CN103070161A - Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) - Google Patents
Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) Download PDFInfo
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Abstract
The invention relates to a cryopreservation liquid and a cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC). The cryopreservation liquid is composed of the following materials: 3 to 10 mg/ml of vitamine C, 2 to 8 mg/ml of vitamin E, 2 to 8 mg/ml of lipoic acid, 5 to 20 volume percent of human autoserum, and DMEM-LG culture medium in balancing amount. The cryopreservation liquid for ADSC does not contain animal serum and DMSO, and is composed of human autoserum, lipoic acid, vitamin E, vitamin C and other main materials. A cell cryopreserved with the cryopreservation liquid has the advantages of high palinesthesia rate, no impact on cell growth characteristic, and maintanience of stem cell pluripotency after palinesthesia.
Description
Technical field
The present invention relates to a kind of stem cell cryopreserving liquid, especially a kind of fat mesenchymal stem cell cryopreserving liquid and preparation method thereof.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is the important member of stem cell family, derives to grow early stage mesoderm and ectoderm.Mescenchymal stem cell is found in marrow at first, because it has the concern that multi-lineage potential, hematopoiesis support and the characteristics such as the implantation of promotion stem cell, immunoregulation and self-replacation are subject to people day by day.Mescenchymal stem cell is in vivo or under the external specific inductive condition, can be divided into the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multi-lineage potential after continuous passage cultivation and the freezing preservation, can be used as desirable seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
The mesenchymal stem cell cryopreserving liquid that uses in the prior art contains hyclone and dimethyl sulfoxide (DMSO) (DMSO) more.Hyclone contains multiple growth factor, is usually used in cell in vitro and cultivates, for the adherent of cell or spread over the growth factor that provides required on the culture matrix; Also can be used for stem cell cryopreserving, the problem that the cytoactive that can slow down descends can be kept Cell viability for a long time.DMSO is usually used in the frozen of cell because it has freeze proof effect.
The employed hyclone of mesenchymal stem cell cryopreserving liquid comes from import mostly in the prior art, and import serum is expensive, and European multinational generation rabid ox disease, and the application of import hyclone also has been subject to certain restriction.There are some researches show that the mescenchymal stem cell that contacts with hyclone for a long time can be to the hyclone generation endocytosis in the solution medium, the variation of some protein expression might occur in the mescenchymal stem cell behind the endocytosis hyclone, be applied to behind the human body immune response that heterogenous animal albumen causes to occur, thereby cause success rate behind the mesenchymal stem cell transplantation to reduce and the generation of bad reaction.DMSO has toxicity, can be to the freeze-stored cell injury, and the external source animal blood serum to cell have pollution may, the long-term use affect cytoactive, follow-up use affects the popularization of cell clinical treatment.
Therefore, also a lot of about the research of stem cell cryopreserving liquid.Be the Chinese invention patent of CN101919379 such as publication number, a kind of cryopreserving liquid for freezing and storing umbilical mesenchymal stem cells for long time is disclosed, made by following steps: 1) Cord blood is placed aseptic centrifuge tube, centrifugal 15-18min under the 1000rpm-1200rpm condition; 2) draw supernatant, be transferred in the new centrifuge tube, centrifugal 18-20min under the 4000g-4200g condition, supernatant are the frozen umbilical cord blood plasma of using; 3) get dimethyl sulfoxide (DMSO) and above-mentioned frozenly mix with 1: 9 by volume ratio of umbilical cord blood plasma, namely get mesenchymal stem cell cryopreserving liquid.Use the blood plasma of Cord Blood-Derived to substitute hyclone in this invention cryopreserving liquid, avoided cell in frozen process, to contact heterogenous animal serum, and reduced the cell cryopreservation cost.
Publication number is the Chinese invention patent application of CN102511471, discloses a kind of mesenchymal stem cell cryopreserving liquid that does not contain animal sources albumen and DMSO, is comprised of trehalose, carbohydrate-electrolyte solution, ATP and human serum albumins.This cryopreserving liquid has high anabiosis rate (〉 90%), do not affect the characteristic of stem cell, cell proliferation does not change, Cell Differentiation does not change; The advantages such as easily use, good stability.
Existing mesenchymal stem cell cryopreserving liquid contains hyclone or DMSO more, even do not contain hyclone or DMSO, frozen effect is also undesirable; More there is not the cryopreserving liquid about fat mesenchymal stem cell.
