Extracting cell activity | 98.20％ | 50.50％ | 34.10％ |
TABLE 2 cryopreservation using the method of the invention
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (10)
1. A cryopreservation method of adipose tissues is characterized in that: the method comprises the following steps:
(1) pretreatment of adipose tissue: centrifuging the collected fat specimen to remove the upper layer of grease, the lower layer of water layer, reserving the middle layer of adipose tissue, and washing the middle layer of adipose tissue with normal saline for 1-3 times to obtain washed adipose tissue;
(2) adding an adipose tissue cryopreservation first-stage reagent into the adipose tissue, and centrifuging and washing; centrifuging, removing washing solution, adding the adipose tissue cryopreservation two-stage reagent into the rest adipose tissue, mixing, pre-cooling at 4 deg.C for 15min, and transferring to a program cooling instrument for program cooling; after the programmed cooling is finished, transferring the frozen fat cells to a liquid nitrogen storage tank at the temperature of-196 ℃ for storage;
the adipose tissue cryopreservation first-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide and 5-8% of trehalose;
the adipose tissue cryopreservation two-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide, 3-8% of trehalose, 2-4% of propylene glycol, 0.5-1% of glutathione and 0.08-0.12% of sodium pyruvate.
2. The method for cryopreserving adipose tissues according to claim 1, wherein: the procedure of the procedure cooling is as follows:
keeping the temperature at 4 ℃ for 5min, and cooling to 0 ℃ at 5 ℃/min;
keeping at 0 deg.C for 5min, and cooling to-15 deg.C at 2 deg.C/min;
keeping at-15 deg.C for 5min, and cooling to-65 deg.C at 1 deg.C/min;
keeping the temperature at minus 65 ℃ for 5min, and cooling to minus 80 ℃ at the temperature of 0.5 ℃/min.
3. The method for cryopreserving adipose tissues according to claim 1, wherein: the specific operation mode of the step (1) is as follows: taking the collected fat sample, subpackaging and transferring the sample into a 50ml centrifuge tube, and centrifuging; absorbing the upper layer of grease, transferring the middle layer of adipose tissue into a new centrifugal tube, adding physiological saline with the same volume, centrifuging, and removing the supernatant; the saline washing of the adipose tissue was repeated twice.
4. The method for cryopreserving adipose tissues according to claim 1, wherein: the specific operation mode of the step (2) is as follows: adding an equal volume of adipose tissue freezing-storing first-stage reagent into the adipose tissue, and centrifugally washing; centrifuging, removing washing liquid, stirring the rest adipose tissue with glass rod, subpackaging into 5ml freezing tubes, adding adipose tissue freezing two-stage reagent 2.5ml per tube, mixing in vortex oscillator, transferring into 4 deg.C refrigerator, pre-cooling for 15min, and transferring into program cooling instrument for program cooling.
5. A method for resuscitating cryopreserved adipose tissue, comprising: taking out the frozen adipose tissues or the adipose tissues frozen by the freezing method according to any one of claims 1 to 4 from liquid nitrogen, and thawing the frozen adipose tissues; adding adipose tissue recovery liquid into the melted liquid, and centrifuging and washing; centrifuging, removing the washing liquid to obtain fat sediment, completing recovery, and performing primary culture on the fat sediment;
the adipose tissue resuscitation solution consists of the following components in percentage by weight: 75-85% of DMEM medium, 15-25% of dextran, 1-2% of L-glutamine and 1-2% of D-glucose.
6. The method for resuscitating frozen adipose tissue according to claim 5, wherein: the adipose tissue resuscitation solution consists of the following components in percentage by weight: 78% of DMEM medium, 19% of dextran, 1.5% of L-glutamine and 1.5% of D-glucose.
7. The method for resuscitating frozen adipose tissue according to claim 5, wherein: the method for performing primary culture comprises the following steps: adding digestive juice and type I collagenase solution into the fat sediment, fully mixing uniformly, sealing, putting into a 37 ℃ shaking table, and digesting for 1 hour at 150 rpm; after digestion, centrifuging, removing upper fat, suspending cell sediment by DMEM/F12 medium basis, and centrifuging; discarding supernatant, resuspending cell pellet with DMEM/F12 culture medium, filtering with 100 μm screen to obtain adipocytes, centrifuging, discarding supernatant, resuspending adipocytes with complete culture medium, inoculating into culture flask to ensure inoculated cell concentration of 2.5 × 104cells/ml, placed at 37 ℃ in CO2Culturing in an incubator, changing the cell culture solution after 24 hours, and removing non-adherent cells; changing the liquid every three days later。
8. The utility model provides an adipose tissue cryopreserved resuscitation kit which characterized in that: comprises an adipose tissue freezing first-stage reagent, an adipose tissue freezing second-stage reagent and an adipose tissue resuscitation solution;
the adipose tissue cryopreservation first-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide and 5-8% of trehalose;
the adipose tissue cryopreservation two-stage reagent consists of the following components in percentage by weight: 75-80% of DMEM culture medium, 5-15% of dimethyl sulfoxide, 3-8% of trehalose, 2-4% of propylene glycol, 0.5-1% of glutathione and 0.08-0.12% of sodium pyruvate;
the adipose tissue resuscitation solution consists of the following components in percentage by weight: 75-85% of DMEM medium, 15-25% of dextran, 1-2% of L-glutamine and 1-2% of D-glucose.
9. The kit for cryopreserving adipose tissues for resuscitation according to claim 8, wherein the kit comprises: the adipose tissue cryopreservation first-stage reagent consists of the following components in percentage by weight: 78% of DMEM medium, 15% of dimethyl sulfoxide and 7% of trehalose;
the adipose tissue cryopreservation two-stage reagent consists of the following components in percentage by weight: 78% of DMEM culture medium, 12% of dimethyl sulfoxide, 6.1% of trehalose, 3% of propylene glycol, 0.8% of glutathione and 0.1% of sodium pyruvate;
the adipose tissue resuscitation solution consists of the following components in percentage by weight: 78% of DMEM medium, 19% of dextran, 1.5% of L-glutamine and 1.5% of D-glucose.
10. Use of the kit for cryopreservation and/or resuscitation of adipose tissue according to claim 8 or 9.
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