CN105586309A - Method for acquiring safe and effective umbilical cord mesenchymal stem cell - Google Patents

Method for acquiring safe and effective umbilical cord mesenchymal stem cell Download PDF

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CN105586309A
CN105586309A CN201610082928.8A CN201610082928A CN105586309A CN 105586309 A CN105586309 A CN 105586309A CN 201610082928 A CN201610082928 A CN 201610082928A CN 105586309 A CN105586309 A CN 105586309A
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cell
umbilical cord
bottle
physiological saline
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CN105586309B (en
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苏爱盟
肖桂清
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FUJIAN YINFENG STEM CELL ENGINEERING Co Ltd
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FUJIAN YINFENG STEM CELL ENGINEERING Co Ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

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Abstract

The invention provides a method for acquiring a safe and effective umbilical cord mesenchymal stem cell. A pollution-free umbilical cord mesenchymal stem cell can be prepared by umbilical cord acquisition, umbilical cord transportation, umbilical cord preparation and methods of detecting, screening, adding antibiotics and the like in a cell culturing process, so that the safety can be guaranteed; and improved culture system and culture method are adopted in the separating and culturing processes, so that the separation efficiency is improved, the culturing cycle is shortened, and a high-purity excellent-characterization cell can be obtained. According to the method, the cost can be effectively reduced, the high-quality umbilical cord mesenchymal stem cell can be quickly acquired, injury on cells can be reduced, the culturing cycle can be shortened, pollution can be reduced, and the cell purity can be improved.

Description

A kind of method that obtains safe and effective umbilical cord mesenchymal stem cells
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method that obtains safe and effective umbilical cord mesenchymal stem cells.
Background technology
Mescenchymal stem cell (mesenchymalstemcell, MSC) is to derive to grow early stage mesoderm and an ectodermic class multipotential stem cell. MSC has immunological regulation, promotes hematopoiesis to recover, can repair the histoorgan of damage or pathology, possess the functions such as multi-lineage potential, is therefore with a wide range of applications.
MSC is found at first in marrow, at present we can be from the tissues such as fat, synovial membrane, bone, muscle, lung, liver, pancreas and placenta, amniotic fluid, umbilical cord separating mesenchymal stem cell. From umbilical cord separating mesenchymal stem cell have gather convenient, be easy to storage and transport, to donor without injury, easily separated, purity is high, heteroplastic transplantation can not trigger the advantages such as immune response.
Method piece adherent method and the enzyme digestion in a organized way of separating mesenchymal stem cell from umbilical cord at present. Enzyme digestion is mainly to adopt one or several in clostridiopetidase A, trypsase, hyaluronidase to digest the tissue block shredding, and it has a fatal shortcoming is damaging cells, affects sign and the function of cell. Although tissue block method's incubation time is longer, it is all better than enzyme digestion at aspects such as cell quantity, cellular morphology, immunophenotype and differentiation potentials.
The immediate technical scheme of present: with the PBS buffer solution containing penicillin and streptomysin, healthy neonatal umbilical cord tissue is fully cleaned, removed blood stains; Umbilical cord scissors is become to the uniform segment of length, carry out Mechanical Method separation, the logical glue of blunt separation China is removed arteria umbilicalis and umbilical vein simultaneously; Logical the China of peeling off glue is evenly shredded; With the logical glue of the resuspended China shredding of MSCs culture medium, be inoculated in the culture dish of gelatin paving quilt, be placed in CO2Incubator is cultivated, and carries out had digestive transfer culture in the time that Growth of Cells merges to 80%-90%.
Technological deficiency at present: prior art is to adopt common culture medium to carry out separation and the cultivation of umbilical cord mesenchymal stem cells, and cultivation cycle is longer; And do not mention coherent detection means, cannot ensure to obtain cell products safely and effectively.
The present invention not only can effectively reduce costs but also can obtain fast the mescenchymal stem cell of high-quality, reduced the injury to cell, had shortened cultivation cycle, reduced and polluted, and can improve cell purity.
Summary of the invention
The invention provides a kind of separating funicle mesenchyme stem cell cultivation of going down to posterity from people's umbilical cord Wharton jelly (Wharton ' sJelly), the final method that obtains cell products, solve the problems such as the separative efficiency that may occur in umbilical cord mesenchymal stem cells separation, incubation is low, the cycle is long, security is not enough.
The technical solution adopted for the present invention to solve the technical problems is:
Thereby the method such as investigation, interpolation antibiotic of taking to detect in umbilical cord acquisition, umbilical cord transport, umbilical cord preparation and cell cultivation process ensures to obtain free of contamination umbilical cord mesenchymal stem cells and guarantees safety, in separation, incubation, adopt cultivating system and the cultural method of improvement to improve separative efficiency, shorten cultivation cycle and also can obtain high-purity, characterize good cell.
