CN1786154A - Human placenta, umbilical cord mesenchyma stemcell stock and its construction method - Google Patents
Human placenta, umbilical cord mesenchyma stemcell stock and its construction method Download PDFInfo
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- CN1786154A CN1786154A CNA2005100154922A CN200510015492A CN1786154A CN 1786154 A CN1786154 A CN 1786154A CN A2005100154922 A CNA2005100154922 A CN A2005100154922A CN 200510015492 A CN200510015492 A CN 200510015492A CN 1786154 A CN1786154 A CN 1786154A
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Abstract
The invention discloses a human placenta, umbilical cord mesenchyme stem cell bank and its making method. The method includes the following steps: the first is taking human placenta, umbilical cord to test; smashing after cleaning by phosphate buffer; adding phosphate buffer to dilute; the second is adding collagenase to digest; the third is adding phosphate buffer; the fourth is adding trypsinization; the fifth is mixing the cell from the second and fourth, centrifuging, removing supernatant, cleaning by the phosphate buffer, centrifuging, and removing supernatant; the mesenchyme stem cell are gained; the sixth is freezing them in liquid nitrogen; saving by ABO/Rh subtype and HLA subtype; setting recallable cell information file. The invention is newborn storage placenta and umbilical cord mesenchyme stem cell. It can offer mesenchyme stem cell to treat the disease for personal, family and others.
Description
Technical field
The present invention relates to a kind of stem cell bank and construction process thereof, specifically relate to a kind of people's placenta, Umbilical Filling stem cell's storehouse library And Construct method.
Technical background:
(mesenchymal stem cells MSCs) is a type of stem cell to mescenchymal stem cell, possesses two key characters of stem cell: very strong self duplication ability and many differentiation potentials.MSCs originates from mesoderm, and theoretically, it can be to other mesoderm tissue differentiation.Recent research shows that MSCs not only can be divided into mesoblastic stroma in suitable body or under the external environment, also maintains endoblastic differentiation potential, can be divided into neurocyte, epithelial cell, myocardial cell and scleroblast etc.Midbrain or striatal cell co-cultivation with MSCs and tire mouse, it can be divided into neurone and spongiocyte, and MSCs is transplanted to around the myocardial ischemia necrotic area, gradually on every side the myocardial cell is similar after 3 weeks, and as seen new vesseles are around newborn myocardial cell in a large number, and these all show the differentiation due of MSCs and microenvironment is closely related on every side.Most scholars thinks that the differentiation of MSCs is determined that by special transcription factor different inductive conditions can determine its differentiation direction now, may be the transcription factor that these inductive conditions have started the decision differentiation direction.
MSCs has the multidirectional differentiation potential of similar embryonic stem cell.Therefore in this sense, perhaps the potential use that MSCs is the most far-reaching is to produce cell and tissue, can be used as a kind of application mode of current " cell therapy ".Numerous disease and functional disorder are often because due to cell dysfunction or the disorganization.Nowadays, the organ of some donations and organizing usually in order to replace sick or destroyed tissue.Regrettably, be subjected to the number of patients of these diseases torments considerably beyond supplying transplanted organ quantity.MSCs can grow after stimulating and be divided into specific cell, make alternative cell and tissue-derived renewal become possibility, thereby can be used for treating countless diseases, uncomfortable situation and deformity, comprise Parkinson's disease, Alzheimer disease (dementia), Spinal injury, apoplexy, burn, heart trouble, diabetes, osteoarthritis and rheumatoid arthritis.Present countries in the world have all dropped into great amount of manpower and material resources and financial resources have been carried out extensive and deep research, the method of utilizing MSCs to transplant, have simple, practical, expense is low and do not have advantage such as immunological rejection, but and MSCs from it source, separation method and differentiated tissues on its special advantages is all arranged, application prospect is very wide.
