CN105647859A - Method for constructing cell transplantation slice by using human umbilical cord mesenchymal stem cells (hUC-MSCs) and amniotic membrane - Google Patents
Method for constructing cell transplantation slice by using human umbilical cord mesenchymal stem cells (hUC-MSCs) and amniotic membrane Download PDFInfo
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Abstract
The invention relates to a method for constructing a cell transplantation slice by using human umbilical cord mesenchymal stem cells (hUC-MSCs) and anthropogenic amniotic membrane. The method comprises the following steps: taking the freshly collected anthropogenic amniotic membrane and umbilical cord, separating chorion from the amniotic membrane, treating the separated amniotic membrane by normal saline, and then cryopreserving for later use; cutting the umbilical cord into small pieces, and tearing Wharton's jelly off; cutting the Wharton's jelly into pieces, putting the cut Wharton's jelly onto a sterile culture vessel, adding a culture solution into the sterile culture vessel, and putting the sterile culture vessel into an incubator for culturing to obtain primary generation hUC-MSCs; adding pancreatin into a culture medium of the primary generation hUC-MSCs, digesting, and continuously culturing in the incubator to obtain the hUC-MSCs; carrying out sterile hydration on the cryopreserved standby amniotic membrane, then flatly laying the amniotic membrane on an amniotic membrane carrier thimble, and then moving into the sterile culture vessel; inoculating the hUC-MSCs in the culture vessel, and then culturing in the incubator to obtain the cell transplantation slice with cell confluence reaching up to 80-90%. The cell transplantation slice prepared by the method is capable of carrying out damage repair at specific parts, so that the defects of using the hUC-MSCs or amniotic membrane alone for treatment are overcome; therefore, a new treatment strategy is provided for refractory diseases, and a new therapy approach is opened up for the clinical application of stem cells.
Description
Technical field
The invention belongs to biomedicine field, it is specifically related to human umbilical cord mesenchymal stem cells and the method for amnion structure Transplanted cells sheet.
Background technology
Stem cell is that a class has different differentiation potential, and under undifferentiated state the cell of self. Stem-cell therapy refers to answers employment stem cell that is autologous or xenogenic origin to input (or implantation) human body after manipulation in vitro, for the process of disease treatment. The multipotent stem cells of adult stem cell, embryonic stem cell and induction is mainly comprised for the stem cell of cell therapy. Carry out multinomial stem cell clinical application research both at home and abroad at present, it relates to multiple stem cell type and multiple disease type. Main disease type comprises bone and joint diseases, liver cirrhosis, graft host rejection reaction (GVHD), Spinal injury and degenerative neural disease, eye surface diseases and diabetes etc. Wherein many stem cell types are the mescenchymal stem cells from marrow, fatty tissue, Cord blood, umbilical cord or placenta tissue source, and they have certain Multidirectional Differentiation potential and anti-inflammation and immunoregulation ability etc.
Amnion was just applied to surgery as a kind of dressing before more than 100 years, was applied in surgical skin transplantation as a kind of donor material at the beginning of last century. Amnion is the innermost layer of placenta, smooth, without blood vessel, nerve and lymph, has certain elasticity. Amnion has the function alleviating inflammation degree, shortening the inflammation time length, suppress new vessel formation and suppress fibrosis, lessen scar formation to be formed. Utilize on clinical amnion have promote epithelial adherence growth and propagation, alleviate inflammation, suppress new vessel formed, reduce the characteristic such as scar proliferation, antiblocking and it is applied to serious acute phase or old Ocular surface burns treatment in go, obtain good clinical effectiveness.
At present, the clinical main application approach of stem cell has the injection of venous re-transfusion, diseased region, lumbar puncture injection, intubation intervention etc., but for some diseases, need stem cell when specific position plays a role as limbal stem cell deficiency, bone and joint diseases, uterine endometrium are adhered etc., these application approach less effective.Needing during these diseases of clinical treatment that stem cell is fixed on diseased region makes it play a role, and the dirt settling of fixing stem cell need to not produce rejection and can degrade voluntarily. The present invention studies for addressing this problem.
