CN103849598A - Method for preparing human adipose tissue-derived stromal cells - Google Patents
Method for preparing human adipose tissue-derived stromal cells Download PDFInfo
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- CN103849598A CN103849598A CN201410078044.6A CN201410078044A CN103849598A CN 103849598 A CN103849598 A CN 103849598A CN 201410078044 A CN201410078044 A CN 201410078044A CN 103849598 A CN103849598 A CN 103849598A
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Abstract
The invention discloses a method for preparing human adipose tissue-derived stromal cells. The method specifically comprises steps of acquiring, transporting and handing over fat, preparing fat microblocks, carrying out cryopreservation and anabiosis on the fat microblocks, carrying out SVF (Stromal Vascular Fraction) extraction, and carrying out primary culture and subculture. The method is simple and convenient in process, low in cost, good in cryopreservation effect, small in cell damage, small in side effect and harmless to human bodies as the residues of a cryopreservation liquid in cells is reduced to the maximum extent, and the prepared human adipose tissue-derived stromal cells can be applied to clinical treatment of various diseases.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method of human adipose mesenchymal stem cells.
Background technology
In fatty tissue, contain the cell that a class has Multidirectional Differentiation potentiality, be fat mesenchymal stem cell (Adipose Derived Msenchymal Stem Cell, AD-MSC), its biological property and mesenchymal stem cells MSCs are similar, can be to various kinds of cell direction differentiation such as fat, bone, cartilage, muscle, endothelium, hematopoiesis, liver, pancreas islet and nerves.
Fatty tissue reserves in human body are abundant, obtain easy wound little, all have broad application prospects at aspects such as organizational project, organ reparation, gene therapies, therefore fat stem cell has become one, the stem cell field focus receiving much concern, has wide potential applicability in clinical practice.In January, 2012, the autologous fat stem cell medicine Cuepistem of Korea S FDA approval successfully goes on the market, and is used for the treatment of the concurrent anal fistula of complicacy clone disease.In October in the same year, U.S. FDA has been ratified the clinical trial of Cytori Therapeutics company fat stem cell to cardiac failure patient validity.
At present, traditional fat mesenchymal stem cell preparation comprises fatty collection, transportation, SVF (Stromal Vascular Fraction, SVF) extraction, former culture, the cultivation of going down to posterity, mesenchymal stem cell cryopreserving and recovery.The mescenchymal stem cell obtaining is applied to clinical after recovery in the short period of time.There is following shortcoming in tradition preparation method: 1, progenitor cells cultivation and cryopreservation resuscitation process expend a large amount of manpower and materials; 2, after cell recovery, need the long period from recovery to complete activity recovery, within a short period of time, applying clinical effect was troubling; 3, frozen storing liquid minimal residue after recovery, can not get rid of at short notice, can cause the untoward reactions such as patient feels sick, vomiting, heating.
Summary of the invention
The problems referred to above that exist in order to overcome prior art field, the object of the invention is to, and a kind of preparation method of human adipose mesenchymal stem cells is provided.
The preparation method of human adipose mesenchymal stem cells provided by the invention, it comprises following concrete steps:
(1), the collection of fat: after the Donor selection of unmanned source specific virus is qualified, aseptic collection fat, can adopt liposuction mode or modus operandi to obtain fat, more than collection capacity 10ml, after collection, fat is placed in and preserves liquid, and 2-8 ℃ of constant temperature is preserved;
(2), the transportation of fat: fat is placed in and preserves liquid, and 2-8 ℃ of constant temperature is kept in vaccine box, delivers to laboratory in 48h;
(3), the handing-over of fat: laboratory receives fat, has registered customer information, and has carried out encoder client;
(4), prepare fatty microlith: with repeatedly rinsing containing the PBS of penicillin and Streptomycin sulphate, remove residual blood, fat is rubbed as 0.5-1mm
3fatty microlith;
(5), fatty microlith is frozen: dimethyl sulfoxide (DMSO) and serum replacement mix and are made into frozen storing liquid according to the ratio of 1:9, fatty microlith is resuspended with frozen storing liquid, divide and install in cryopreservation tube, put into freezing storing box, be placed in after-80 ℃ of refrigerator overnight, transfer to liquid nitrogen next day and preserve;
(6), the recovery of fatty microlith: take out the fatty tissue microlith that will recover from liquid nitrogen, drop into immediately in 37 ℃ of sterilized water baths, at the uniform velocity jiggle, it was melted within 2 minutes;
(7) SVF extracts: 0.1% collagenase I digests fatty microlith, the centrifugal upper strata that discards, and 100um strainer filtering, obtains SVF;
(8), former culture: according to 1 × 10
5individual/ml is inoculated into culturing bottle, culturing bottle is positioned over to carbonic acid gas fixed temperature and humidity incubator, culture condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is 5 ± 0.2%, cultivates: human mesenchymal stem cell serum free medium;
(9), the cultivation of going down to posterity: former culture is through 7d left and right, when cytogamy reaches 70%～80%, according to 5000～6000/cm
2density pass P1, through 3d, when P1 reaches 80%～90% for fusion, the same, pass P2, by that analogy.