At present the main source of MSCs is adult's marrow, but adult's bone marrow MSCs cell quantity and proliferation and differentiation potential descends with the increase at age, and viral infection rate is higher, and the collection of donor MSCs must the row bone marrow puncture, and the source is restricted.Discover that multiple tissue extensively exists MSCs at human fetal with after giving birth to, and comprises periosteum, fat, amniotic fluid, corium, tooth, skeletal muscle, tire lung, tire liver and bleeding of the umbilicus etc.But, extracts MSCs in the fetus body and the bodily tissue of aborted fetus need be destroyed, so this kind The Application of Technology is subjected to the query of moral ethics and the restriction of traditional concept again.Therefore, the source of seeking new MSCs becomes at present the focus of research both at home and abroad.We have carried out the research to placenta and umbilical cord source MSCs.According to document, adopt traditional endothelium digestion method, during from the subcutaneous separation MSCs like cell of fetal cord endothelium/interior, we find institute's cell count that obtains seldom, and success ratio is low, has only 30% sample can obtain MSCs, and 70% sample can not obtain the MSCs that can repeatedly go down to posterity.
Stem cell bank is to be about the place that (profound hypothermia) stores stem cell or collect storage stem cell resource related data in-196 ℃ of liquid nitrogen.A perfect stem cell bank should possess the ability that healthy stem cell is provided clinical use whenever and wherever possible.At present worldwide, by stored source of human stem cell and acquisition mode, stem cell bank mainly is divided into cord blood stem cell bank, marrow and peripheral hematopoietic stem cells storehouse.The work that cord blood stem cell bank is done be with behind the neonatal cord blood collection through separating freezing and storing.In every part of bleeding of the umbilicus, approximately contain millions of stem cells, enough use for a child or juvenile patient.If in adult sequela, the stem cell population that a bleeding of the umbilicus provided is just not enough, but can be resolved by the amplification technique of stem cell in the future.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 marrow or the peripheral hematopoietic stem cells 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 but the patient's that autologous stem cells is not saved demand to satisfy the demand.Then be the part stem cell that is stored in oneself birth or gathers when healthy from the body storehouse, use when own sick.Still do not have placenta and umbilical cord mesenchyma stem cell at present in the world, annual a large amount of neonatal placentas and umbilical cord go out of use, and stem cell resource wherein runs off in vain.
Summary of the invention:
The object of the present invention is to provide and a kind ofly make full use of neonatal placenta and umbilical cord is people's placenta, the umbilical cord mesenchyma stem cell of resource.
Second purpose of the present invention provides the construction process of a kind of people's placenta, umbilical cord mesenchyma stem cell.
Technical scheme of the present invention is summarized as follows:
A kind of people's placenta, umbilical cord mesenchyma stem cell are to be built up by following step:
(1) get people's placenta, umbilical cord carries out the detection of ABO/Rh blood group, the detection of HLA somatotype, microorganism immunodetection, with pH is the phosphoric acid buffer wash and remove residual blood of 7.2-7.4, pulverize, add 1～1.5 times of phosphoric acid buffer dilution (pH is 7.2-7.4) to the crushed material volume;
(2) 1/2～1/3 of adding step (1) liquor capacity that obtains mass/volume is than the collagenase phosphoric acid buffer liquor digestion that is 0.05%～0.2%, temperature is 37 ℃, digested 40～70 minutes, preferred 60 minutes, cross the 100-200 mesh sieve, filterable cell suspension and digestion tissue are not separated;
(3) in digestion is not organized, add 1～1.5 times to the phosphoric acid buffer dilution (pH is 7.2-7.4) that does not digest tissue volume;
(4) 1/2～1/3 of adding step (3) liquor capacity that obtains mass/volume is than the pancreatin phosphoric acid buffer liquor digestion that is 0.05%～0.2%, the digestion temperature is 37 ℃, digestion time is 10～30 minutes, preferred 20 minutes, the serum that adds 1-2g in this 100ml solution stops the pancreatin effect, cross the 100-200 mesh sieve, filtered solution is a cell suspension;
(5) cell suspension that step (2) and step (4) are obtained mixes, and is centrifugal, and the rotating speed of whizzer is 1500 to change～2500 rev/mins, time is 10～20 minutes, abandoning supernatant is removed residual enzyme 1～3 time with phosphoric acid buffer (pH is 7.2-7.4) washing, and is centrifugal again, the rotating speed of whizzer is 1500 rev/mins～2500 rev/mins, time is 10～20 minutes, and abandoning supernatant promptly obtains mescenchymal stem cell, the two times centrifugal rotating speed is preferably 2000 rev/mins, and the time is 15 minutes;
(6) mescenchymal stem cell is put liquid nitrogen freezing, the temperature of liquid nitrogen freezing is-196 ℃, preserves by its ABO/Rh somatotype and HLA somatotype, and foundation can promptly be constructed people's placenta, umbilical cord mesenchyma stem cell for the cell news file of retrieval.
