CN101922048A - Method for constructing public library of umbilical mesenchymal stem cells - Google Patents
Method for constructing public library of umbilical mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a method for constructing a public library of umbilical mesenchymal stem cells, which overcomes the defects of long time consumption and high cost, simplifies the operation steps, lowers the intervention errors of the manual operation and improves the efficiency.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of banking process of umbilical cord mesenchymal stem cells public library.
Technical background
(mesenchymal stem cells is the important member of stem cell family MSC) to mescenchymal stem cell, derives to grow early stage mesoderm and ectoderm.MSC finds in marrow at first, because of it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to be subjected to people's attention day by day.As mescenchymal stem cell in vivo or under the external specific inductive condition, can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
The mescenchymal stem cell clinical application is in solving multiple disease in the blood system, cardiovascular disorder, liver cirrhosis, nervous system disorders, meniscus of knee joint partly excises injury repairing, and aspects such as autoimmune disorder have obtained important breakthrough, has saved the life of a lot of sufferers.In addition, mescenchymal stem cell also is used for the treatment research of diabetes and complication, lower limb ischemia pathology, cardiovascular and cerebrovascular diseases, liver cirrhosis, bone and muscle decline property disease, brain and autoimmune disorders such as neurologic defict, senile dementia and lupus erythematosus and scleroderma, and the clinical test results of having obtained is encouraging.
Mescenchymal stem cell is distributed widely in the marrow, periosteum, spongy bone, fat, synovial membrane, skeletal muscle, tire liver, deciduous teeth, umbilical cord, Cord blood of fetus and adult, wherein the mescenchymal stem cell quality height in umbilical cord source, pure, quantity is many.The mescenchymal stem cell in umbilical cord source not only can become the desirable surrogate of mesenchymal stem cells MSCs, and has bigger application potential.Umbilical cord mesenchymal stem cells is expressed the peculiar molecular marker of multiple embryonic stem cell, have that differentiation potential is big, multiplication capacity is strong, immunogenicity is low, draw materials conveniently, the restriction of amoral ethics problem, be easy to feature such as preparation of industrialization, therefore might become the multipotential stem cell of tool potential applicability in clinical practice.
Stem cell bank is to be about the place that stores stem cell or related data in-196 ℃ of liquid nitrogen.Marrow and peripheral hematopoietic stem cells storehouse can be divided into database and storehouse in kind.The work that database is done is that marrow that healthy person is provided or peripheral hematopoietic stem cells carry out HLA and join type and data registration, gathers its marrow when need waiting again.Storehouse in kind then is to carry out when cell joins type and data registration, and gathering and store healthy supplier can be for the stem cell of transplanting usefulness.Stem cell bank is divided into public library again by presentation mode and from the body storehouse.The stored stem cell of public library is other people stem cell, transplants to satisfy the demand but autologous stem cells not have the patient's of preservation demand.
The construction process of existing umbilical cord mesenchyma stem cell, be made up of following step: (1) is got people's umbilical cord and is detected, and uses the damping fluid wash and remove residual blood, shreds, and obtains human umbilical tissue; (2) add collagenase digesting; (3) add damping fluid dilution digestion back tissue, mixing, centrifugal; (4) abandon supernatant, add the resuspended precipitation of damping fluid, sieve, collect filtrate, centrifugal, repeat twice, promptly get human umbilical cord mesenchymal stem cells; (5) human umbilical cord mesenchymal stem cells that obtains is put liquid nitrogen and preserve, preserve by ABO/Rh somatotype and HLA somatotype, foundation can promptly be constructed human umbilical cord mesenchyma stem cell for the cell news file of retrieval.
Existing umbilical cord mesenchyma stem cell construction process has following deficiency.The one, length consuming time.Public library is frozen after stem cell is prepared, and every part of stem cell warehouse-in cycle is long, complex steps.The 2nd, the cost height.After the donor umbilical cord is donated, there is public library, waits for that the receptor who needs uses, it is frozen again that every part of umbilical cord is all prepared stem cell before use, consume reagent, material, artificial etc. more, be not that each part stem cell all can be used, this just makes the cost up of public library.
Summary of the invention
In order to solve the deficiencies in the prior art, overcome length consuming time, defect of high cost, the step that simplifies the operation reduces manually-operated intervention mistake, raises the efficiency.