The preparation method of human adipose mesenchymal stem cells provided by the invention, its beneficial effect is, simple process, cost is low, and frozen effective, cell injury is little, at utmost reduce frozen storing liquid residual in cell, side effect is little, harmless, and the human adipose mesenchymal stem cells of preparing can be for clinical treatment various diseases.
Accompanying drawing explanation
Fig. 1 is frozen fatty microlith of the present invention source human adipose mesenchymal stem cells growth curve;
Fig. 2 is frozen fatty microlith of the present invention source human adipose mesenchymal stem cells (P0 Dai-3 days-40 times);
Fig. 3 is frozen fatty microlith of the present invention source human adipose mesenchymal stem cells (P0 Dai-7 days-40 times);
Fig. 4 is frozen fatty microlith of the present invention source human adipose mesenchymal stem cells (P3 Dai-3 days-100 times);
Fig. 5 is 2 weeks rear oil red O stain positive findings figure of the frozen fatty microlith of the present invention source human adipose mesenchymal stem cells Adipogenic induction;
Fig. 6 is that human adipose mesenchymal stem cells osteogenic induction rear alkaline phosphatase staining in 2 weeks in the frozen fatty microlith of the present invention source is strong positive reaction result figure.
Embodiment
With reference to the accompanying drawings,, to the preparation method of human adipose mesenchymal stem cells provided by the invention, be described in detail in conjunction with the embodiments.
Embodiment mono-
The preparation method of the human adipose mesenchymal stem cells of the present embodiment, comprises following concrete steps:
(1), the collection of fat: donor is carried out to unmanned source specific virus and comprises the detection of HIV, HBV, HCV, HTLV, EBV, CMV etc., detects without infection by Treponema pallidum, detect qualified after, gather fat in aseptic place; Can adopt liposuction mode, liposuction position comprises: waist abdomen, buttocks, shank etc., also can adopt modus operandi, and gather abdominal cavity or pelvic cavity fat lump, fat quantity is with more than 10ml; After collection, fat is placed in and preserves liquid, 2-8 ℃ of constant temperature is preserved; Fat is preserved liquid and is made up of μ g/ml Streptomycin sulphate+5, μ g/ml penicillin+200, DMEM substratum+200 μ g/ml amphotericin;
(2), the transportation of fat: fat is placed in and preserves liquid, and 2-8 ℃ of constant temperature is kept in vaccine box, delivers to laboratory in 48h;
(3), the handing-over of fat: laboratory receives fat, has registered customer information, and has carried out encoder client;
(4), prepare fatty microlith: with repeatedly rinsing containing the PBS of penicillin and Streptomycin sulphate, remove residual blood, fat is rubbed as 0.5-1mm
3fatty microlith;
(5), fatty microlith is frozen: collect fatty tissue microlith, at the centrifugal 10min of 700g, the a small amount of liquid of Xi Qi lower floor, by dimethyl sulfoxide (DMSO) and serum replacement (KnockOut Serum Replacement for fatty tissue microlith, KSR, article No. 10828-028, the Gibco of producer) resuspended according to the frozen storing liquid of the ratio mixing of 1:9; Divide and install in cryopreservation tube, put into freezing storing box ,-80 ℃ of refrigerator overnight, transfer to liquid nitrogen next day and preserve;
(6), the recovery of fatty microlith: take out the fatty tissue microlith that will recover from liquid nitrogen, drop into immediately in 37 ℃ of sterilized water baths, at the uniform velocity jiggle, it was melted within 2 minutes; The fatty tissue microlith taking out joins in DMEM substratum and mixes, the centrifugal 10min of 700g, the DMEM of Xi Qi lower floor substratum;
(7), SVF extracts (fatty microlith collagenase digesting): fatty microlith adds collagenase I solution (the 0.1% collagenase I compound method: take 0.1g collagenase I powder dissolution and do not add in the substratum of any factor in 100ml of the preheating of the new preparation of equivalent, with 37 ℃ of preheatings before), sealed membrane sealing, acutely rock culturing bottle 5-10 second, be placed in vibration gas bath pot, 37 ℃, 200rpm, digestion 30-60min, acutely rock culturing bottle 5-10 second every 15min, until seem comparatively level and smooth; Centrifugal 10 minutes of room temperature 400g, discards upper strata adipocyte and oil, adds physiological saline resuspended, by aseptic 100um strainer filtering for the throw out obtaining, and centrifugal 10 minutes of 400g again, the precipitation obtaining is SVF;