With collagenase and trysinization the time, available rotor stirs simultaneously, improves the degree of digestion
The construction process of a kind of people's placenta, umbilical cord mesenchyma stem cell, form by following step:
(1) get people's placenta, umbilical cord carries out the detection of ABO/Rh blood group, the detection of HLA somatotype, microorganism immunodetection, with phosphoric acid buffer (pH is 7.2-7.4) wash and remove residual blood, pulverize, add 1～1.5 times of phosphoric acid buffer dilution (pH is 7.2-7.4) to the crushed material volume;
(2) 1/2～1/3 of adding step (1) liquor capacity that obtains mass/volume is than the collagenase phosphoric acid buffer liquor digestion that is 0.05%～0.2%, temperature is 37 ℃, digested 40～70 minutes, preferred 50-60 minute, cross the 100-200 mesh sieve, filterable cell suspension and digestion tissue are not separated;
(3) in digestion is not organized, add 1～1.5 times to the phosphoric acid buffer dilution (pH is 7.2-7.4) that does not digest tissue volume;
(4) 1/2～1/3 of adding step (3) liquor capacity that obtains mass/volume is than the pancreatin phosphoric acid buffer liquor digestion that is 0.05%～0.2%, the digestion temperature is 37 ℃, digestion time is 10～30 minutes, preferred 15-20 minute, the serum that adds 1-2g in this 100ml solution stops the pancreatin effect, cross the 100-200 mesh sieve, filtered solution is a cell suspension;
(5) cell suspension that step (2) and step (4) are obtained mixes, and is centrifugal, and the rotating speed of whizzer is 1500 to change～2500 rev/mins, time is 10～20 minutes, abandoning supernatant is removed residual enzyme 1～3 time with phosphoric acid buffer (pH is 7.2-7.4) washing, and is centrifugal again, the rotating speed of whizzer is 1500 rev/mins～2500 rev/mins, time is 10～20 minutes, and abandoning supernatant promptly obtains mescenchymal stem cell, the two times centrifugal rotating speed is preferably 2000 rev/mins, and the time is 15 minutes;
(6) mescenchymal stem cell is put liquid nitrogen freezing, the temperature of liquid nitrogen freezing is-196 ℃, preserves by its ABO/Rh somatotype and HLA somatotype, and foundation can promptly be constructed people's placenta, umbilical cord mesenchyma stem cell for the cell news file of retrieval.
With collagenase and trysinization the time, available rotor stirs simultaneously, improves the degree of digestion.
Above-mentioned all processes is all operated in gnotobasis.
Obtain people's placenta, umbilical cord mesenchymal stem cells among the present invention by 1～2 * 10
4/ cm
2Be inoculated in the plastic culture bottle, (Sigma, USA), (Gibco BRL, substratum USA) are put 37 ℃, 5%CO to 5%FCS with containing DMEM-LG/F12
2Incubator is cultivated, and changes liquid after 4 days, discards non-adherent cell, and amount was changed liquid in later per 3 days half, treats that cell 80% merges, and 0.25% pancreatin (Sigma, USA) went down to posterity by 1: 3, can obtain a large amount of (1 * 10 by digestion
10) 1 people's placenta, umbilical cord mesenchymal stem cells be used for clinical and scientific research.
A kind of people's placenta of the present invention, Umbilical Filling stem cell's storehouse library And Construct method, can from people's placenta, umbilical cord, obtain a large amount of MSCs, set up the system engineering technology storage vault, it is a kind of effective ways that existing resource is made full use of, the method according to this invention, be stored in the mescenchymal stem cell in the storehouse, can obtain 1 * 10 through Short-term Culture
10MSCs can be rich in active placenta and umbilical cord mesenchymal stem cells in a large number, and can preserve for a long time and do not lose its activity, and operation is simple, and this is cheap to build Kucheng, is rich in application prospect.