The present invention takes following technical scheme:
A kind of construction process of umbilical cord mesenchyma stem cell may further comprise the steps:
1. get umbilical cord with the PBS damping fluid that contains penicillin and Streptomycin sulphate, wash umbilical cord gently;
2. get the long umbilical cord of 6-8cm, add the DMEM substratum that 2ml contains volume ratio 10% foetal calf serum, shear umbilical cord as early as possible under 4 ℃, size is to 0.5-1mm
3
3. collect umbilical cord tissue piece, 4 ℃ of centrifugal 10min under the 850g condition;
4. abandon supernatant, sedimentary tissue block is resuspended with the frozen storing liquid of dimethyl sulfoxide (DMSO) and 2: 8 by volume mixed of foetal calf serum;
5. divide in the frozen pipe of packing into, be cooled to-196 ℃, change liquid nitrogen over to and preserve;
6. preserve by donor ABO/Rh blood group and sex, foundation can be for the cell news file of retrieval;
7. carrying out the go down to posterity cultivation, stem cell cryopreserving of recovery of umbilical cord tissue piece and former foster, the stem cell of being commissioned to train when using again operates.
The recovery of above-mentioned umbilical cord tissue piece, former be commissioned to train to support can adopt following operation:
1. from liquid nitrogen, take out to need the tissue block of recovery, put into 42 ℃ of sterilized water bath cabinets immediately and rock at the uniform velocity repeatedly, make its in 2min in thawing;
2. the tissue block of taking out wherein joins in the DMEM substratum that contains volume ratio 10% foetal calf serum, fully mixing;
3. at 4 ℃, under the 850g condition, centrifugal 10min abandons supernatant liquor, and with the resuspended tissue block of DMEM substratum that contains volume ratio 10% foetal calf serum, inoculation is cultivated in incubator;
4. to 24 hours, supplemented medium changed a subculture in later per 3 days;
5. to 12-17 days, can select stem cell to go down to posterity to cultivate or frozen.
Above-mentioned freezing and storing umbilical tissue block source MSC goes down to posterity to cultivate and can adopt following operation:
1. abandon residual substratum in the most culture dish, with twice of PBS damping fluid flushing;
2. culture dish adds 2ml and contains volume ratio 0.25% tryptic PBS damping fluid digestion, treats that the intercellular substance increases, and cell shortens and then trypsinase discarded; See under the inverted microscope cell rounding then each culture dish respectively add the DMEM substratum that 6ml contains volume ratio 10% foetal calf serum, stop also piping and druming cell of digestion;
With the buoyant cell harvesting in centrifuge tube, the mixing cell;
4. cell suspension is according to 8000-10000/cm
2Density be inoculated in culture dish, place incubator to cultivate.
Above-mentioned freezing and storing umbilical tissue block source MSC is frozen to take following operation:
1. abandon the substratum of each culture dish, with twice of PBS damping fluid flushing;
2. add 2ml and contain volume ratio 0.25% tryptic PBS damping fluid digestion, treat that the intercellular substance increases, cell shortens and then trypsinase is discarded; See under the inverted microscope cell rounding then each culture dish respectively add the DMEM substratum that 6ml contains volume ratio 10%FBS, stop also piping and druming cell of digestion;
With the buoyant cell harvesting in centrifuge tube, the mixing cell;
4. get the part cell and be inoculated in culture dish, place incubator to cultivate, when treating cytogamy 50-80% under 4 ℃, 600g condition centrifugal 10min, abandon supernatant liquor, cell is upspring, the frozen storing liquid that adds precooling in advance is frozen.
Description of drawings
Fig. 1: umbilical cord tissue piece source mescenchymal stem cell former generation (10 *)
Fig. 2: umbilical cord tissue piece source mescenchymal stem cell P1, P2 generation (10 *)
Fig. 3: umbilical cord tissue piece source mescenchymal stem cell streaming detected result
Technique effect
The method of structure umbilical cord mesenchyma stem cell of the present invention is simplified operating procedure, reduces the intervention mistake of manually-operated, raises the efficiency, and saves cost.
Embodiment
One, the construction step of umbilical cord mesenchyma stem cell
1. get umbilical cord and carry out blood group detection (comprising that the ABO/Rh blood group detects), microorganism immunodetection, umbilical cord washes umbilical cord gently with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate.Described penicillin/streptomycin solution is available from U.S. ScienCell company, P/S (Penicillin/Streptomycin Solution).
2. select umbilical cord sample light good brightness, do not have breakage, no oedema zone, it is long to get 6-8cm.
