Summary of the invention
The objective of the invention is to solve that prior art is frozen, the defective of recovery umbilical cord tissue method, provide a kind of simple and effective umbilical cord tissue frozen method, and the method for the freezing and storing umbilical tissue recovery of the use that matches, and the method that after recovery, mescenchymal stem cell separates and increases.The present invention finds to use frozen, the recovery of specific operation step and separates and amplification, can effectively obtain mescenchymal stem cell.The present invention is based on this discovery and be accomplished.
Therefore, first aspect present invention provides the method for processing umbilical cord tissue, and the method comprises followingly carries out frozen step to the umbilical cord flesh tissue that exsomatizes:
(1) preparation umbilical cord tissue frozen storing liquid: comprise human serum albumin and DMSO (dimethyl sulfoxide (DMSO)) in described umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃);
(2) sterilization and clean: with thimerosal (for example alcohol), the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling is cleaned umbilical cord tissue by damping fluid (for example PBS damping fluid), with red corpuscle on the minimizing umbilical cord tissue;
(3) umbilical cord tissue is processed: the umbilical cord tissue that step (2) is obtained is cut into tissue block;
(4) umbilical cord tissue is cold deposits: at 0-15 ℃ (for example 1 ℃ to 7 ℃, for example 4 ℃) low temperature environment under, tissue block and frozen storing liquid are added in frozen container, then frozen container is put into the programmed cooling device, first under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃) deepfreeze 0.2-2 hour (for example 0.5 hour), under the temperature condition of-10 ℃ to-150 ℃ (for example-80 ℃) freezing 0.25-3 days (for example 1 day) again, then frozen container is freezing in liquid nitrogen, standby.
According to the method for first aspect present invention, the method also comprises the following step that the stripped flesh tissue of frozen umbilical cord is recovered:
(5) freezing and storing umbilical tissue recovery: the freezing umbilical cord tissue of step (4) is taken out from liquid nitrogen, thaw to 20%-70% (for example 50%) frozen storing liquid and begin to melt, utilize the mescenchymal stem cell substratum to clean umbilical cord tissue, filter and remove waste liquid, so that the recovery of frozen umbilical cord tissue.
According to the method for first aspect present invention, the method comprises that also following umbilical cord tissue to recovery carries out the step that mescenchymal stem cell separates and increases:
(6) the adherent processing of umbilical cord tissue: separately get the cell cultures plate, tissue block is tiled in plate, the tissue block quantity in each plate maintains 5-20 piece (for example 10-15 piece), makes the air-dry 2-50 of tissue block minute until tissue is attached on plate;
(7) umbilical cord tissue is cultivated: slowly add the mescenchymal stem cell substratum to flood to tissue along the plate edge and get final product; Put plate into CO2Concentration is that 37 ℃ of incubators of 5% are cultivated, and be cultured to 3-7 days (for example 4-6 days, for example the 5th day) time takes out plate from incubator, adds appropriate (tissue is flooded to be got final product) mescenchymal stem cell substratum; 9-11 days (for example the 10th day) times, the substratum in plate is shifted out, add appropriate (tissue is flooded to be got final product) fresh mescenchymal stem cell substratum, continue to cultivate; Remove all umbilical cord tissue pieces and continue when 11-13 days (for example the 12nd day) and cultivate; After this (for example every 2 days) once changed liquid entirely in every 1-3 days;
(8) passage: when the attached cell fusion rate in plate reaches the 50-70% left and right, use digestive ferment (for example TrypLE Express, invitrogen product) to make attached cell break away from the plate bottom; Centrifugal, remove supernatant liquor, add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in the T25 Tissue Culture Flask and go down to posterity and carry out amplification cultivation; After this (for example every 2 days) changed liquid once in every 1-3 days, until after fusion rate reaches 70-90%, namely get umbilical cord mesenchymal stem cells, went down to posterity in case of necessity;
And optional following one or more steps:
(9) for step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(10) umbilical cord mesenchymal stem cells after step (8) gained is gone down to posterity is frozen in liquid nitrogen, standby.