Description of drawings:
The cellular form of the MSCs in Fig. 1, placenta and umbilical cord source: (A-C) be the cellular form of MSCs cultivation after 7 days that adopts method of the present invention to obtain; (D is that MSCs passes the cellular form of the first-generation after 14-20 days E); (F, G are that MSCs passes the 1st, 3,5 days cellular form of the s-generation H); (I-L), traditional endothelium digestion method obtains MSCs cultured cells form, passed for two generations after cellular form change;
The cell growth curve of the MSCs in Fig. 2, placenta and umbilical cord source: third generation MSCs is by 2 * 10
4Cells/well is seeded in 24 orifice plates, every 3 holes of 24h digestion, and collecting cell, and expect blue dyeing back living cell counting with 0.4% platform, get the mean value of 5 experimental results, the drafting growth curve;
Embodiment:
The present invention is further illustrated below in conjunction with the drawings and specific embodiments, but the present invention is not limited to following embodiment.
Embodiment 1:
The preparation of people's placenta umbilical cord mesenchymal stem cells:
(1) after (detection of the detection of ABO/Rh blood group, HLA somatotype, syphilis antibody detection, HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test etc.) determines security through strict trace routine with people's placenta, umbilical cord, by pH is that 7.2 PBS (phosphoric acid buffer) washes repeatedly, remove remained blood, be cut to about 1mm with aseptic cutter
3-1.5mm
3Fine grained chippings or with other aseptic instruments it is crushed to meat gruel shape (1mm
3-1.5mm
3), getting meat gruel 25ml, adding pH is 7.2 PBS to 50ml;
(2) add mass/volume than the collagenase phosphoric acid buffer liquor 20ml that is 0.1%, put 37 ℃ of digestion 1 hour, this mixed solution is continued magnetic agitation therebetween; This mixture is filtered with 100 eye mesh screens, filterable cell suspension and digestion tissue are not separated, filterable cell suspension is put in the supercentrifuge, 2000 rev/mins, centrifugal 15 minutes, abandoning supernatant placed sedimentary cell on the vibrator and to vibrate, make cell loose, it is standby to collect this cell;
(3) the rapid not digestion of collecting of previous step being organized 20ml add pH is 7.2 PBS to 40ml;
(4) add mass/volume again than the pancreatin phosphoric acid buffer liquor 20ml that is 0.125%, digest 20min down at 37 ℃, this mixed solution is continued magnetic agitation therebetween, the serum that adds mixture final concentration 0.6g afterwards stops the effect of pancreatin, filter with 100 eye mesh screens, filterable cell suspension discards indigested tissue; With the cell suspension high speed centrifugation, centrifuge speed is 2000 rev/mins, centrifugal 15 minutes, and abandoning supernatant, the vibration cell makes it loose, and it is standby to collect this cell;
(5) cell that step (2) and (4) are obtained places the 50ml pipe, adds pH and be 7.2 PBS to full, high speed centrifugation, centrifuge speed is 2000 rev/mins, centrifugal 15 minutes, and abandoning supernatant, the vibration cell makes it loose, and this step repeats 2 times to clean residual enzyme; With the cell harvesting after centrifugal, promptly prepare and build storehouse required people's placenta, umbilical cord mesenchymal stem cells at last.
Aforesaid operations all carries out in strict gnotobasis, and not contaminated for guaranteeing cell, the PBS of all uses has all added 1% penicillin and Streptomycin sulphate.
Morphological observation: adopt the isolating primary cell of present method more than adherent in 24-48 hour, cultivate 1-2 week, the visible attached cell of inverted microscope is fusiformis, polygon, rate of growth is very fast after 2 weeks, become the spindle cell of the relative homogeneous of form, be be arranged in parallel growth or swirl shape growth, merge the attached cell layer in forming 80% 2-3 week.Passed for 20 generations continuously, cellular form does not have obvious change.And adopt the endothelium digestion method of bibliographical information to have only 30% sample can isolate MSCs, 70% sample attached cell is gone down to posterity and was no more than for 2 generations.(Fig. 1) cell count of present method acquisition and success ratio are apparently higher than the usual way according to the digestion of document endothelium.
The mensuration of cell growth curve with attached cell with 0.25% trysinization after, by 2 * 10
4Cells/well is seeded in 24 orifice plates, every 3 holes of 24h digestion, and collecting cell, and expect blue living cell counting, the drafting growth curve with 0.4% tire.Cell proliferation is very fast, and at logarithmic phase, cell doubling time all is about 30h (Fig. 2).The every biography generation of cell, cell count increases by 2 times approximately.