Summary of the invention
In sum, in order to overcome the deficiency of prior art problem, the present invention provides a kind of end user's umbilical cord mesenchymal stem cells and the method for people source amnion structure Transplanted cells sheet. Human umbilical cord mesenchymal stem cells English name is: hUC-MSCs, human umbilical cord mesenchymal stem cells (hUC-MSCs) is inoculated on amnion by the present invention, injury repairing can be carried out at privileged site, compensate for the deficiency being used alone hUC-MSCs or amniotic membrane for treatment, for some refractory diseases provide new therapeutic strategy, also for the clinical application of stem cell opens new therapy approach.
In order to achieve the above object, the technical solution used in the present invention is:
A kind of end user's umbilical cord mesenchymal stem cells (hUC-MSCs) and people source amnion build the method for Transplanted cells sheet, and it comprises the steps:
(1) get people's amnion and the umbilical cord of fresh collection, after cleaning, umbilical cord is put into physiological saline and soaks for subsequent use;
(2) amnion process: first the clean amnion blunt separation of cleaning is fallen chorion, re-use the moistening gauze of physiological saline by amnion wiped clean, then amnion is soaked in the physiological saline containing penicillin 50��200U/ml and Vetstrep 50��200U/ml and soaks after 0.5��1 hour, put into sterile glycerol 2��8 DEG C of dehydrations, dewatering time is no more than 12 hours, then proceeds in sterile glycerol and save backup at-20 DEG C;
(3) segment that step (1) soaked umbilical cord is cut into 1��2cm length, cleans, is then transferred in culture dish by umbilical cord tissue, adds physiological saline to flooding umbilical cord tissue 1/2 place in culture dish; By umbilical cord one end along vein blood vessel parallel direction clip, with organizing tweezer to tear along cut-out direction vein blood vessel edge longitudinal, 1 vein blood vessel and two arteries are cutd open from, remove amnion with organizing tweezer, tear and get China's Tong Shi glue, be placed in the centrifuge tube being added with physiological saline;
(4) by China's Tong Shi glue, fully wash with physiological saline, then shred to 1��3mmSize, is evenly placed into the tissue block shredded in sterile petri dish, paves, and slowly adds 5��10ml nutrient solution along culture dish edge, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, within every three days, change a nutrient solution, cultivate after 15��20 days, until observing when forming multiple not of uniform size, cell island that cell quantity is intensive at the bottom of ware, then obtaining primary hUC-MSCs;
(5) nutrient solution is moved out completely, pancreatin 1��3ml that mass percent is 0.05��0.25% is added in primary hUC-MSCs culture dish, put into the digestion of 37 DEG C of incubators, when observing attached cell circle contracting, and at the bottom of having departed from bottle after, add 5��10ml nutrient solution and terminate digestion, suspension is moved in centrifuge tube, centrifugal abandons supernatant, centrifuge tube adds new nutrient solution, repeatedly blow and beat and be separated into individual cells suspension to it, then cell suspension be adjusted to 4000��6000 cells/2System be inoculated in culturing bottle to be placed in 37 DEG C, saturated humidity, volume fraction be 5% case resume of incubating cultivate, within every 3��4 days, change nutrient solution once, reach 80��90% to degrees of fusion, obtain hUC-MSCs, by frozen for subsequent use after cell harvesting;
(6) get amnion frozen in step (2), put into stroke-physiological saline solution aquation 10��30 minutes, amnion good for aquation is moved to sterile petri dish for subsequent use;
(7) amnion prepared by step (6) is laid on the amnion carrier collar, then moves in sterile petri dish, put into the CO of 37 DEG CIncubate in case stand-by;
(8) frozen hUC-MSCs is recovered, cell density is adjusted to 4000��6000 cells/2System is inoculated in the culture dish with step (6), be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating after cultivating 3��5 days in case, cytogamy degree reaches 80��90% and namely obtains Transplanted cells sheet.
Further, the physiological saline containing penicillin 50��200U/ml and Vetstrep 50��200U/ml in described step (1) is dissolved into injection penicillin, Vetstrep in the sodium chloride injection of 0.9% according to concentration ratio 1:1 to obtain.
Further, the sodium chloride injection that the cleaning in described step (1), (2) and step (3) is 0.9% cleans.
Further, the described nutrient solution in step (4) is the foetal calf serum gained that DMEM/F12 nutrient solution (purchased from Gibco company) adds 10��20%.
The streaming Phenotypic examination of the present invention comprises the following steps:
1. get four streaming pipes, it is labeled as separation passage cell sample tube 1 respectively and is separated passage cell sample tube 2; Transplanted cells sheet digests cell sample pipe 3 and Transplanted cells sheet digest cell sample pipe 4.