According to the method for first aspect present invention, the method comprises:
(A) below, the stripped flesh tissue of umbilical cord is carried out frozen step:
(1) preparation umbilical cord tissue frozen storing liquid: comprise human serum albumin and DMSO (dimethyl sulfoxide (DMSO)) in described umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃);
(2) sterilization and clean: with thimerosal (for example alcohol), the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling is cleaned umbilical cord tissue by damping fluid (for example PBS damping fluid), with red corpuscle on the minimizing umbilical cord tissue;
(3) umbilical cord tissue is processed: the umbilical cord tissue that step (2) is obtained is cut into tissue block;
(4) umbilical cord tissue is cold deposits: at 0-15 ℃ (for example 1 ℃ to 7 ℃, for example 4 ℃) low temperature environment under, tissue block and frozen storing liquid are added in frozen container, then frozen container is put into the programmed cooling device, first under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃) deepfreeze 0.2-2 hour (for example 0.5 hour), under the temperature condition of-10 ℃ to-150 ℃ (for example-80 ℃) freezing 0.25-3 days (for example 1 day) again, then frozen container is freezing in liquid nitrogen, standby;
(B) the following step that the stripped flesh tissue of frozen umbilical cord is recovered:
(5) freezing and storing umbilical tissue recovery: the freezing umbilical cord tissue of step (4) is taken out from liquid nitrogen, thaw to 20%-70% (for example 50%) frozen storing liquid and begin to melt, utilize the mescenchymal stem cell substratum to clean umbilical cord tissue, filter and remove waste liquid, so that the recovery of frozen umbilical cord tissue;
(C) following umbilical cord tissue to recovery carries out the step that mescenchymal stem cell separates and increases:
(6) the adherent processing of umbilical cord tissue: separately get the cell cultures plate, tissue block is tiled in plate, the tissue block quantity in each plate maintains 5-20 piece (for example 10-15 piece), makes the air-dry 2-50 of tissue block minute until tissue is attached on plate;
(7) umbilical cord tissue is cultivated: slowly add the mescenchymal stem cell substratum to flood to tissue along the plate edge and get final product; Put plate into CO2Concentration is that 37 ℃ of incubators of 5% are cultivated, and be cultured to 3-7 days (for example 4-6 days, for example the 5th day) time takes out plate from incubator, adds appropriate (tissue is flooded to be got final product) mescenchymal stem cell substratum; 9-11 days (for example the 10th day) times, the substratum in plate is shifted out, add appropriate (tissue is flooded to be got final product) fresh mescenchymal stem cell substratum, continue to cultivate; Remove all umbilical cord tissue pieces and continue when 11-13 days (for example the 12nd day) and cultivate; After this (for example every 2 days) once changed liquid entirely in every 1-3 days;
(8) passage: when the attached cell fusion rate in plate reaches the 50-70% left and right, use digestive ferment (for example TrypLE Express, invitrogen product) to make attached cell break away from the plate bottom; Centrifugal, remove supernatant liquor, add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in the T25 Tissue Culture Flask and go down to posterity and carry out amplification cultivation; After this (for example every 2 days) changed liquid once in every 1-3 days, until after fusion rate reaches 70-90%, namely get umbilical cord mesenchymal stem cells, went down to posterity in case of necessity;
And optional
(D) following one or more step:
(9) for step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(10) umbilical cord mesenchymal stem cells after step (8) gained is gone down to posterity is frozen in liquid nitrogen, standby.
According to the method for first aspect present invention, wherein said umbilical cord is the Fresh tissue of umbilical cord.
According to the method for first aspect present invention, comprise human serum albumin and DMSO in wherein said umbilical cord tissue frozen storing liquid.In one embodiment, the human serum albumin that contains the 55-95 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the human serum albumin that contains the 70-90 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the human serum albumin that contains 80 weight parts in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 4-20 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 7-15 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 10 weight parts in described umbilical cord tissue frozen storing liquid.In one embodiment, contain the human serum albumin of 80 weight parts of having an appointment, the about DMSO of 10 weight parts in described umbilical cord tissue frozen storing liquid.In above-mentioned embodiment, in the umbilical cord tissue frozen storing liquid, the summation of each component concentration is 100%.The inventor finds; contain the human serum albumin of 80 weight parts of having an appointment, approximately the umbilical cord tissue frozen storing liquid of the DMSO of 10 weight parts (namely wherein the weight ratio of human serum albumin and DMSO is 80:10) is particularly preferred, than the formula that changes 10% or more in the arbitrary component content that makes this umbilical cord tissue frozen storing liquid protecting umbilical cord tissue not to be subjected to have significant advantage aspect freezing destroyed texts.In the present invention, owing to also may containing those skilled in the art general other dosing solvent or solute in frozen storing liquid, therefore above-mentioned is a kind of relative quantity with the human serum albumin of " weight part " expression and the amount of DMSO, and it can be milligram, gram, kilogram etc.
According to the method for first aspect present invention, wherein the described refrigeration of step (1) is to preserve in refrigerator.