(8), former culture: according to 1 × 10
5individual/ml is inoculated into culturing bottle, and culturing bottle is positioned over to carbonic acid gas fixed temperature and humidity incubator; Culture condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is 5 ± 0.2%.Substratum: human mesenchymal stem cell serum free medium (LONZA, 00190632), former culture the 24th hour, carries out full dose and changes liquid, after this changes liquid every 3 days full doses;
(9), about cultivation: 7d of going down to posterity, when the area percentage of primary cultured cells cloning cluster arrives 70%～80%, digestion results; In culturing bottle, add digestive ferment, digestive ferment is 0.125%Trypsin～0.01% EDTA solution, and digestion time is 1.5～2.5min, at the bottom of adding substratum 2～3ml repeatedly to blow and beat bottle, come off to cell major part, move in 50ml centrifuge tube, the centrifugal 8min of 1000rpm, observes remaining cell precipitation amount in single centrifuge tube, suitably merge in several centrifuge tubes in cell precipitation to 1 centrifuge tube, add appropriate substratum, blow and beat gently resuspension cell, be settled to 30ml, piping and druming mixes, sampling counting; 1000rpm after counting, 8min secondary centrifuging; Remove supernatant liquor, add substratum appropriate in centrifuge tube, blow and beat gently resuspension cell, be seeded in new culture vessel after constant volume, passage cell density is 5000～6000/cm
2, on culture vessel, indicate the information such as cell algebraically and incubation time.Culture vessel is positioned over to carbonic acid gas fixed temperature and humidity incubator to start to cultivate; Culture condition: carbonic acid gas fixed temperature and humidity incubator, condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is about 5 ± 0.2%, 3d, reaches and merge while reaching 80%～90% until cell, again goes down to posterity, and breaks with this type of.
Embodiment two
The evaluation of fat mesenchymal stem cell
1, the detection of fluidic cell surface markers: get above-mentioned P3 generation frozen fatty microlith source human adipose mesenchymal stem cells, flow cytometer detection cell surface marker, streaming result is as table 1:
The detection of table 1 fat mesenchymal stem cell surface marker
Wherein, positive mark's thing CD29, CD73, CD90, CD49d express and are greater than 95%, and negative marker thing CD14, CD34, CD45, HLA-DR express lower than 2%, and proving this cell is mescenchymal stem cell.
2, fat mesenchymal stem cell becomes fat induction and identifies: get above-mentioned P3 generation frozen fatty microlith source human adipose mesenchymal stem cells, with 2 × 10
4/ cm
2density be inoculated in 24 orifice plates, every hole adds 15%FBS, the low sugar DMEM nutrient solution 0.8ml of 1ng/ml bFGF; Treat that cell reaches 80% fusion, be changed to inducing culture liquid (DMEM in high glucose, FBS 15%, dexamethasone 1uM, INDOMETHACIN 200uM, IBMX 0.5uM, Regular Insulin 10ng/ul), later every 3～4d changes a not good liquor; Induce after 2 weeks and carry out oil red O stain evaluation.
Result: induce after 2 weeks, the fat producing in oil red O stain visible cell is dyed redness by specificity, and control group is unchanged, proves that it becomes fat differentiation capability.
3, fat mesenchymal stem cell osteogenic induction and evaluation: get above-mentioned P3 generation frozen fatty microlith source human adipose mesenchymal stem cells, with 2 × 10
4/ cm
2density be inoculated in 24 orifice plates, every hole adds 15%FBS, the low sugar DMEM nutrient solution 0.8ml of 1ng/ml bFGF; Treat that cell reaches 80% fusion, be changed to inducing culture liquid (DMEM in high glucose, dexamethasone 0.1uM, β-phospho-glycerol 10mM, xitix 50uM), later every 3～4d changes a not good liquor; Induce after 2 weeks and carry out alkaline phosphatase staining evaluation.