The structure of people's placenta, umbilical cord mesenchyma stem cell:
The mescenchymal stem cell that is obtained is put in the freezer bag, after the frozen solution pre-treatment, extracting air; the stem cell Storage Box is put in sealing into, adopts 1010 type refrigeration systems (Forma Scientific Inc; USA) freezing, then Storage Box is kept in-196 ℃ of cryogenic liquid nitrogen containers.Leaving and taking the small portion cell is stored in-80 ℃ of refrigerators, as the check sample of inventory information.With its details (comprise the donor name, donor father and mother name, address, contact method, stem cell join type information etc.) the input computer data, set up the inquiry for future reference of perfect data archival.
Embodiment 2:
(1) after (detection of the detection of ABO/Rh blood group, HLA somatotype, syphilis antibody detection, HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test etc.) determines security through strict trace routine with people's placenta, umbilical cord, by pH is that 7.4 PBS washes repeatedly, remove remained blood, be cut to about 1mm with aseptic cutter
3-1.5mm
3Fine grained chippings or with other aseptic instruments it is crushed to meat gruel shape (1mm
3-1.5mm
3), getting meat gruel 20ml, adding pH is 7.4 PBS to 50ml;
(2) add mass/volume than the collagenase phosphoric acid buffer liquor 25ml that is 0.05%, put 37 ℃ of digestion 70 minutes, this mixed solution is continued magnetic agitation therebetween; This mixture is filtered with 200 eye mesh screens, filterable cell suspension and digestion tissue are not separated, filterable cell suspension is put in the supercentrifuge, 1500 rev/mins, centrifugal 20 minutes, abandoning supernatant placed sedimentary cell on the vibrator and to vibrate, make cell loose, it is standby to collect this cell;
(3) the rapid not digestion of collecting of previous step being organized 20ml add pH is 7.4 PBS to 50ml;
(4) add mass/volume again than the pancreatin phosphoric acid buffer liquor 25ml that is 0.05%, digest 30min down at 37 ℃, this mixed solution is continued magnetic agitation therebetween, the effect that adds 1.5g serum termination pancreatin afterwards, filter with 200 eye mesh screens, filterable cell suspension discards indigested tissue; With the cell suspension high speed centrifugation, centrifuge speed is 1500 rev/mins, centrifugal 20 minutes, and abandoning supernatant, the vibration cell makes it loose, and it is standby to collect this cell;
(5) cell that step (2) and (4) are obtained places the 50ml pipe, adds pH and be 7.4 PBS to full, high speed centrifugation, centrifuge speed is 1500 rev/mins, centrifugal 20 minutes, and abandoning supernatant, the vibration cell makes it loose, and this step repeats 3 times to clean residual enzyme; With the cell harvesting after centrifugal, promptly prepare and build storehouse required people's placenta, umbilical cord mesenchymal stem cells at last.
Aforesaid operations all carries out in strict gnotobasis, and is not contaminated for guaranteeing cell, and the pH of all uses is that 7.4 PBS has all added 1% penicillin and Streptomycin sulphate.
(6) before the stem cell warehouse-in, by 2 * 10
4/ cm
2Be inoculated in the plastic culture bottle, (Sigma, USA), (Gibco BRL, substratum USA) are put 37 ℃, 5%CO to 5%FCS with containing DMEM-LG/F12
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges; 0.25% pancreatin (Sigma; USA) digestion; went down to posterity by 1: 3; obtain a large amount of stem cells after the amplification, institute is obtained cell be divided in 5 parts of threading freezer bags, after 50%DMSO and 5% dextran cryoprotectant pre-treatment; put it in the small-sized storage tank of preserving the cell special use, adopt BioArchive
TMSystem's substep is freezing, it is kept in-196 ℃ of cryogenic liquid nitrogen containers.Leaving and taking the small portion cell is stored in-80 ℃ of refrigerators, as the check sample of inventory information.With its details (comprise the donor name, donor father and mother name, address, contact method, stem cell join type information etc.) the input computer data, set up the inquiry for future reference of perfect data archival.