Summary of the invention
In view of this, be necessary for the problems referred to above, a kind of fat mesenchymal stem cell cryopreserving liquid and cryopreservation methods that does not contain animal blood serum and DMSO is provided.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
A kind of fat mesenchymal stem cell cryopreserving liquid is comprised of the following component of following content:
As a kind of preferred embodiment of fat mesenchymal stem cell cryopreserving liquid of the present invention, described fat mesenchymal stem cell cryopreserving liquid is comprised of the following component of following content:
A kind of method of frozen fat mesenchymal stem cell may further comprise the steps:
1) aseptic absorption liposuction procedures extraction of lipid liquid with the PBS flushing for several times, is removed used medicine and haemocyte in the liposuction procedures; Use the type i collagen enzymic digestion, centrifugal, draw upper strata fat and filter.The cell suspension that obtains is centrifugal, after cell precipitation washs with PBS, suspend with containing volume fraction 10% autoserous DMEM-LG culture fluid, be inoculated in the culture dish, place incubator to cultivate after 48 hours, sucking-off suspension cell liquid more renews medium; Changed liquid in 3 days 1 time, when cultivating 10 days left and right sides cells and reaching more than 80% degree of converging, the cultivation of going down to posterity;
2) with pancreatin cell is digested, discard medium, PBS flushing twice is used trypsin digestion cell, centrifugal collecting cell;
3) with the cell of fat mesenchymal stem cell cryopreserving liquid of the present invention frozen acquisition, be placed on-80 ℃ of refrigerator overnight, put into the liquid nitrogen container long preservation on the 2nd day.
As a kind of preferred embodiment of the method for the frozen fat mescenchymal stem cell of the present invention fat, described is to digest 1 hour with 37 ℃ of 0.1%I Collagenase Types with the type i collagen enzymic digestion.
Compared with prior art, the present invention is a kind of fat mesenchymal stem cell cryopreserving liquid that does not contain animal blood serum and DMSO, by Human autologous serum, lipoic acid, vitamin E, the Main Ingredients and Appearances such as vitamin C form, and utilize the frozen cell of this cryopreserving liquid to have high anabiosis rate, do not affect cell growth characteristics, cell keeps the characteristics such as versatility of stem cell after the recovery.Without the external source animal blood serum, the Human autologous serum of interpolation, lipoic acid, vitamin E and vitamin C do not affect the application of follow-up clinical cell therapy.Human autologous serum without foreign protein, and has multiple growth factor from the stem cell cryopreserving person, and the ratio of each growth factor conforms to normal ratio in the body, after using cell recovery is had everything to gain and nothing to lose.
Description of drawings
Fig. 1 uses the Growth of Cells form behind the frozen cell recovery of different mesenchymal stem cell cryopreserving liquids.Figure 1A is for using the frozen cell of fat mesenchymal stem cell cryopreserving liquid of the embodiment of the invention 1, and Figure 1B is for using the frozen cell of common stem cell cryopreserving liquid.
Fig. 2 uses many differentiation potentials of cell the result behind the frozen cell recovery of different mesenchymal stem cell cryopreserving liquids; Fig. 2 A and Fig. 2 C become fat and Osteoblast Differentiation the result after for the frozen cell recovery of the fat mesenchymal stem cell cryopreserving liquid that uses the embodiment of the invention 1; Fig. 2 B becomes fat and Osteoblast Differentiation the result with Fig. 2 D after using the frozen cell recovery of common stem cell cryopreserving liquid.
Embodiment
Explanation the present invention for good is described further below in conjunction with specific embodiments and the drawings.
A kind of fat mesenchymal stem cell cryopreserving liquid is comprised of the following component of following content:
Lipoic acid not only tool is water-soluble but also tool is fat-soluble, so it can be deep into each position in the cell and play antioxidation, lipoic acid is mutually coordinated sugar, fat, metabolism of amino acid by participating in the oxidative deamination reaction, and can be separated removing heavy metals to containing the murder by poisoning of sulfydryl enzyme as a kind of coenzyme.Also has clinically the effect of anti-fatty liver and reduction cholesterolemia.
Vitamin E is lipovitamin; not only biomembranous 26S Proteasome Structure and Function there is preferably protective effect; the vitamin e free radical of Mulberry Extract generation can further generate the non-free radical product with another molecule radical reaction again rapidly: the fertility quinone, therefore its oxidation resistance is stronger.
Vitamin C also participates in hydroxylation reaction not only as the antioxidant protection cell, thus with body in collagen generate, proteometabolism, anti-inflammatory, anti-infective, detoxifcation waits effect directly relevant.
Lipoic acid, vitamin E and vitamin C all have very strong antioxidation activity, can effectively remove interior free yl to the toxic action of cell, thereby play the effect of Cell protection, are applied in the cell cryopreservation that effectively Cell protection is active.