2. by the step (5) of identical amount, separation passage cell and step (8) Transplanted cells sheet digest cell (the number of nucleated cells 2-5*10 got off respectively5Individual) join in each streaming pipe of corresponding mark, tube wall can not be adhered to.
3. it is separated passage cell sample tube 1 and adds CD34-FITC, CD29-PE, CD45-percp, CD44-APC; Separation passage cell sample tube 2 adds CD105-PE, CD45-percp, HLA_DR-APC; Transplanted cells sheet digests cell sample pipe 3 and adds CD34-FITC, CD29-PE, CD45-percp, CD44-APC; Transplanted cells sheet digests cell sample pipe 4 and adds each 5ul of CD105-PE, CD45-percp, HLA_DR-APC, mixed even.
4. room temperature lucifuge places 15 minutes, adds 2mlPBS/ pipe, 1400rpm/min, centrifugal 5min.
5. abandon supernatant, pipe inner cell is upspring, adds 0.5mlPBS, mixed even, upper machine testing.
6. go up machine testing result to obtain: CD29, CD44, CD105 expression rate is greater than 95%, it is the positive; CD34, CD45, HLA-DR expression rate is less than 2%, is feminine gender.
7. above-mentioned explanation: according to the preparation method hUC-MSCs that obtains of separation be inoculated in amnion after stem cell meet CD29, CD44, CD105 expression rate and be greater than 95%, be the positive; CD34, CD45, HLA-DR expression rate is less than 2%, is the result of feminine gender, through being accredited as mescenchymal stem cell.
The Sterility testing of the present invention be the nutrient solution got in step (8) Transplanted cells sheet culture dish as trial-product, get sulphur glycollate culture medium 6, pancreas junket soya peptone liquid nutrient medium 4 (often prop up substratum amount and be 15 milliliters). Inoculating 1ml trial-product respectively for 4 in 6 sulphur glycollate culture mediums, remain two 1 inoculations and be not less than 100cfu streptococcus aureus and do positive control, 1 is done negative control. Inoculating 1ml trial-product respectively for two in 4 pancreas junket soya peptone substratum, remain two 1 inoculations and be not less than 100cfu Candida albicans, 1 is done negative control. The container bifurcation of 4 THIOGLYCOLLIC ACID salt fluid substratum of inoculation trial-product is placed in 30��35 �� of C and cultivates, and two are placed in 20��25 �� of C and cultivate, and positive control and negative control are placed in 30��35 �� of C and cultivate.Pancreas junket soya peptone substratum 4 is all placed in 20��25 �� of C and cultivates. Incubation time is 14 days. Should observe day by day between incubation period and record and whether have bacteria growing. Positive control should have bacteria growing in 24 hours, and sample hose does not have bacterium and fungal growth. (THIOGLYCOLLIC ACID salt fluid substratum in Sterility testing and pancreas junket soya peptone substratum are all according to the Pharmacopoeia of the People's Republic of China 2015 editions preparation).
Useful effect:
1, multinomial stem cell (referring to non-hematopoietic stem cell) clinical application research has been carried out both at home and abroad at present, it relates to multiple stem cell type and multiple disease type. Main disease type comprises bone and joint diseases, liver cirrhosis, graft host rejection reaction (GVHD), Spinal injury and degenerative neural disease, eye surface diseases and diabetes etc. The mescenchymal stem cell in umbilical cord tissue source, has certain Multidirectional Differentiation potential and anti-inflammation and immunoregulation ability etc., and wide material sources, belong to Biohazard Waste and easily obtain. But it is when repairing such as eye surface diseases, joint repair and uterine endometrium for some diseases, it may also be useful to current therapy approach less effective, limit its application. Amnion can alleviate inflammation degree, shortens the inflammation time length, suppress new vessel formed and suppress the function of fibrosis, lessen scar formation formation. Clinical upper amnion is used alone and is applied to a table, joint and endometrial surface and has obtained some gratifying clinical effectiveness, but owing to lacking seed cell for the serious patient's limited efficiency of tissue injury. HUC-MSCs is inoculated on amnion by the present invention so that it is the fixing injury repairing carrying out privileged site again, compensate for the deficiency being used alone hUC-MSCs or amniotic membrane for treatment so that it is effect is more remarkable.