3. add the DMEM substratum that 2ml contains volume ratio 10%FBS (foetal calf serum), shear umbilical cord as early as possible under 4 ℃, size is to 0.5-1mm
3
4. collect umbilical cord tissue piece, 4 ℃ of centrifugal 10min under the 850g condition.
5. abandon supernatant, sedimentary tissue block is resuspended with the frozen storing liquid of 2: 8 by volume mixed of FBS with DMSO (dimethyl sulfoxide (DMSO)).
6. divide in the frozen pipe of 5ml of packing into, (program control) is cooled to-196 ℃, changes liquid nitrogen over to and preserves.
7. preserve by donor ABO/Rh blood group and sex, foundation can be for the cell news file of retrieval.
8. carry out the operations such as cultivation, stem cell cryopreserving of going down to posterity of recovery of umbilical cord tissue piece and former foster, the stem cell of being commissioned to train when using again.
Two, umbilical cord tissue piece recovery, former being commissioned to train are supported the operation (see figure 1)
1. from liquid nitrogen, take out to need the tissue block of recovery, put into 42 ℃ of sterilized water bath cabinets immediately and rock at the uniform velocity repeatedly, make its in 2min in thawing.
2. the tissue block of taking out wherein joins in the DMEM substratum (available from Hyclone company) that contains volume ratio 10%FBS, fully mixing.
3. at 4 ℃, under the 850g condition, centrifugal 10min abandons supernatant liquor, with the resuspended tissue block of DMEM substratum (3-4ml tissue block precipitation add the 1ml substratum resuspended) that contains volume ratio 10%FBS, inoculation, cultivates in 37 ℃, the incubator of 5%CO2.
To 24h (hour), supplemented medium, each culture dish is added the fresh DMEM substratum (do not abandon original substratum, just add fresh substratum) that contains volume ratio 10%FBS of 2ml, changes a subculture (discard old substratum and add the equivalent fresh culture) in later per 3 days.
5. to about 15 days, cell clone quantity increased, and clone's inner cell merges 60-90%, can select passage or frozen.
Three, freezing and storing umbilical tissue block source MSC goes down to posterity and cultivates operation (Fig. 2)
1. abandon residual substratum in the most culture dish, with twice of PBS damping fluid flushing.
2. culture dish adds 2ml and contains volume ratio 0.25% tryptic PBS damping fluid digestion, treats that the intercellular substance increases, cell shorten and then trypsinase is discarded (not having to discard trypsinase before floating at cell); See under the inverted microscope cell rounding then each culture dish respectively add the DMEM substratum that 6ml contains volume ratio 10%FBS, stop also piping and druming cell of digestion.
With the buoyant cell harvesting in the 50ml centrifuge tube, mixing cell, meter cell quantity and vigor.
4. cell suspension is according to 8000-10000/cm
2Density be inoculated in culture dish, place 37 ℃, 5%CO
2Incubator cultivate.
Four, the frozen operation of freezing and storing umbilical tissue block source MSC (Fig. 3)
1. each culture dish is left and taken the 2ml substratum, in order to the usefulness of aerophil and fungi detection, abandons remaining substratum, with twice of PBS damping fluid flushing.
2. add 2ml and contain volume ratio 0.25% tryptic PBS damping fluid digestion, treat that the intercellular substance increases, cell shorten and then trypsinase is discarded (not having to discard trypsinase before floating) at cell; See under the inverted microscope cell rounding then each culture dish respectively add the DMEM substratum that 6ml contains volume ratio 10%FBS, stop also piping and druming cell of digestion.
With the buoyant cell harvesting in the 50ml centrifuge tube, mixing cell, meter cell quantity and vigor.
4. the substratum of leaving and taking in above-mentioned each culture dish is squeezed into aerophil with asepsis injector and detect in the bottle, put in the bacteria culture instrument and cultivated 15 days, detect aerophil and fungi.
5. get the part cell and be inoculated in culture dish, place 37 ℃, cultivate in the incubator of 5%CO2, be used for detection of mycoplasma when treating cytogamy 50-80%; Leave and take 5 * 10
5-1 * 10
6Cell identifies that to be used for flow cytometer identification of indicator is CD34, CD45, CD44, CD105, CD146, HLA-DR.
6. remaining cell suspension, centrifugal 10min under 4 ℃, 600g condition abandons supernatant liquor, and cell is upspring, and adds the frozen storing liquid (with the frozen storing liquid of DMSO (dimethyl sulfoxide (DMSO)) with 2: 8 by volume mixed of FBS) of precooling in advance, and is frozen.