Described method comprises as follows:
1), in gnotobasis, by being placed in the accumulating bottle that has added accumulating liquid after umbilical cord sterilization, be sent to laboratory and separate preparation; α-MEM that wherein accumulating liquid is 40ml, and added gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml; Transport temperature is 4-25 DEG C, and haulage time is no more than 24 hours;
2) umbilical cord bottle is with putting into Biohazard Safety Equipment after alcohol wipe;
3) in Tissue Culture Dish, use cleaning solution fully to wash umbilical cord 3-6 time, umbilical cord two ends are respectively cut off to 1cm length, abandon; Wash umbilical cord tissue 2-4 time again; Wherein cleaning solution is for having added 100U/ml gentamicin, 50mg/ml penicillin, the medical saline of 5ug/ml amphotericin B;
4) umbilical cord is cut into the segment of 2cm length, cleans 3 times, shift umbilical cord tissue to new culture dish, in culture dish, add physiological saline to flooding umbilical cord tissue 1/2 place; Remove vein blood vessel and arteries, tear and get the logical glue of China; The logical glue of China is placed in the 50ml centrifuge tube that adds 10ml cleaning solution;
5) the logical glue of China fully washs 3 times with cleaning solution, then with physiological saline washing 2 times, is transferred in new 50ml centrifuge tube, fully shreds to 1mm3～3mm3Size;
6) the 10ml pipette that spends leading portion is divided logical China blob of viscose in 4 bottles of 175cm2 blake bottles that added 25ml complete medium equally, at the bottom of jiggling it being uniformly distributed in bottle; Wherein, complete medium is in α-MEM, to add 10%FBS and following combination of cytokines: EGF5 ~ 10ng/ml+PDGF5 ~ 10ng/ml+TNF-α 3 ~ 10ng/ml+IFN-γ 3 ~ 10ng/ml;
7) carefully blake bottle is put into 37 DEG C, 5%CO2In incubator; Cultivate initial 7 days, keep blake bottle absolute rest;
8) the 8th day according to growing state, with 75% alcohol disinfecting medium bottle outer wall, is placed in Biohazard Safety Equipment;
9) inhale and abandon old culture medium with the pipette of decaptitating, change pipette, add 10ml physiological saline in acellular cultivation face, light shaking washing is organized adherent, discards, and repeats 2 times;
10) every bottle adds digestive juice 3ml, and homogeneous immersion cell attachment face is hatched 1min or incubated at room 3min for 37 DEG C; After cell rounding, every bottle adds stop buffer 10ml, concussion fast, and with 10ml pipette piping and druming cell attachment face, in sucking-off cell suspension to a 2 50ml centrifuge tube, every bottle, each blake bottle adds 10ml physiological saline, and purge once, imports in 50ml centrifuge tube;
11) 1200rpm, centrifugal 6min, abandons supernatant; Merge and be precipitated to 1 pipe, add 40ml physiological saline centrifuge washing again, precipitation is resuspended with 10ml complete medium, filters through cell sieve, and 5ml complete medium rinses screen cloth, counting;
12) according to cell quantity paving bottle, making cell concentration is 3 ~ 5 × 104/ml, and mark is put in 37 DEG C, 5%CO2 incubator and cultivated;
13) observation of cell, gathers in the crops frozen when Growth of Cells extremely approaches healing.
Described frozen concrete steps are:
1) configuration stop buffer, before digestion, 3min is placed into digestive juice in 37 DEG C of water-baths, and frozen mother liquor is equilibrated to 4 DEG C;
2) Tissue Culture Flask is taken out, at the bottom of 75% alcohol disinfecting bottle, put into Biohazard Safety Equipment;
3) inhale and abandon old culture medium with pipette, change pipette, add 10ml physiological saline in acellular cultivation face, jiggle and make it cover the whole bottle end, inhale and abandon cell washing liquid, repeated washing once;
4) every bottle adds 3ml digestive juice, and 37 DEG C of digestion 1min, after cell obviously bounces back, add stop buffer 10ml/ bottle, and termination digestion is collected all liq in 50ml centrifuge tube, more every bottle adds 10ml physiological saline, soft piping and druming in the rear 50ml of remittance centrifuge tube;
5) 1200 turn/the centrifugal 6min of min, abandon supernatant, and cell precipitation suspends with 16ml physiological saline, mixes to get that 1ml counts and flow cytometer detection;
6) add physiological saline to 40ml, get 500 μ l supernatants and be endotoxin detection, 1200rpm, centrifugal 6min;
7) supernatant is poured in clean centrifuge tube, done bacterium inspection and detection of mycoplasma, centrifugation suspends with 2.5mlFBS, more slowly adds the frozen mother liquor of 2.5ml, divides and installs in cryopreservation tube, every pipe 1ml after mixing; Freeze-stored cell number is 5-10 × 106/ ml, if cell should increase and go down to posterity very little;
8) labeled cell algebraically, density, bar code and operating time are outside tube wall;
9) cryopreservation tube is placed in to the program temperature reduction box of 4 DEG C of processing in advance, places 24h in-80 DEG C of side by side combination refrigerators, then sample is proceeded in liquid nitrogen container, record storage location.