Result: induce after 2 weeks, alkaline phosphatase staining is strong positive reaction, and control group is negative, and proves its Osteoblast Differentiation ability.
By fluidic cell surface markers and become fat, Osteoblast Differentiation ability, prove that frozen fatty microlith source cell is mescenchymal stem cell.
4, the drafting of growth curve: get the frozen fatty microlith of above-mentioned P3 source human adipose mesenchymal stem cells, with serum free medium weaker concn to 1 × 10
4/ ml kind is planted in 8 96 orifice plates, adds 200 μ l cell suspensions in every hole, establishes 8 multiple holes, and blank row (this is listed as every hole and adds 200 μ l serum free mediums) are set in each 96 orifice plates; 96 orifice plates are placed in to 37 ℃, 5%CO2, saturated humidity incubator to be cultivated.Adopt randomly assigne choose at every turn a plate in 96 plates plantation 0,24,48,72,96,120,144,168h detects the OD value of cell.Method: every hole adds MTT(5g/L) 20 μ l, be placed in 37 ℃, 5%CO2, saturated humidity incubator continuation cultivation 4h, the cell centrifugation in hole to be collected, every hole adds 150 μ lDMSO, after vibration 10min, automatic detection OD value under microplate reader 492nm wavelength.Experiment repeats 3 times.After statistical analysis, draw growth curve, growth curve demonstration, this cell doubling time is 48h, rises in value in good condition, without abnormal.
Claims (1)
1. a preparation method for human adipose mesenchymal stem cells, is characterized in that: it comprises following concrete steps:
(1), the collection of fat: after the Donor selection of unmanned source specific virus is qualified, aseptic collection fat, can adopt liposuction mode or modus operandi to obtain fat, more than collection capacity 10ml, after collection, fat is placed in and preserves liquid, and 2-8 ℃ of constant temperature is preserved;
(2), the transportation of fat: fat is placed in and preserves liquid, and 2-8 ℃ of constant temperature is kept in vaccine box, delivers to laboratory in 48h;
(3), the handing-over of fat: laboratory receives fat, has registered customer information, and has carried out encoder client;
(4), prepare fatty microlith: with repeatedly rinsing containing the PBS of penicillin and Streptomycin sulphate, remove residual blood, fat is rubbed as 0.5-1mm
3fatty microlith;
(5), fatty microlith is frozen: dimethyl sulfoxide (DMSO) and serum replacement mix and are made into frozen storing liquid according to the ratio of 1:9, fatty microlith is resuspended with frozen storing liquid, divide and install in cryopreservation tube, put into freezing storing box, be placed in after-80 ℃ of refrigerator overnight, transfer to liquid nitrogen next day and preserve;
(6), the recovery of fatty microlith: take out the fatty tissue microlith that will recover from liquid nitrogen, drop into immediately in 37 ℃ of sterilized water baths, at the uniform velocity jiggle, it was melted within 2 minutes;
(7) SVF extracts: 0.1% collagenase I digests fatty microlith, the centrifugal upper strata that discards, and 100um strainer filtering, obtains SVF;
(8), former culture: according to 1 × 10
5individual/ml is inoculated into culturing bottle, culturing bottle is positioned over to carbonic acid gas fixed temperature and humidity incubator, culture condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is 5 ± 0.2%, cultivates: human mesenchymal stem cell serum free medium;
(9), the cultivation of going down to posterity: former culture is through 7d left and right, when cytogamy reaches 70%～80%, according to 5000～6000/cm
2density pass P1, through 3d, when P1 reaches 80%～90% for fusion, the same, pass P2, by that analogy.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357383A (en) * | 2014-10-11 | 2015-02-18 | 张炳强 | Preparation method of human adipose-derived MSCs (mesenchymal stem cells) and application of human adipose-derived mesenchymal stem cell in preparation of medicine for treating diseases |