Claims (4)
1. the construction process of a umbilical cord mesenchyma stem cell is characterized in that may further comprise the steps:
1) gets umbilical cord with the PBS damping fluid that contains penicillin and Streptomycin sulphate, wash umbilical cord gently;
2) get the long umbilical cord of 6-8cm, add the DMEM substratum that contains volume ratio 10% foetal calf serum, shear umbilical cord as early as possible under 4 ℃, size is to 0.5-1mm
3
3) collect umbilical cord tissue piece, 4 ℃ of centrifugal 10min under the 850g condition;
4) abandon supernatant, sedimentary tissue block is resuspended with the frozen storing liquid of dimethyl sulfoxide (DMSO) and 2: 8 by volume mixed of foetal calf serum;
5) divide in the frozen pipe of packing into, be cooled to-196 ℃, change liquid nitrogen over to and preserve;
6) preserve by donor ABO/Rh blood group and sex, foundation can be for the cell news file of retrieval;
Carrying out the go down to posterity cultivation, stem cell cryopreserving of recovery of umbilical cord tissue piece and former foster, the stem cell of being commissioned to train when 7) using again operates.
2. the method for claim 1 is characterized in that described umbilical cord tissue piece recovery and former foster may further comprise the steps of being commissioned to train:
1) from liquid nitrogen, take out to need the tissue block of recovery, put into 42 ℃ of bath cabinets immediately and rock at the uniform velocity repeatedly, make its in 2min in thawing;
2) tissue block of taking out wherein joins in the DMEM substratum that contains volume ratio 10% foetal calf serum, fully mixing;
3) at 4 ℃, under the 850g condition, centrifugal 10min abandons supernatant liquor, and with the resuspended tissue block of DMEM substratum that contains volume ratio 10% foetal calf serum, inoculation is cultivated in incubator;
4) to 24 hours, supplemented medium changed a subculture in later per 3 days;
5), can select stem cell to go down to posterity to cultivate or frozen to 12-17 days.
3. the method for claim 1 is characterized in that described stem cell goes down to posterity to cultivate may further comprise the steps:
1) abandons residual substratum in the most culture dish, with twice of PBS damping fluid flushing;
2) culture dish adds and contains volume ratio 0.25% tryptic PBS damping fluid digestion, treats that the intercellular substance increases, and cell shortens and then trypsinase discarded; See under the inverted microscope cell rounding then each culture dish each add the DMEM substratum contain volume ratio 10% foetal calf serum, stop also piping and druming cell of digestion;
3) with the buoyant cell harvesting in centrifuge tube, the mixing cell;
4) cell suspension is according to 8000-10000/cm
2Density be inoculated in culture dish, place incubator to cultivate.
4. the method for claim 1 is characterized in that described stem cell cryopreserving may further comprise the steps:
1) abandons the substratum of each culture dish, with twice of PBS damping fluid flushing;
2) adding contains volume ratio 0.25% tryptic PBS damping fluid digestion, treats that the intercellular substance increases, and cell shortens and then trypsinase discarded; See under the inverted microscope cell rounding then each culture dish each add the DMEM substratum contain volume ratio 10% foetal calf serum, stop also piping and druming cell of digestion;
3) with the buoyant cell harvesting in centrifuge tube, the mixing cell;
4) get cell inoculation in culture dish, place incubator to cultivate, when treating cytogamy 50-80% under 4 ℃, 600g condition centrifugal 10min, abandon supernatant liquor, cell is upspring, the frozen storing liquid that adds precooling in advance is frozen.