Preferred described combination of cytokines: EGF6ng/ml+PDGF7ng/ml+TNF-α 5ng/ml+IFN-γ 5ng/ml.
Described digestive juice is containing 0.25wt.% pancreatin, 0.03wt.%EDTA.
The invention has the advantages that:
Thereby the method such as investigation, interpolation antibiotic of taking to detect in umbilical cord acquisition, umbilical cord transport, umbilical cord preparation and cell cultivation process ensures to obtain free of contamination umbilical cord mesenchymal stem cells and guarantees safety, in separation, incubation, adopt cultivating system and the cultural method of improvement to improve separative efficiency, shorten cultivation cycle and also can obtain high-purity, characterize good cell.
In culture medium, add EGF, PDGF, TNF-α and IFN-γ to add the propagation that can effectively promote MSC in cultivating system simultaneously, thereby effectively shorten incubation time, and can maintain its phenotypic characteristic.
Static cultivation 7 days, helps lend some impetus to the climbing out of of adherent and cell of tissue block. In accumulating liquid and cleaning solution, add gentamicin, penicillin, amphotericin B can effectively prevent and control bacterium, fungal contamination. When cell harvesting, count, flow cytometer detection, endotoxin detection, detection of mycoplasma, Bacteria Culture detection etc., only have all qualified just permission warehouse-in storages of every detection, characterize and have or not the many-sides such as microbial contamination to ensure validity and the security of cell products from cell quantity, cell.
Brief description of the drawings
Fig. 1. the state (40 power microscopes under observe) of embodiment 1 umbilical cord mesenchymal stem cells in separation, incubation. A) tissue block paving bottle 7 days cell states afterwards; B) tissue block paving bottle 12 days cell states afterwards; C) P0 passes the P1 cell state of the 2nd day; D) P0 passes the P1 cell state of the 4th day; E) P0 passes the P1 cell state of the 6th day.
Fig. 2. embodiment 1 umbilical cord mesenchymal stem cells flow cytometer detection result figure.
Fig. 3. embodiment 2 umbilical cord mesenchymal stem cells pass the P1 cell state of the 4th day at P0.
Fig. 4. embodiment 3 umbilical cord mesenchymal stem cells pass the P1 cell state of the 4th day at P0.
Fig. 5. the umbilical cord mesenchymal stem cells of commercially available medium culture passes the P1 cell state of the 4th day at P0.
Fig. 6. the incubation time statistics of different culture media formula.
Detailed description of the invention
Embodiment 1
1 umbilical cord preparation:
1) in gnotobasis, when third stage of labor, gather umbilical cord, note draining the residual blood of umbilical cord arteriovenous ligation; After umbilical cord sterilization after collection, be placed in the accumulating bottle that has added accumulating liquid, be sent to laboratory and separate preparation. α-MEM that wherein accumulating liquid is 40ml, and added gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml. Before collection, guarantee five detections of pregnant woman virus all negative, that ALT detects is qualified. Transport temperature is 4 DEG C, and haulage time is no more than 24 hours.
2) umbilical cord bottle is with putting into Biohazard Safety Equipment after alcohol wipe.
3) at 150cm2In Tissue Culture Dish, use cleaning solution fully to wash umbilical cord 3 times, umbilical cord two ends are respectively cut off to 1cm length with tip staight scissors, abandon. Wash again umbilical cord tissue 2 times. Wherein cleaning solution is for having added 100U/ml gentamicin, 50mg/ml penicillin, the medical saline of 5ug/ml amphotericin B.
4) with tip staight scissors by umbilical cord scissors the segment into about 2cm length, clean 3 times. Shift in the culture dish that umbilical cord tissue to is new, in culture dish, add physiological saline to flooding umbilical cord tissue 1/2 place. Remove vein blood vessel and arteries, tear and get the logical glue of China. The logical glue of China is placed in the 50ml centrifuge tube that adds a small amount of cleaning solution.
5) the logical glue of China fully washs 3 times with cleaning solution, then with physiological saline washing 2 times, is transferred in new 50ml centrifuge tube, fully shreds to 1mm3～3mm3Size.
6) the 10ml pipette that spends leading portion is divided logical China blob of viscose equally 4 bottles of 175cm that added 25ml complete medium2In blake bottle, at the bottom of jiggling it being uniformly distributed in bottle, wherein, complete medium is in α-MEM, to add 10%FBS and 6ng/mlEGF+7ng/mlPDGF+5ng/mlTNF-α+5ng/mlIFN-gamma cells combinations of factors.
7) tag-related on blake bottle, comprises sample strip shape code, operation item, operating time, operator etc.
8) carefully blake bottle is put into 37 DEG C, 5%CO2In incubator.
9) cultivate initial 7 days, keep blake bottle absolute rest.