2, hUC-MSCs and people source amnion are combined by the present invention, learn from other's strong points to offset one's weaknesses, for some refractory diseases provide new therapeutic strategy, also for the clinical application of stem cell opens new therapy approach.
3, the present invention uses our unit's patent of invention " the amnion carrier collar ", adds the operability of technology, and after making graft, Clinical practice is convenient, for this new therapy approach of stem cell clinical application adds feasibility.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated:
Embodiment 1
Using hUC-MSCs and amnion to build a method for Transplanted cells sheet, it comprises the steps:
(1) getting people's amnion and the umbilical cord of fresh collection, the rear umbilical cord of cleaning is put into physiological saline and is soaked for subsequent use;
(2) amnion process: the clean amnion blunt separation of cleaning is fallen chorion, re-use the moistening gauze of physiological saline by amnion wiped clean, then amnion is soaked in the physiological saline containing penicillin 150U/ml and Vetstrep 150U/ml and soaks after 0.5 hour, put into sterile glycerol 4 DEG C of dehydrations, dewatering time is no more than 12 hours, then proceeds in sterile glycerol and save backup at-20 DEG C;
(3) segment that soaked umbilical cord is cut into about 1��2cm length, cleans, is then transferred in culture dish by umbilical cord tissue, adds physiological saline to flooding umbilical cord tissue 1/2 place in culture dish; By umbilical cord one end along vein blood vessel parallel direction clip, with organizing tweezer to tear along cut-out direction vein blood vessel edge longitudinal, 1 vein blood vessel and 2 arteries are cutd open from, remove amnion with organizing tweezer, tear and get China's Tong Shi glue, be placed in the centrifuge tube being added with physiological saline;
(4) China Tong Shi glue physiological saline is fully washed, then shred to 1��3mmSize, is evenly placed into the tissue block shredded in sterile petri dish, paves, and slowly adds 5ml nutrient solution along culture dish edge, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, within every three days, change a nutrient solution, cultivate after 15 days, until observing when forming multiple not of uniform size, cell island that cell quantity is intensive at the bottom of ware, then obtaining primary hUC-MSCs;
(5) nutrient solution is moved out completely, the pancreatin 1ml that mass percent is 0.05% is added in original cuiture ware, put into the digestion of 37 DEG C of incubators, when observing attached cell circle contracting, and at the bottom of having departed from bottle after, add 5ml nutrient solution and terminate digestion, suspension is moved in centrifuge tube, centrifugal abandons supernatant, centrifuge tube adds new nutrient solution, repeatedly blow and beat and be separated into individual cells suspension to it, then cell suspension be adjusted to 4000 cells/2System be inoculated in culturing bottle to be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubate case resume to cultivate, within every 3-4 days, change nutrient solution once, reach 80-90% to degrees of fusion, obtain hUC-MSCs, by frozen for subsequent use after cell harvesting;
(6) get amnion frozen in step (2), put into stroke-physiological saline solution aquation 10 minutes, good for aquation amnion is moved to sterile petri dish for subsequent use;
(7) amnion prepared by step (6) is laid on the amnion carrier collar, then moves in sterile petri dish, put into 37 DEG C of COIncubate in case stand-by;
(8) frozen hUC-MSCs is recovered, cell density is adjusted to 4000 cells/2System is inoculated in the culture dish with step (7), be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, after 3-5 days, cytogamy degree reaches 80-90% and namely obtains Transplanted cells sheet.
The streaming Phenotypic examination of the present embodiment comprises the following steps:
1. get four streaming pipes, it is labeled as separation passage cell sample tube 1 respectively and is separated passage cell sample tube 2; Transplanted cells sheet digests cell sample pipe 3 and Transplanted cells sheet digest cell sample pipe 4.
2. by the step (5) of identical amount, separation passage cell and step (8) Transplanted cells sheet digest cell (the number of nucleated cells 2-5*10 got off respectively5Individual) join in each streaming pipe of corresponding mark, tube wall can not be adhered to.
3. it is separated passage cell sample tube 1 and adds CD34-FITC, CD29-PE, CD45-percp, CD44-APC; Separation passage cell sample tube 2 adds CD105-PE, CD45-percp, HLA_DR-APC; Transplanted cells sheet digests cell sample pipe 3 and adds CD34-FITC, CD29-PE, CD45-percp, CD44-APC; Transplanted cells sheet digests cell sample pipe 4 and adds each 5ul of CD105-PE, CD45-percp, HLA_DR-APC, mixed even.