A kind of method of frozen fat mesenchymal stem cell may further comprise the steps:
1) aseptic absorption liposuction procedures extraction of lipid liquid with the PBS flushing for several times, is removed used medicine and haemocyte in the liposuction procedures; Use the type i collagen enzymic digestion, centrifugal, draw upper strata fat and filter.The cell suspension that obtains is centrifugal, after cell precipitation washs with PBS, suspend with containing volume fraction 10% autoserous DMEM-LG culture fluid, be inoculated in the culture dish, place 37 ℃, volume fraction 5%CO
2Cultivate in the incubator; After 48 hours, sucking-off suspension cell liquid more renews medium; Changed liquid in 3 days 1 time, when cultivating 10 days left and right sides cells and reaching more than 80% degree of converging, the cultivation of going down to posterity;
2) with pancreatin cell is digested, discard medium, PBS flushing twice is used trypsin digestion cell, centrifugal collecting cell;
3) with the cell of fat mesenchymal stem cell cryopreserving liquid of the present invention frozen acquisition, be placed on-80 ℃ of refrigerator overnight, put into the liquid nitrogen container long preservation on the 2nd day.
Embodiment 1
A kind of fat mesenchymal stem cell cryopreserving liquid is comprised of the following component of following content:
Make the fat mesenchymal stem cell cryopreserving liquid according to aforementioned proportion and set of dispense.Described fat mesenchymal stem cell cryopreserving liquid is by following method freeze-stored cell.
1) the extraction of lipid liquid of aseptic absorption liposuction procedures with the PBS flushing for several times, is removed used medicine and haemocyte in the liposuction procedures; With 37 ℃ of digestion of 0.1%I Collagenase Type 1 hour, the centrifugal 5min of 400g/min, behind the fat of absorption upper strata, mixing filters with 200 eye mesh screens; With the cell suspension 1200r/min that obtains, after centrifugal 8min, cell precipitation washed with PBS, transferring cell concentration was 1 * 10
9Individual/L, suspend with containing volume fraction 10% autoserous DMEM-LG medium, be inoculated in the 10cm culture dish, place 37 ℃, volume fraction 5%CO
2Cultivate in the incubator; After 48 hours, sucking-off suspension cell liquid more renews medium; Changed liquid in 3 days 1 time, when cultivating 10 days left and right sides cells and reaching more than 80% degree of converging, the cultivation of going down to posterity;
2) with pancreatin cell is digested, remove the medium in the blake bottle or carefully suck medium in the culture plate with rifle, PBS flushing twice is with trypsin digestion cell (gentle as far as possible), centrifugal collecting cell;
3) be mixed with fat mesenchymal stem cell cryopreserving liquid freeze-stored cell, cell density is 1 * 10
6Individual/mL, mark freeze-stored cell title after the sealing, cryopreserving liquid kind and frozen date; Adopt the programmed cooling device to be placed on-80 ℃ of refrigerator overnight, second day is put into the liquid nitrogen container long preservation.
The present invention is a kind of fat mesenchymal stem cell cryopreserving liquid that does not contain animal blood serum and DMSO, by Human autologous serum, lipoic acid, vitamin E, the Main Ingredients and Appearances such as vitamin C form, utilize the frozen cell of this cryopreserving liquid to have high anabiosis rate, do not affect cell growth characteristics, cell keeps the characteristics such as versatility of stem cell after the recovery.Without the external source animal blood serum, the Human autologous serum of interpolation, lipoic acid, vitamin E and vitamin C do not affect the application of follow-up clinical cell therapy.Human autologous serum without foreign protein, and has multiple growth factor from the stem cell cryopreserving person, and the ratio of each growth factor conforms to normal ratio in the body, after using cell recovery is had everything to gain and nothing to lose.
Use common stem cell cryopreserving liquid (surplus is DMEM-LG for hyclone 10%, DMSO10%) in contrast.High anabiosis rate to freeze-stored cell does not affect cell growth characteristics, and cell keeps the versatility of stem cell to investigate after the recovery.
Cryopreservation tube is taken out from liquid nitrogen, drop into immediately in 37 ℃ of water-baths, slightly shake.After liquid all melts (general 1-1.5min), take out a small amount of alcohol of spray and be put in the superclean bench.Above-mentioned cell suspension is drawn onto in the centrifuge tube of 15mL of dress 10mL medium (with medium cryopreservation tube is washed one time, the cell that is bonded on the wall is all washed), 1000 leave heart 5min.Supernatant is outwelled, added 1mL DEME-LG perfect medium, cell is suspended.Be drawn onto in the 10cm culture dish that the 10mL medium is housed and all around shake gently, the cell in the culture dish is evenly distributed.Mark cell category and date, kinds of culture medium people etc., be put into CO
2Cultivate in the incubator, behind the cell attachment, trypsinization collecting cell again.