10) within the 8th day, according to growing state, foundation is changed liquid, is gone down to posterity, gathers in the crops frozen standard practice instructions execution.
2 passages:
1) with 75% alcohol disinfecting medium bottle outer wall, be placed in Biohazard Safety Equipment.
2) inhale and abandon old culture medium with the pipette of decaptitating, change pipette, add 10ml physiological saline in acellular cultivation face.
3) light shaking washing is organized adherent, discards, and repeats 2 times.
4) every bottle adds digestive juice 3ml, and digestive juice is 0.25% pancreatin+0.03%EDTA, and homogeneous immersion cell attachment face is hatched 1min for 37 DEG C. After cell rounding, every bottle adds stop buffer 10ml, concussion fast, and with 10ml pipette piping and druming cell attachment face, in sucking-off cell suspension to a 2 50ml centrifuge tube, every bottle, each blake bottle adds 10ml physiological saline, and purge once, imports in 50ml centrifuge tube.
5) 1200rpm, centrifugal 6min, abandons supernatant. Merge and be precipitated to 1 pipe, add 40ml physiological saline centrifuge washing again, precipitation is resuspended with 10ml complete medium, filters through cell sieve, and 5ml complete medium rinses screen cloth, counting.
6) according to cell quantity paving bottle, making cell concentration is 3 × 104/ ml, mark, puts 37 DEG C, 5%CO2In incubator, cultivate.
3 cell harvestings:
1) observation of cell, gathers in the crops frozen when Growth of Cells extremely approaches healing.
2) configuration stop buffer, before digestion, 3min is placed into digestive juice in 37 DEG C of water-baths, and frozen mother liquor is equilibrated to 4 DEG C.
3) Tissue Culture Flask is taken out, at the bottom of 75% alcohol disinfecting bottle, put into Biohazard Safety Equipment.
4) inhale and abandon old culture medium with pipette, change pipette, add 10ml physiological saline in acellular cultivation face, jiggle and make it cover the whole bottle end, inhale and abandon cell washing liquid, repeated washing once.
5) every bottle adds 3ml digestive juice, and 37 DEG C of digestion 1min, after cell obviously bounces back, add stop buffer 10ml/ bottle, and termination digestion is collected all liq in 50ml centrifuge tube, more every bottle adds 10ml physiological saline, soft piping and druming in the rear 50ml of remittance centrifuge tube.
6) 1200 turn/the centrifugal 6min of min, abandon supernatant, and cell precipitation suspends with 16ml physiological saline, mixes to get that 1ml counts and flow cytometer detection.
7) add physiological saline to 40ml, get 500 μ l supernatants and be endotoxin detection, 1200rpm, centrifugal 6min.
8) supernatant is poured in clean centrifuge tube, done bacterium inspection and detection of mycoplasma, centrifugation suspends with 2.5mlFBS, more slowly adds the frozen mother liquor of 2.5ml, divides and installs in cryopreservation tube, every pipe 1ml after mixing. Freeze-stored cell concentration is 5 × 106 /ml，
9) labeled cell algebraically, density, bar code and operating time are outside tube wall.
10) cryopreservation tube is placed in to the program temperature reduction box of 4 DEG C of processing in advance, places 24h in-80 DEG C of side by side combination refrigerators, then coordinate frozen personnel that sample is proceeded in liquid nitrogen container, record storage location.
Embodiment 2
1 umbilical cord preparation:
1) in gnotobasis, when third stage of labor, gather umbilical cord, note draining the residual blood of umbilical cord arteriovenous ligation; After umbilical cord sterilization after collection, be placed in the accumulating bottle that has added accumulating liquid, be sent to laboratory and separate preparation. α-MEM that wherein accumulating liquid is 40ml, and added gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml. Before collection, guarantee five detections of pregnant woman virus all negative, that ALT detects is qualified. Transport temperature is 4 DEG C, and haulage time is no more than 24 hours.
2) umbilical cord bottle is with putting into Biohazard Safety Equipment after alcohol wipe.
3) at 150cm2In Tissue Culture Dish, use cleaning solution fully to wash umbilical cord 3 times, umbilical cord two ends are respectively cut off to 1cm length with tip staight scissors, abandon. Wash again umbilical cord tissue 2 times. Wherein cleaning solution is for having added 100U/ml gentamicin, 50mg/ml penicillin, the medical saline of 5ug/ml amphotericin B.
4) with tip staight scissors by umbilical cord scissors the segment into about 2cm length, clean 3 times. Shift in the culture dish that umbilical cord tissue to is new, in culture dish, add physiological saline to flooding umbilical cord tissue 1/2 place. Remove vein blood vessel and arteries, tear and get the logical glue of China. The logical glue of China is placed in the 50ml centrifuge tube that adds a small amount of cleaning solution.
5) the logical glue of China fully washs 3 times with cleaning solution, then with physiological saline washing 2 times, is transferred in new 50ml centrifuge tube, fully shreds to 1mm3～3mm3Size.