Embodiment 3:
(1) after (detection of the detection of ABO/Rh blood group, HLA somatotype, syphilis antibody detection, HIV immunodetection, CMV antibody test, Australia antigen(AA) antibody test etc.) determines security through strict trace routine with people's placenta, umbilical cord, wash repeatedly by PBS (pH is 7.4), remove remained blood, be cut to about 1mm with aseptic cutter
3-1.5mm
3Fine grained chippings or with other aseptic instruments it is crushed to meat gruel shape (1mm
3-1.5mm
3), get meat gruel 25ml, add PBS (pH is 7.4) to 50ml;
(2) add mass/volume than the collagenase phosphoric acid buffer liquor 16.7ml that is 0.2%, put 37 ℃ of digestion 40 minutes, this mixed solution is continued magnetic agitation therebetween; This mixture is filtered with 100 eye mesh screens, filterable cell suspension and digestion tissue are not separated, filterable cell suspension is put in the supercentrifuge, 2500 rev/mins, centrifugal 10 minutes, abandoning supernatant placed sedimentary cell on the vibrator and to vibrate, make cell loose, it is standby to collect this cell;
(3) organize 20ml to add PBS (pH is 7.4) the rapid not digestion of collecting of previous step to 40ml;
(4) add mass/volume again than the pancreatin phosphoric acid buffer liquor 16.7ml that is 0.2%, digest 10min down at 37 ℃, this mixed solution is continued magnetic agitation therebetween, the effect that adds 0.7g serum termination pancreatin afterwards, filter with 100 eye mesh screens, filterable cell suspension discards indigested tissue; With the cell suspension high speed centrifugation, centrifuge speed is 2500 rev/mins, centrifugal 10 minutes, and abandoning supernatant, the vibration cell makes it loose, and it is standby to collect this cell;
(5) cell that step (2) and (4) are obtained places the 50ml pipe, adds (pH is 7.4) PBS to full, and high speed centrifugation, centrifuge speed are 2500 rev/mins, and centrifugal 10 minutes, abandoning supernatant, the vibration cell makes it loose, cleans residual enzyme; With the cell harvesting after centrifugal, promptly prepare and build storehouse required people's placenta, umbilical cord mesenchymal stem cells at last.
Aforesaid operations all carries out in strict gnotobasis, and not contaminated for guaranteeing cell, (pH is 7.4) PBS of all uses has all added 1% penicillin and Streptomycin sulphate.
The preparation method of people's placenta, umbilical cord mesenchymal stem cells (with embodiment 1); People's placenta, umbilical cord mesenchyma stem cell make up: institute is obtained cell put in the freezer bag, after 50%DMSO and 5% dextran cryoprotectant pre-treatment, put it into the sealing of transparent container bag, reinstall the stem cell Storage Box, adopt BioArchive
TMSystem is freezing, then it is kept in-196 ℃ of cryogenic liquid nitrogen containers.Leave and take the small portion cell and be stored in-80 ℃ of refrigerators, join the check sample of type as HLA before using.With its specifying information input computer data, set up the inquiry for future reference of perfect data archival.
Before to be used, the cell taking-up is thawed, by 2 * 104/cm
2Be inoculated in the plastic culture bottle, (Sigma, USA), (Gibco BRL, substratum USA) are put 37 ℃, 5%CO to 5%FCS with containing DMEM-LG/F12
2Incubator is cultivated, and changes liquid after 4-5 days, discards non-adherent cell, and half amount was changed liquid in later every 3-4 days.Treat that cell 80% merges, 0.25% pancreatin (Sigma, USA) went down to posterity by 1: 3 by digestion.Obtain a large amount of stem cells after the amplification, be applied to prepare various stem cell medicines.
Collagenase I/II type, GIBCO company, 100mg packing is dissolved in that can to get the mass/volume ratio among the 100mlPBS be 0.1% enzyme solution, and then dilution is used; Trypsin is a pancreatin), GIBCO company, the 100mg packing also is recently to prepare by mass/volume during use.
Claims (7)
1. people's placenta, umbilical cord mesenchyma stem cell is characterized in that being built up by following step:
(1) get people's placenta, umbilical cord carries out the detection of ABO/Rh blood group, the detection of HLA somatotype, microorganism immunodetection, uses the phosphoric acid buffer wash and remove residual blood, pulverizes, adds 1～1.5 times of phosphoric acid buffer and dilutes to the crushed material volume;
(2) 1/2～1/3 of adding step (1) liquor capacity that obtains mass/volume is than the collagenase phosphoric acid buffer liquor digestion that is 0.05%～0.2%, temperature is 37 ℃, digested 40～70 minutes, and crossed the 100-200 mesh sieve, filterable cell suspension and digestion are not organized separated;
(3) in digestion is not organized, add 1～1.5 times to the phosphoric acid buffer dilution that does not digest tissue volume;
(4) 1/2～1/3 of adding step (3) liquor capacity that obtains mass/volume is than the pancreatin phosphoric acid buffer liquor digestion that is 0.05%～0.2%, the digestion temperature is 37 ℃, digestion time is 10～30 minutes, the serum that adds 1-2g in this 100ml solution stops the pancreatin effect, cross the 100-200 mesh sieve, filtered solution is a cell suspension;
(5) cell suspension that step (2) and step (4) are obtained mixes, and is centrifugal, and abandoning supernatant is removed residual enzyme 1～3 time with the phosphoric acid buffer washing, and centrifugal again, abandoning supernatant promptly obtains mescenchymal stem cell;
(6) mescenchymal stem cell is put liquid nitrogen freezing, preserve by its ABO/Rh somatotype and HLA somatotype, foundation can promptly be constructed people's placenta, umbilical cord mesenchyma stem cell for the cell news file of retrieval.