| CN104396940A (en) * | 2014-10-11 | 2015-03-11 | 张炳强 | Tissue sample preservative solution and preparation method thereof |
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN106754676A (en) * | 2016-12-21 | 2017-05-31 | 广东康仹生命科技有限公司 | Composition for protecting adipose tissue and preparation method and application thereof |
| CN106719602A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of frozen stock solution and its application with adipose tissue is frozen |
| CN107475190A (en) * | 2017-10-13 | 2017-12-15 | 上海莱馥医疗科技有限公司 | A kind of method that fatty SVF cells clinical grade is efficiently prepared and frozen and application thereof |
| CN107828726A (en) * | 2017-12-15 | 2018-03-23 | 北京焕生汇生物技术研究院 | A kind of method of the fractionation of fatty mescenchymal stem cell from fat |
| CN110791477A (en) * | 2019-11-21 | 2020-02-14 | 陕西九州细胞基因工程有限公司 | Culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes |
| CN112823799A (en) * | 2019-11-18 | 2021-05-21 | 安迪博斯生命医学研发(天津)有限公司 | Preparation method and application of pharmaceutical composition for treating knee osteoarthritis |
| CN113215092A (en) * | 2021-05-26 | 2021-08-06 | 山东博森医学工程技术有限公司 | Rapid adipose-derived mesenchymal stem cell adipogenic differentiation method |
| CN114568422A (en) * | 2020-11-30 | 2022-06-03 | 北京瑷格干细胞科技有限公司 | Fat preservation liquid and application thereof |
-
2014
- 2014-03-05 CN CN201410078044.6A patent/CN103849598A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 原博等: "脂肪组织中血管基质部分（SVF）的细胞成分及应用", 《组织工程与重建外科杂志》 * |
| 李春明: "以脂肪间充质干细胞为种子细胞构建组织工程脂肪的实验研究", 《中国优秀硕士学位论文全文数据库》 * |
| 谢甬淋等: "人脂肪间充质干细胞的分离培养及冻存前后生物学变化", 《中国神经免疫学和神经病学杂志》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104396940A (en) * | 2014-10-11 | 2015-03-11 | 张炳强 | Tissue sample preservative solution and preparation method thereof |
| CN104396940B (en) * | 2014-10-11 | 2016-07-13 | 张炳强 | A kind of tissue samples preserves liquid and preparation method thereof |
| CN104357383A (en) * | 2014-10-11 | 2015-02-18 | 张炳强 | Preparation method of human adipose-derived MSCs (mesenchymal stem cells) and application of human adipose-derived mesenchymal stem cell in preparation of medicine for treating diseases |
| CN104770363A (en) * | 2015-04-21 | 2015-07-15 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution and cryopreservation method for mesenchymal stem cells |
| CN106719602A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of frozen stock solution and its application with adipose tissue is frozen |
| CN106754676B (en) * | 2016-12-21 | 2018-02-13 | 广东康仹生命科技有限公司 | Composition for protecting adipose tissue and preparation method and application thereof |
| CN106754676A (en) * | 2016-12-21 | 2017-05-31 | 广东康仹生命科技有限公司 | Composition for protecting adipose tissue and preparation method and application thereof |
| CN107475190A (en) * | 2017-10-13 | 2017-12-15 | 上海莱馥医疗科技有限公司 | A kind of method that fatty SVF cells clinical grade is efficiently prepared and frozen and application thereof |
| CN107475190B (en) * | 2017-10-13 | 2020-05-19 | 上海莱馥医疗科技有限公司 | Method for clinical-level efficient preparation and cryopreservation of fat SVF cells and application thereof |
| CN107828726A (en) * | 2017-12-15 | 2018-03-23 | 北京焕生汇生物技术研究院 | A kind of method of the fractionation of fatty mescenchymal stem cell from fat |
| CN112823799A (en) * | 2019-11-18 | 2021-05-21 | 安迪博斯生命医学研发(天津)有限公司 | Preparation method and application of pharmaceutical composition for treating knee osteoarthritis |
| CN110791477A (en) * | 2019-11-21 | 2020-02-14 | 陕西九州细胞基因工程有限公司 | Culture method of mesenchymal stem cells after cryopreservation and recovery of adipocytes |
| CN114568422A (en) * | 2020-11-30 | 2022-06-03 | 北京瑷格干细胞科技有限公司 | Fat preservation liquid and application thereof |
| CN113215092A (en) * | 2021-05-26 | 2021-08-06 | 山东博森医学工程技术有限公司 | Rapid adipose-derived mesenchymal stem cell adipogenic differentiation method |
| CN113215092B (en) * | 2021-05-26 | 2022-04-05 | 广州峰缘生物科技有限公司 | Rapid adipose-derived mesenchymal stem cell adipogenic differentiation method |
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