6) the 10ml pipette that spends leading portion is divided logical China blob of viscose equally 4 bottles of 175cm that added 25ml complete medium2In blake bottle, at the bottom of jiggling it being uniformly distributed in bottle, wherein, complete medium is in α-MEM, to add 10%FBS and 5ng/mlEGF+10ng/mlPDGF+5ng/mlTNF-α+5ng/mlIFN-gamma cells combinations of factors.
7) tag-related on blake bottle, comprises sample strip shape code, operation item, operating time, operator etc.
8) carefully blake bottle is put into 37 DEG C, 5%CO2In incubator.
9) cultivate initial 7 days, keep blake bottle absolute rest.
10) within the 8th day, according to growing state, foundation is changed liquid, is gone down to posterity, gathers in the crops frozen standard practice instructions execution.
2 passages:
1) with 75% alcohol disinfecting medium bottle outer wall, be placed in Biohazard Safety Equipment.
2) inhale and abandon old culture medium with the pipette of decaptitating, change pipette, add 10ml physiological saline in acellular cultivation face.
3) light shaking washing is organized adherent, discards, and repeats 2 times.
4) every bottle adds digestive juice 3ml, and digestive juice is 0.25% pancreatin+0.03%EDTA, and homogeneous immersion cell attachment face is hatched 1min for 37 DEG C. After cell rounding, every bottle adds stop buffer 10ml, concussion fast, and with 10ml pipette piping and druming cell attachment face, in sucking-off cell suspension to a 2 50ml centrifuge tube, every bottle, each blake bottle adds 10ml physiological saline, and purge once, imports in 50ml centrifuge tube.
5) 1200rpm, centrifugal 6min, abandons supernatant. Merge and be precipitated to 1 pipe, add 40ml physiological saline centrifuge washing again, precipitation is resuspended with 10ml complete medium, filters through cell sieve, and 5ml complete medium rinses screen cloth, counting.
6) according to cell quantity paving bottle, making cell concentration is 4.4 × 104/ ml, mark, puts 37 DEG C, 5%CO2In incubator, cultivate.
3 cell harvestings:
1) observation of cell, gathers in the crops frozen when Growth of Cells extremely approaches healing.
2) configuration stop buffer, before digestion, 3min is placed into digestive juice in 37 DEG C of water-baths, and frozen mother liquor is equilibrated to 4 DEG C.
3) Tissue Culture Flask is taken out, at the bottom of 75% alcohol disinfecting bottle, put into Biohazard Safety Equipment.
4) inhale and abandon old culture medium with pipette, change pipette, add 10ml physiological saline in acellular cultivation face, jiggle and make it cover the whole bottle end, inhale and abandon cell washing liquid, repeated washing once.
5) every bottle adds 3ml digestive juice, and 37 DEG C of digestion 1min, after cell obviously bounces back, add stop buffer 10ml/ bottle, and termination digestion is collected all liq in 50ml centrifuge tube, more every bottle adds 10ml physiological saline, soft piping and druming in the rear 50ml of remittance centrifuge tube.
6) 1200 turn/the centrifugal 6min of min, abandon supernatant, and cell precipitation suspends with 16ml physiological saline, mixes to get that 1ml counts and flow cytometer detection.
7) add physiological saline to 40ml, get 500 μ l supernatants and be endotoxin detection, 1200rpm, centrifugal 6min.
8) supernatant is poured in clean centrifuge tube, done bacterium inspection and detection of mycoplasma, centrifugation suspends with 2.5mlFBS, more slowly adds the frozen mother liquor of 2.5ml, divides and installs in cryopreservation tube, every pipe 1ml after mixing. Freeze-stored cell concentration is 5 × 106 /ml，
9) labeled cell algebraically, density, bar code and operating time are outside tube wall.
10) cryopreservation tube is placed in to the program temperature reduction box of 4 DEG C of processing in advance, places 24h in-80 DEG C of side by side combination refrigerators, then coordinate frozen personnel that sample is proceeded in liquid nitrogen container, record storage location.
Embodiment 3
1 umbilical cord preparation:
1) in gnotobasis, when third stage of labor, gather umbilical cord, note draining the residual blood of umbilical cord arteriovenous ligation; After umbilical cord sterilization after collection, be placed in the accumulating bottle that has added accumulating liquid, be sent to laboratory and separate preparation. α-MEM that wherein accumulating liquid is 40ml, and added gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml. Before collection, guarantee five detections of pregnant woman virus all negative, that ALT detects is qualified. Transport temperature is 4 DEG C, and haulage time is no more than 24 hours.
2) umbilical cord bottle is with putting into Biohazard Safety Equipment after alcohol wipe.
3) at 150cm2In Tissue Culture Dish, use cleaning solution fully to wash umbilical cord 3 times, umbilical cord two ends are respectively cut off to 1cm length with tip staight scissors, abandon. Wash again umbilical cord tissue 2 times. Wherein cleaning solution is for having added 100U/ml gentamicin, 50mg/ml penicillin, the medical saline of 5ug/ml amphotericin B.