2. the construction process of people's placenta, umbilical cord mesenchyma stem cell, form by following step:
(1) get people's placenta, umbilical cord carries out the detection of ABO/Rh blood group, the detection of HLA somatotype, microorganism immunodetection, uses the phosphoric acid buffer wash and remove residual blood, pulverizes, adds 1～1.5 times of phosphoric acid buffer and dilutes to the crushed material volume;
(2) 1/2～1/3 of adding step (1) liquor capacity that obtains mass/volume is than the collagenase phosphoric acid buffer liquor digestion that is 0.05%～0.2%, temperature is 37 ℃, digested 40～70 minutes, and crossed the 100-200 mesh sieve, filterable cell suspension and digestion are not organized separated;
(3) in digestion is not organized, add 1～1.5 times to the phosphoric acid buffer dilution that does not digest tissue volume;
(4) 1/2～1/3 of adding step (3) liquor capacity that obtains mass/volume is than the pancreatin phosphoric acid buffer liquor digestion that is 0.05%～0.2%, the digestion temperature is 37 ℃, digestion time is 10～30 minutes, the serum that adds 1-2g in this 100ml solution stops the pancreatin effect, cross the 100-200 mesh sieve, filtered solution is a cell suspension;
(5) cell suspension that step (2) and step (4) are obtained mixes, and is centrifugal, and abandoning supernatant is removed residual enzyme 1～3 time with the phosphoric acid buffer washing, and centrifugal again, abandoning supernatant promptly obtains mescenchymal stem cell;
(6) mescenchymal stem cell is put liquid nitrogen freezing, preserve by its ABO/Rh somatotype and HLA somatotype, foundation can promptly be constructed people's placenta, umbilical cord mesenchyma stem cell for the cell news file of retrieval.
3. according to the construction process of the described a kind of people's placenta of claim 2, umbilical cord mesenchyma stem cell, it is characterized in that the described collagenase digesting time is 60 minutes.
4. according to the construction process of the described a kind of people's placenta of claim 2, umbilical cord mesenchyma stem cell, it is characterized in that the described trysinization time is 20 minutes.
5. according to the construction process of the described a kind of people's placenta of claim 2, umbilical cord mesenchyma stem cell, when it is characterized in that the two times centrifugal of described step (5), the rotating speed of whizzer is 1500 rev/mins～2500 rev/mins, and the time is 10～20 minutes.
6. according to the construction process of the described a kind of people's placenta of claim 5, umbilical cord mesenchyma stem cell, the rotating speed that it is characterized in that described step whizzer is 2000 rev/mins, and the time is 15 minutes.
7. according to the construction process of the described a kind of people's placenta of claim 2, umbilical cord mesenchyma stem cell, it is characterized in that the temperature of described liquid nitrogen freezing is-196 ℃.
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Cited By (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Assignee: Beijing Hanshi United Biological Technology Co., Ltd. Assignor: Tianjin AmCellGene Engineering Co., Ltd. Contract fulfillment period: 2007.11.1 to 2015.10.31 contract change Contract record no.: 2007990000088 Denomination of invention: Human placenta, umbilical cord mesenchyma stemcell stock and its construction method Granted publication date: 20071024 License type: exclusive license Record date: 20071127 |
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Free format text: EXCLUSIVE LICENCE; TIME LIMIT OF IMPLEMENTING CONTACT: 2007.11.1 TO 2015.10.31 Name of requester: BEIJING HANSHI JOINT BIOLOGICAL TECHNOLOGY CO., L Effective date: 20071127 |