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Cited By (12)
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| CN102119936A (en) * | 2011-03-07 | 2011-07-13 | 李荣旗 | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection |
| CN102660502A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell |
| CN102960334A (en) * | 2011-09-01 | 2013-03-13 | 生宝生物科技股份有限公司 | Umbilical cord tissue cryopreservation suitable for umbilical cord tissue-derived stem cells |
| CN103451151A (en) * | 2013-06-19 | 2013-12-18 | 武汉道培胎盘干细胞生物技术有限公司 | Method for culturing human umbilical cord mesenchymal stem cells |
| CN103651331A (en) * | 2013-12-13 | 2014-03-26 | 青岛中天干细胞工程有限公司 | Construction method of pool of adipose-derived stem cells |
| CN106434547A (en) * | 2016-12-17 | 2017-02-22 | 重庆市干细胞技术有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN107177545A (en) * | 2017-06-13 | 2017-09-19 | 安徽安龙基因医学检验所有限公司 | A kind of method of Improved inbred-line from people umbilical cord separating mesenchymal stem cell |
| WO2018045914A1 (en) * | 2016-09-09 | 2018-03-15 | 深圳市茵冠生物科技有限公司 | Recombinant lentivirus-modified human umbilical cord mesenchymal stem cell carrying pigment epithelial-derived factor gene and preparation method therefor |
| CN109859803A (en) * | 2019-02-15 | 2019-06-07 | 和携科技(北京)有限公司 | A kind of production inventory management system and method for stem cell medicine |
| CN111896340A (en) * | 2020-06-24 | 2020-11-06 | 四川大学华西医院 | Simple PBMC separation method for flow cytometry detection |
| CN112522791A (en) * | 2020-12-09 | 2021-03-19 | 河北医科大学 | Construction method of human umbilical cord mesenchymal stem cell bank |
| CN113812400A (en) * | 2021-11-06 | 2021-12-21 | 刘春玲 | Cryopreservation protection solution for constructing umbilical cord mesenchymal stem cell bank and construction method of cell bank |
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| CN102119936B (en) * | 2011-03-07 | 2012-12-12 | 李荣旗 | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection |
| CN102119936A (en) * | 2011-03-07 | 2011-07-13 | 李荣旗 | Method for preparing injection for treating ischemic brain damage by using human amniotic mesenchymal cells and injection |
| US8703411B2 (en) | 2011-09-01 | 2014-04-22 | Healthbanks Biotech Co., Ltd. | Cryopreservation of umbilical cord tissue for cord tissue-derived stem cells |
| CN102960334A (en) * | 2011-09-01 | 2013-03-13 | 生宝生物科技股份有限公司 | Umbilical cord tissue cryopreservation suitable for umbilical cord tissue-derived stem cells |
| CN102660502A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell |
| CN103451151A (en) * | 2013-06-19 | 2013-12-18 | 武汉道培胎盘干细胞生物技术有限公司 | Method for culturing human umbilical cord mesenchymal stem cells |
| CN103451151B (en) * | 2013-06-19 | 2016-01-20 | 武汉道培胎盘干细胞生物技术有限公司 | A kind of method of cultivator umbilical cord mesenchymal stem cells |
| CN103651331A (en) * | 2013-12-13 | 2014-03-26 | 青岛中天干细胞工程有限公司 | Construction method of pool of adipose-derived stem cells |
| WO2018045914A1 (en) * | 2016-09-09 | 2018-03-15 | 深圳市茵冠生物科技有限公司 | Recombinant lentivirus-modified human umbilical cord mesenchymal stem cell carrying pigment epithelial-derived factor gene and preparation method therefor |
| CN106434547A (en) * | 2016-12-17 | 2017-02-22 | 重庆市干细胞技术有限公司 | Method for preparing umbilical cord mesenchymal stem cells |
| CN107177545A (en) * | 2017-06-13 | 2017-09-19 | 安徽安龙基因医学检验所有限公司 | A kind of method of Improved inbred-line from people umbilical cord separating mesenchymal stem cell |
| CN109859803A (en) * | 2019-02-15 | 2019-06-07 | 和携科技(北京)有限公司 | A kind of production inventory management system and method for stem cell medicine |
| CN111896340A (en) * | 2020-06-24 | 2020-11-06 | 四川大学华西医院 | Simple PBMC separation method for flow cytometry detection |
| CN112522791A (en) * | 2020-12-09 | 2021-03-19 | 河北医科大学 | Construction method of human umbilical cord mesenchymal stem cell bank |
| CN113812400A (en) * | 2021-11-06 | 2021-12-21 | 刘春玲 | Cryopreservation protection solution for constructing umbilical cord mesenchymal stem cell bank and construction method of cell bank |
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Inventor after: Huang Yuxiang Inventor after: Gao Hong Inventor after: Wang Li Inventor after: Hu Jianxia Inventor after: Zhang Xuefeng Inventor after: Zhang Meirong Inventor before: Wei Sili Inventor before: Gao Hong Inventor before: Wang Li Inventor before: Hu Jianxia Inventor before: Zhang Xuefeng Inventor before: Zhang Meirong |
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Free format text: CORRECT: INVENTOR; FROM: WEI SILI GAO HONG WANG LI HU JIANXIA ZHANG XUEFENG ZHANG MEIRONG TO: HUANGYUXIANG GAO HONG WANG LI HU JIANXIA ZHANG XUEFENG ZHANG MEIRONG |
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