4) with tip staight scissors by umbilical cord scissors the segment into about 2cm length, clean 3 times. Shift in the culture dish that umbilical cord tissue to is new, in culture dish, add physiological saline to flooding umbilical cord tissue 1/2 place. Remove vein blood vessel and arteries, tear and get the logical glue of China. The logical glue of China is placed in the 50ml centrifuge tube that adds a small amount of cleaning solution.
5) the logical glue of China fully washs 3 times with cleaning solution, then with physiological saline washing 2 times, is transferred in new 50ml centrifuge tube, fully shreds to 1mm3～3mm3Size.
6) the 10ml pipette that spends leading portion is divided logical China blob of viscose equally 4 bottles of 175cm that added 25ml complete medium2In blake bottle, at the bottom of jiggling it being uniformly distributed in bottle, wherein, complete medium is in α-MEM, to add 10%FBS and 6ng/mlEGF+7ng/mlPDGF+10ng/mlTNF-α+10ng/mlIFN-gamma cells combinations of factors.
7) tag-related on blake bottle, comprises sample strip shape code, operation item, operating time, operator etc.
8) carefully blake bottle is put into 37 DEG C, 5%CO2In incubator.
9) cultivate initial 7 days, keep blake bottle absolute rest.
10) within the 8th day, according to growing state, foundation is changed liquid, is gone down to posterity, gathers in the crops frozen standard practice instructions execution.
2 passages:
1) with 75% alcohol disinfecting medium bottle outer wall, be placed in Biohazard Safety Equipment.
2) inhale and abandon old culture medium with the pipette of decaptitating, change pipette, add 10ml physiological saline in acellular cultivation face.
3) light shaking washing is organized adherent, discards, and repeats 2 times.
4) every bottle adds digestive juice 3ml, and digestive juice is 0.25% pancreatin+0.03%EDTA, and homogeneous immersion cell attachment face is hatched 1min for 37 DEG C. After cell rounding, every bottle adds stop buffer 10ml, concussion fast, and with 10ml pipette piping and druming cell attachment face, in sucking-off cell suspension to a 2 50ml centrifuge tube, every bottle, each blake bottle adds 10ml physiological saline, and purge once, imports in 50ml centrifuge tube.
5) 1200rpm, centrifugal 6min, abandons supernatant. Merge and be precipitated to 1 pipe, add 40ml physiological saline centrifuge washing again, precipitation is resuspended with 10ml complete medium, filters through cell sieve, and 5ml complete medium rinses screen cloth, counting.
6) according to cell quantity paving bottle, making cell concentration is 4.3 × 104/ ml, mark, puts 37 DEG C, 5%CO2In incubator, cultivate.
3 cell harvestings:
1) observation of cell, gathers in the crops frozen when Growth of Cells extremely approaches healing.
2) configuration stop buffer, before digestion, 3min is placed into digestive juice in 37 DEG C of water-baths, and frozen mother liquor is equilibrated to 4 DEG C.
3) Tissue Culture Flask is taken out, at the bottom of 75% alcohol disinfecting bottle, put into Biohazard Safety Equipment.
4) inhale and abandon old culture medium with pipette, change pipette, add 10ml physiological saline in acellular cultivation face, jiggle and make it cover the whole bottle end, inhale and abandon cell washing liquid, repeated washing once.
5) every bottle adds 3ml digestive juice, and 37 DEG C of digestion 1min, after cell obviously bounces back, add stop buffer 10ml/ bottle, and termination digestion is collected all liq in 50ml centrifuge tube, more every bottle adds 10ml physiological saline, soft piping and druming in the rear 50ml of remittance centrifuge tube.
6) 1200 turn/the centrifugal 6min of min, abandon supernatant, and cell precipitation suspends with 16ml physiological saline, mixes to get that 1ml counts and flow cytometer detection.
7) add physiological saline to 40ml, get 500 μ l supernatants and be endotoxin detection, 1200rpm, centrifugal 6min.
8) supernatant is poured in clean centrifuge tube, done bacterium inspection and detection of mycoplasma, centrifugation suspends with 2.5mlFBS, more slowly adds the frozen mother liquor of 2.5ml, divides and installs in cryopreservation tube, every pipe 1ml after mixing. Freeze-stored cell concentration is 5 × 106 /ml，
9) labeled cell algebraically, density, bar code and operating time are outside tube wall.
10) cryopreservation tube is placed in to the program temperature reduction box of 4 DEG C of processing in advance, places 24h in-80 DEG C of side by side combination refrigerators, then coordinate frozen personnel that sample is proceeded in liquid nitrogen container, record storage location.
Carry out result detection of the present invention and contrast by following 2 aspects.