4. room temperature lucifuge places 15 minutes, adds 2mlPBS/ pipe, 1400rpm/min, centrifugal 5min.
5. abandon supernatant, pipe inner cell is upspring, adds 0.5mlPBS, mixed even, upper machine testing.
6. go up machine testing result to obtain: CD29, CD44, CD105 expression rate is greater than 95%, it is the positive; CD34, CD45, HLA-DR expression rate is less than 2%, is feminine gender.
7. above-mentioned explanation: according to the preparation method hUC-MSCs that obtains of separation be inoculated in amnion after stem cell meet CD29, CD44, CD105 expression rate and be greater than 95%, be the positive;CD34, CD45, HLA-DR expression rate is less than 2%, is the result of feminine gender, through being accredited as mescenchymal stem cell.
The Sterility testing of the present embodiment be the nutrient solution got in step (8) Transplanted cells sheet culture dish as trial-product, get sulphur glycollate culture medium 6, pancreas junket soya peptone liquid nutrient medium 4 (often prop up substratum amount and be 15 milliliters). Inoculating 1ml trial-product respectively for 4 in 6 sulphur glycollate culture mediums, remain two 1 inoculations and be not less than 100cfu streptococcus aureus and do positive control, 1 is done negative control. Inoculating 1ml trial-product respectively for two in 4 pancreas junket soya peptone substratum, remain two 1 inoculations and be not less than 100cfu Candida albicans, 1 is done negative control. The container bifurcation of 4 THIOGLYCOLLIC ACID salt fluid substratum of inoculation trial-product is placed in 30��35 �� of C and cultivates, and two are placed in 20��25 �� of C and cultivate, and positive control and negative control are placed in 30��35 �� of C and cultivate. Pancreas junket soya peptone substratum 4 is all placed in 20��25 �� of C and cultivates. Incubation time is 14 days. Should observe day by day between incubation period and record and whether have bacteria growing. Positive control should have bacteria growing in 24 hours, and sample hose does not have bacterium and fungal growth. (THIOGLYCOLLIC ACID salt fluid substratum in Sterility testing and pancreas junket soya peptone substratum are all according to the Pharmacopoeia of the People's Republic of China 2015 editions preparation).
Embodiment 2
Using hUC-MSCs and amnion to build a method for Transplanted cells sheet, it comprises the steps:
(1) getting people's amnion and the umbilical cord of fresh collection, the rear umbilical cord of cleaning is put into physiological saline and is soaked for subsequent use;
(2) amnion process: the clean amnion blunt separation of cleaning is fallen chorion, re-use the moistening gauze of physiological saline by amnion wiped clean, then amnion is soaked in the physiological saline containing penicillin 200U/ml and Vetstrep 200U/ml and soaks after 1 hour, put into sterile glycerol 8 DEG C of dehydrations, dewatering time is no more than 12 hours, then proceeds in sterile glycerol and save backup at-20 DEG C;
(3) segment that soaked umbilical cord is cut into about 1��2cm length, cleans, is then transferred in culture dish by umbilical cord tissue, adds physiological saline to flooding umbilical cord tissue 1/2 place in culture dish; By umbilical cord one end along vein blood vessel parallel direction clip, with organizing tweezer to tear along cut-out direction vein blood vessel edge longitudinal, 1 vein blood vessel and 2 arteries are cutd open from, remove amnion with organizing tweezer, tear and get China's Tong Shi glue, be placed in the centrifuge tube being added with physiological saline;
(4) China Tong Shi glue physiological saline is fully washed, then shred to 1��3mmSize, is evenly placed into the tissue block shredded in sterile petri dish, paves, and slowly adds 10ml nutrient solution along culture dish edge, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, within every three days, change a nutrient solution, cultivate after 17 days, until observing when forming multiple not of uniform size, cell island that cell quantity is intensive at the bottom of ware, then obtaining primary hUC-MSCs;
(5) nutrient solution is moved out completely, the pancreatin 3ml that mass percent is 0.25% is added in original cuiture ware, put into the digestion of 37 DEG C of incubators, when observing attached cell circle contracting, and at the bottom of having departed from bottle after, add 10ml nutrient solution and terminate digestion, suspension is moved in centrifuge tube, centrifugal abandons supernatant, centrifuge tube adds new nutrient solution, repeatedly blow and beat and be separated into individual cells suspension to it, then cell suspension be adjusted to 6000 cells/2System be inoculated in culturing bottle to be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubate case resume to cultivate, within every 3-4 days, change nutrient solution once, reach 80-90% to degrees of fusion, obtain hUC-MSCs, by frozen for subsequent use after cell harvesting;
(6) get amnion frozen in step (2), put into stroke-physiological saline solution aquation 30 minutes, good for aquation amnion is moved to sterile petri dish for subsequent use;
(7) amnion prepared by step (6) is laid on the amnion carrier collar, then moves in sterile petri dish, put into 37 DEG C of COIncubate in case stand-by;
(8) frozen hUC-MSCs is recovered, cell density is adjusted to 6000 cells/2System is inoculated in the culture dish with step (7), be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, after 3-5 days, cytogamy degree reaches 80-90% and namely obtains Transplanted cells sheet.