Get 50uL cell sap and expect blue the mixing with the 50uL platform, get a little cell mixture and add haemocytometer, and be beneficial to microscopically calculating cell number.Living cells is not dyeed, and dead cell can be dyed to blueness.Calculate the total cell number of 4 large grid of large nine grids, except in 4, take advantage of again in extension rate again, take advantage of in 10 again
4, namely be cell number number among every mL.Be every mL cell number=always calculate cell number/4 * extension rate * 10
4After using the frozen cell recovery of the fat mesenchymal stem cell cryopreserving liquid of the embodiment of the invention 1, viable count 2 * 10
6Individual/mL, anabiosis rate reaches 90%; Use the frozen cell viable count 1 * 10 of common stem cell cryopreserving liquid
6Individual/mL, anabiosis rate is 70%.Use the frozen cell of fat mesenchymal stem cell cryopreserving liquid of the present invention, anabiosis rate is high.
Observe respectively and use the frozen cell of embodiment 1 described fat mesenchymal stem cell cryopreserving liquid and use the frozen cell growth characteristics of common stem cell cryopreserving liquid and versatility to find, use the frozen cell of embodiment 1 described fat mesenchymal stem cell cryopreserving liquid better to keep the activity of fat stem cell, cell has high anabiosis rate, do not affect cell growth characteristics (seeing Fig. 1), (see Fig. 2, cell keeps many differentiation potentials to the characteristics such as versatility of cell maintenance stem cell after the recovery after the recovery.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (4)
1. a fat mesenchymal stem cell cryopreserving liquid is characterized in that, is comprised of the following component of following content:
2. fat mesenchymal stem cell cryopreserving liquid according to claim 1 is characterized in that, is comprised of the following component of following content:
3. the method for a frozen fat mesenchymal stem cell is characterized in that, may further comprise the steps:
1) bacterium draws liposuction procedures extraction of lipid liquid, with the PBS flushing for several times, removes used medicine and haemocyte in the liposuction procedures; Use the type i collagen enzymic digestion, centrifugal, draw upper strata fat and filter.The cell suspension that obtains is centrifugal, after cell precipitation washs with PBS, suspend with containing volume fraction 10% autoserous DMEM-LG culture fluid, be inoculated in the culture dish, place incubator to cultivate after 48 hours, sucking-off suspension cell liquid more renews medium; Changed liquid in 3 days 1 time, when cultivating 10 days left and right sides cells and reaching more than 80% degree of converging, the cultivation of going down to posterity;
2) with pancreatin cell is digested: discard medium, PBS flushing twice is used trypsin digestion cell, centrifugal collecting cell;
3) with the cell of fat mesenchymal stem cell cryopreserving liquid of the present invention frozen acquisition, be placed on-80 ℃ of refrigerator overnight, put into the liquid nitrogen container long preservation on the 2nd day.
4. the method for frozen fat mesenchymal stem cell according to claim 3 is characterized in that, described is with 37 ℃ of digestion of 0.1%I Collagenase Type 1 hour with the type i collagen enzymic digestion.
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| CN105211051A (en) * | 2015-10-29 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | Cultured NK cell freezing medium and preparation method thereof |
| CN105532649A (en) * | 2016-02-16 | 2016-05-04 | 崔长友 | Freezing medium for long-term storage of adult stem cells and preparing method thereof |
| CN105624114A (en) * | 2016-02-04 | 2016-06-01 | 广东盈生力健康科技有限公司 | Stem cell differentiation medium and application thereof |
| CN106222134A (en) * | 2016-07-29 | 2016-12-14 | 中卫华医（北京）生物科技有限公司 | The cultural method of effective acquisition fat mesenchymal stem cell |
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| CN109090102A (en) * | 2018-09-05 | 2018-12-28 | 成都汇欣生命科技有限公司 | A kind of general serum-free frozen stock solution of mescenchymal stem cell and preparation method thereof |
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| CN110839614A (en) * | 2019-11-27 | 2020-02-28 | 西南医科大学 | Autologous umbilical cord mesenchymal stem cell cryopreservation liquid and preparation and cryopreservation methods thereof |
| CN111011363A (en) * | 2019-12-16 | 2020-04-17 | 广东唯泰生物科技有限公司 | Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and recovery method |
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| CN113455498A (en) * | 2021-08-12 | 2021-10-01 | 郑州贝因生物科技有限公司 | Adipose mesenchymal stem cell cryopreservation liquid and cryopreservation method |
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