1. the Growth of Cells form comparison that different culture media is cultivated:
Adopt the culture medium of different formulations to cultivate human umbilical cord mesenchymal stem cells, the comparison of taking pictures under inverted microscope, acquired results is as shown in Fig. 1 .d and Fig. 3 ~ 5, wherein Fig. 1 .d is the product of embodiment 1, Fig. 3 is the product of embodiment 2, Fig. 4 is the product of embodiment 3, and Fig. 5 is the cell products of commercially available MSC medium culture. Can find out from Fig. 1 .d and Fig. 3 ~ 5, umbilical cord mesenchymal stem cells form is desirable fusiformis and spindle; But the cell density of embodiment 1 is larger, the cell of embodiment 2 and embodiment 3 takes second place, and the cell density of commercially available MSC medium culture is minimum.
2. different culture media is cultivated cell number and cultivation cycle comparison:
Adopt the culture medium of different formulations to cultivate human umbilical cord mesenchymal stem cells, P0 is counted for cell, result is as follows: the cell number of embodiment 1, embodiment 2, embodiment 3 and commercially available MSC medium culture is respectively: 1.50 × 106Individual, 1.10 × 106Individual, 1.08 × 106Individual, 0.65 × 106Individual. Adopt the medium culture umbilical cord mesenchymal stem cells of different formulations, finally all gather in the crops 2.50 × 107Individual cell, adds up the incubation time in per generation, and as shown in Figure 6, embodiment 1, embodiment 2, embodiment 3 and the total incubation time of commercially available MSC culture medium are respectively: 21 days, and 24 days, 25 days, 28 days.
Result shows, the present invention adopts cultivating system and the cultural method of improvement can improve separative efficiency separating, in incubation, shortens cultivation cycle and can obtain high-purity, characterizes good cell.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

Claims (4)

1. a method that obtains safe and effective umbilical cord mesenchymal stem cells, is characterized in that: described method comprises as follows:
1), in gnotobasis, by being placed in the accumulating bottle that has added accumulating liquid after umbilical cord sterilization, be sent to laboratory and separate preparation; α-MEM that wherein accumulating liquid is 40ml, and added gentamicin 100U/ml, penicillin 50mg/ml, amphotericin B 5ug/ml; Transport temperature is 4-25 DEG C, and haulage time is no more than 24 hours;
2) umbilical cord bottle is with putting into Biohazard Safety Equipment after alcohol wipe;
3) in Tissue Culture Dish, use cleaning solution fully to wash umbilical cord 3-6 time, umbilical cord two ends are respectively cut off to 1cm length, abandon; Wash umbilical cord tissue 2-4 time again; Wherein cleaning solution is for having added 100U/ml gentamicin, 50mg/ml penicillin, the medical saline of 5ug/ml amphotericin B;
4) umbilical cord is cut into the segment of 2cm length, cleans 3 times, shift umbilical cord tissue to new culture dish, in culture dish, add physiological saline to flooding umbilical cord tissue 1/2 place; Remove vein blood vessel and arteries, tear and get the logical glue of China; The logical glue of China is placed in the 50ml centrifuge tube that adds 10ml cleaning solution;
5) the logical glue of China fully washs 3 times with cleaning solution, then with physiological saline washing 2 times, is transferred in new 50ml centrifuge tube, fully shreds to 1mm3～3mm3Size;
6) the 10ml pipette that spends leading portion is divided logical China blob of viscose equally 4 bottles of 175cm that added 25ml complete medium2In blake bottle, at the bottom of jiggling it being uniformly distributed in bottle; Wherein, complete medium is in α-MEM culture medium, to add 10wt.%FBS and following combination of cytokines: EGF5 ~ 10ng/ml+PDGF5 ~ 10ng/ml+TNF-α 3 ~ 10ng/ml+IFN-γ 3 ~ 10ng/ml;
7) carefully blake bottle is put into 37 DEG C, 5%CO2In incubator; Cultivate initial 7 days, keep blake bottle absolute rest;
8) the 8th day according to growing state, with 75% alcohol disinfecting medium bottle outer wall, is placed in Biohazard Safety Equipment;
9) inhale and abandon old culture medium with the pipette of decaptitating, change pipette, add 10ml physiological saline in acellular cultivation face, light shaking washing is organized adherent, discards, and repeats 2 times;
10) every bottle adds digestive juice 3ml, and homogeneous immersion cell attachment face is hatched 1min or incubated at room 3min for 37 DEG C; After cell rounding, every bottle adds stop buffer 10ml, concussion fast, and with 10ml pipette piping and druming cell attachment face, in sucking-off cell suspension to a 2 50ml centrifuge tube, every bottle, each blake bottle adds 10ml physiological saline, and purge once, imports in 50ml centrifuge tube;
11) 1200rpm, centrifugal 6min, abandons supernatant; Merge and be precipitated to 1 pipe, add 40ml physiological saline centrifuge washing again, precipitation is resuspended with 10ml complete medium, filters through cell sieve, and 5ml complete medium rinses screen cloth, counting;
12) according to cell quantity paving bottle, making cell concentration is 3 ~ 5 × 104/ ml, mark, puts 37 DEG C, 5%CO2In incubator, cultivate;
13) observation of cell, gathers in the crops frozen when Growth of Cells extremely approaches healing.