The streaming Phenotypic examination step of the present embodiment is identical with embodiment 1.
The Sterility testing step of the present embodiment is identical with embodiment 1.
Embodiment 3
Using hUC-MSCs and amnion to build a method for Transplanted cells sheet, it comprises the steps:
(1) getting people's amnion and the umbilical cord of fresh collection, the rear umbilical cord of cleaning is put into physiological saline and is soaked for subsequent use;
(2) amnion process: the clean amnion blunt separation of cleaning is fallen chorion, re-use the moistening gauze of physiological saline by amnion wiped clean, then amnion is soaked in the physiological saline containing penicillin 50U/ml and Vetstrep 50U/ml and soaks 0.5-1 after individual hour, put into sterile glycerol 2 DEG C of dehydrations, dewatering time is no more than 12 hours, then proceeds in sterile glycerol bottle and save backup at-20 DEG C;
(3) segment that soaked umbilical cord is cut into about 1��2cm length, cleans, is then transferred in culture dish by umbilical cord tissue, adds physiological saline to flooding umbilical cord tissue 1/2 place in culture dish; By umbilical cord one end along vein blood vessel parallel direction clip, with organizing tweezer to tear along cut-out direction vein blood vessel edge longitudinal, 1 vein blood vessel and 2 arteries are cutd open from, remove amnion with organizing tweezer, tear and get China's Tong Shi glue, be placed in the centrifuge tube being added with physiological saline;
(4) China Tong Shi glue physiological saline is fully washed, then shred to 1��3mmSize, is evenly placed into the tissue block shredded in sterile petri dish, paves, and slowly adds 8ml nutrient solution along culture dish edge, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubate in case and cultivate, within every three days, change a nutrient solution, cultivate after 20 days, until observing when forming multiple not of uniform size, cell island that cell quantity is intensive at the bottom of ware, then acquisition hUC-MSCs primary cell;
(5) nutrient solution is moved out completely, pancreatin 1��2ml that mass percent is 0.25% is added in original cuiture ware, put into the digestion of 37 DEG C of incubators, when observing attached cell circle contracting, and at the bottom of having departed from bottle after, add 8ml nutrient solution and terminate digestion, suspension is moved in centrifuge tube, centrifugal abandons supernatant, centrifuge tube adds new nutrient solution, repeatedly blow and beat and be separated into individual cells suspension to it, then cell suspension be adjusted to 5000 cells/2System be inoculated in culturing bottle to be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubate case resume to cultivate, within every 3-4 days, change nutrient solution once, reach 80-90% to degrees of fusion, obtain hUC-MSCs, by frozen for subsequent use after cell harvesting;
(6) get amnion frozen in step (2), put into stroke-physiological saline solution aquation 20 minutes, good for aquation amnion is moved to sterile petri dish for subsequent use;
(7) amnion prepared by step (6) is laid on the amnion carrier collar, then moves in sterile petri dish, put into 37 DEG C of COIncubate in case stand-by;
(8) frozen hUC-MSCs is recovered, cell density is adjusted to 5000 cells/2System is inoculated in the culture dish with step (7), be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, after 3-5 days, cytogamy degree reaches 80-90% and namely obtains Transplanted cells sheet.
The streaming Phenotypic examination step of the present embodiment is identical with embodiment 1.
The Sterility testing step of the present embodiment is identical with embodiment 1.
Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it being made some modifications or improvements, this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (4)
1. end user's umbilical cord mesenchymal stem cells and people source amnion build the method for Transplanted cells sheet, it is characterised in that, comprise the steps:
(1) get people's amnion and the umbilical cord of fresh collection, after cleaning, umbilical cord is put into physiological saline and soaks for subsequent use;
(2) amnion process: first the clean amnion blunt separation of cleaning is fallen chorion, re-use the moistening gauze of physiological saline by amnion wiped clean, then amnion is soaked in the physiological saline containing penicillin 50��200U/ml and Vetstrep 50��200U/ml and soaks after 0.5��1 hour, put into sterile glycerol 2��8 DEG C of dehydrations, dewatering time is no more than 12 hours, then proceeds in sterile glycerol and save backup at-20 DEG C;
(3) segment that step (1) soaked umbilical cord is cut into 1��2cm length, cleans, is then transferred in culture dish by umbilical cord tissue, adds physiological saline to flooding umbilical cord tissue 1/2 place in culture dish; By umbilical cord one end along vein blood vessel parallel direction clip, with organizing tweezer to tear along cut-out direction vein blood vessel edge longitudinal, 1 vein blood vessel and two arteries are cutd open from, remove amnion with organizing tweezer, tear and get China's Tong Shi glue, be placed in the centrifuge tube being added with physiological saline;
(4) by China's Tong Shi glue, fully wash with physiological saline, then shred to 1��3mmSize, is evenly placed into the tissue block shredded in sterile petri dish, paves, and slowly adds 5��10ml nutrient solution along culture dish edge, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating in case and cultivate, within every three days, change a nutrient solution, cultivate after 15��20 days, until observing when forming multiple not of uniform size, cell island that cell quantity is intensive at the bottom of ware, then obtaining primary hUC-MSCs;
(5) nutrient solution is moved out completely, pancreatin 1��3ml that mass percent is 0.05��0.25% is added in primary hUC-MSCs culture dish, put into the digestion of 37 DEG C of incubators, when observing attached cell circle contracting, and at the bottom of having departed from bottle after, add 5��10ml nutrient solution and terminate digestion, suspension is moved in centrifuge tube, centrifugal abandons supernatant, centrifuge tube adds new nutrient solution, repeatedly blow and beat and be separated into individual cells suspension to it, then cell suspension be adjusted to 4000��6000 cells/2System be inoculated in culturing bottle to be placed in 37 DEG C, saturated humidity, volume fraction be 5% case resume of incubating cultivate, within every 3��4 days, change nutrient solution once, reach 80��90% to degrees of fusion, obtain hUC-MSCs, by frozen for subsequent use after cell harvesting;
(6) get amnion frozen in step (2), put into stroke-physiological saline solution aquation 10��30 minutes, amnion good for aquation is moved to sterile petri dish for subsequent use;
(7) amnion prepared by step (6) is laid on the amnion carrier collar, then moves in sterile petri dish, put into the CO of 37 DEG CIncubate in case stand-by;
(8) frozen hUC-MSCs is recovered, cell density is adjusted to 4000��6000 cells/2System is inoculated in the culture dish with step (6), be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%Incubating after cultivating 3��5 days in case, cytogamy degree reaches 80��90% and namely obtains Transplanted cells sheet.
2. a kind of end user's umbilical cord mesenchymal stem cells according to claim 1 and people source amnion build the method for Transplanted cells sheet, it is characterised in that: the physiological saline containing penicillin 50��200U/ml and Vetstrep 50��200U/ml in described step (1) is dissolved into injection penicillin, Vetstrep in the sodium chloride injection of 0.9% according to concentration ratio 1:1 to obtain.
3. a kind of end user's umbilical cord mesenchymal stem cells according to claim 1 and people source amnion build the method for Transplanted cells sheet, it is characterised in that: the cleaning in described step (1), (2) and step (3) is the sodium chloride injection cleaning of 0.9%.
4. a kind of end user's umbilical cord mesenchymal stem cells according to claim 1 and people source amnion build the method for Transplanted cells sheet, it is characterised in that: the described nutrient solution in step (4) be add purchased from Gibco company DMEM/F12 nutrient solution 10��20% foetal calf serum gained.
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