2. a kind of method that obtains safe and effective umbilical cord mesenchymal stem cells according to claim 1, is characterized in that: described frozen concrete steps are:
1) configuration stop buffer, before digestion, 3min is placed into digestive juice in 37 DEG C of water-baths, and frozen mother liquor is equilibrated to 4 DEG C;
2) Tissue Culture Flask is taken out, at the bottom of 75% alcohol disinfecting bottle, put into Biohazard Safety Equipment;
3) inhale and abandon old culture medium with pipette, change pipette, add 10ml physiological saline in acellular cultivation face, jiggle and make it cover the whole bottle end, inhale and abandon cell washing liquid, repeated washing once;
4) every bottle adds 3ml digestive juice, and 37 DEG C of digestion 1min, after cell obviously bounces back, add stop buffer 10ml/ bottle, and termination digestion is collected all liq in 50ml centrifuge tube, more every bottle adds 10ml physiological saline, soft piping and druming in the rear 50ml of remittance centrifuge tube;
5) 1200 turn/the centrifugal 6min of min, abandon supernatant, and cell precipitation suspends with 16ml physiological saline, mixes to get that 1ml counts and flow cytometer detection;
6) add physiological saline to 40ml, get 500 μ l supernatants and be endotoxin detection, 1200rpm, centrifugal 6min;
7) supernatant is poured in clean centrifuge tube, done bacterium inspection and detection of mycoplasma, centrifugation suspends with 2.5mlFBS, more slowly adds the frozen mother liquor of 2.5ml, divides and installs in cryopreservation tube, every pipe 1ml after mixing; Freeze-stored cell number is answered 5-10 × 106/ ml, if cell should increase and go down to posterity very little;
8) labeled cell algebraically, density, bar code and operating time are outside tube wall;
9) cryopreservation tube is placed in to the program temperature reduction box of 4 DEG C of processing in advance, places 24h in-80 DEG C of side by side combination refrigerators, then sample is proceeded in liquid nitrogen container, record storage location.
3. a kind of method that obtains safe and effective umbilical cord mesenchymal stem cells according to claim 1, is characterized in that: described combination of cytokines: EGF6ng/ml+PDGF7ng/ml+TNF-α 5ng/ml+IFN-γ 5ng/ml.
4. a kind of method that obtains safe and effective umbilical cord mesenchymal stem cells according to claim 1 and 2, is characterized in that: described digestive juice is containing 0.25wt.% pancreatin, 0.03wt.%EDTA.
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CN109628394A (en) * 2019-01-29 2019-04-16 劳敏翔 A method of extraction umbilical cord mesenchymal stem cells of the grinding in conjunction with mixed enzyme
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CN112342191A (en) * 2019-08-07 2021-02-09 路春光 hMSC production kit development and quality management standard optimization and system establishment
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CN106264558A (en) * 2016-08-01 2017-01-04 北京世纪劲得生物技术有限公司 A kind of umbilical cord, umbilical blood, Placenta Hominis collection suit
CN107114356A (en) * 2016-08-01 2017-09-01 北京世纪劲得生物技术有限公司 A kind of placenta and umbilical cord cells protect liquid
CN106190837B (en) * 2016-08-11 2018-07-20 北京昱龙盛世生物科技有限公司 A kind of kit and cultural method for cultivating mescenchymal stem cell
CN106190837A (en) * 2016-08-11 2016-12-07 北京昱龙盛世生物科技有限公司 A kind of test kit for cultivating mescenchymal stem cell and cultural method
CN106244532A (en) * 2016-09-08 2016-12-21 石家庄融和生物科技有限责任公司 The preparation method of people source 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
CN107467014A (en) * 2017-10-12 2017-12-15 北京臻惠康生物科技有限公司 A kind of method that umbilical cord tissue freezes, recovered
CN108048298A (en) * 2017-12-01 2018-05-18 何加铭 A kind of umbilical cord tissue mescenchymal stem cell separator and its control method
CN109628394A (en) * 2019-01-29 2019-04-16 劳敏翔 A method of extraction umbilical cord mesenchymal stem cells of the grinding in conjunction with mixed enzyme
CN109897818A (en) * 2019-03-20 2019-06-18 江苏瑞思坦生物科技有限公司 A kind of umbilical cord tissue cleaning solution and preparation method
CN112342191A (en) * 2019-08-07 2021-02-09 路春光 hMSC production kit development and quality management standard optimization and system establishment
CN112111450A (en) * 2020-11-02 2020-12-22 贵州北科生物科技有限公司 Culture method and application of umbilical cord mesenchymal stem cells
CN113025568A (en) * 2021-03-10 2021-06-25 云南圣衍干细胞技术开发应用股份有限公司 Culture method and application of umbilical cord mesenchymal stem cells

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