According to the method for first aspect present invention, wherein said umbilical cord is the Fresh tissue of umbilical cord.
According to the method for first aspect present invention, wherein the described umbilical cord tissue of step (2) is to process in Biohazard Safety Equipment.
According to the method for first aspect present invention, wherein the step of the described tiling of step (2) is that umbilical cord tissue is tiled in culture dish, and described culture dish diameter is the culture dish of 5-20cm, and preferred culture dish diameter is the culture dish of 10cm.
According to the method for first aspect present invention, the concentration of wherein said alcohol is 25%-95%, preferred 75%.
According to the method for first aspect present invention, the sodium salt that wherein said PBS damping fluid is phosphoric acid and/or sylvite preparation, its pH is 5.0-8.0, and preferred pH is 5.5-76, and preferred pH is 6.0-7.0.In one embodiment, in described PBS damping fluid, the concentration of phosphate radical is 0.01-0.5M, preferred 0.02-0.1M.In the present invention hereinafter tested, PBS damping fluid used was sodium phosphate salt, and wherein the concentration of phosphate radical is 0.025M, and pH is 6.5.Need to prove, inventor's discovery, PBS buffer concentration and pH value in above-mentioned scope are little for the influential effect of the inventive method.
Method according to first aspect present invention, wherein step (3) is that umbilical cord tissue is transferred in another cell cultures plate, then carry out umbilical cord tissue and be cut into tissue block, described culture dish diameter is the culture dish of 5-20cm, and preferred culture dish diameter is the culture dish of 10cm.
According to the method for first aspect present invention, wherein in step (3), organize block size to be about the 0.2-2.5 cubic centimetre, preferably be about the 0.5-1.5 cubic centimetre, the preferred approximately cube bulk of 1 cubic centimetre.
According to the method for first aspect present invention, wherein in step (3), the tissue block size is at the 0.5-1.5 cubic centimetre, preferred 0.5-1.0 cubic centimetre, and particularly big or small is approximately very preferred 1 cubic centimetre the time.although the little realization that is conducive to the inventive method of intended tissue fragment, yet the inventor finds in test at 0.2 cubic centimetre, 0.5 cubic centimetre, under 1 cubic centimetre of three kinds of state, they are to improving the cold storage effect of umbilical cord tissue, resuscitation effect, increase the adherent effect of umbilical cord tissue, the time equivalence fruit aspect that shortens that attached cell climbs out of from tissue is basically identical, and volume is later to improving the cold storage effect of umbilical cord tissue greater than 1.5 cubic centimetres, resuscitation effect, increase the adherent effect of umbilical cord tissue, shorten attached cell and from the time equivalence that tissue climbs out of, disadvantageous effect is arranged really.
According to the method for first aspect present invention, wherein the described low temperature environment of step (4) is 0-15 ℃, preferred 1 ℃ to 7 ℃, and preferred 4 ℃.
According to the method for first aspect present invention, wherein the described frozen container of step (4) is cryopreservation tube.
According to the method for first aspect present invention, wherein the described programmed cooling device of step (4) is program temperature reduction box.
Method according to first aspect present invention; wherein the density of the described tissue block of step (4) in frozen container all has enough DMSO to provide protection as the upper limit to guarantee arbitrary tissue block; described density refers to the tissue block number of individuals in unit space, preferred 0.1-0.8/cm3, preferred 0.5/cm3
According to the method for first aspect present invention, wherein the described deepfreeze of step (4) and cryogenic freezing are put into the refrigerator realization with programmed cooling device (for example program temperature reduction box).
According to the method for first aspect present invention, wherein the described sample of step (4) refrigeration flow process is under the temperature condition of 1 ℃ to 7 ℃ deepfreeze 0.2-2 hour.In one embodiment, described deepfreeze is under the temperature condition of 2 ℃ to 6 ℃.In one embodiment, described deepfreeze is under the temperature condition of 3 ℃ to 5 ℃.In one embodiment, described deepfreeze is under the temperature condition of 5 ℃.In one embodiment, the described deepfreeze time is 0.3-1.5 hour.In one embodiment, the described deepfreeze time is 0.4-1 hour.In one embodiment, the described deepfreeze time is 0.5 hour.Inventor's discovery, deepfreeze was particularly preferred in 0.5 hour under the temperature condition of 4 ℃, can guarantee that DMSO and umbilical cord tissue fully merge.Above-mentioned other refrigerating temperature conditions and cold preservation time also can make DMSO and umbilical cord tissue merge, and its syncretizing effect can satisfy primary demand of the present invention, but under the temperature condition of 4 ℃, the deepfreeze syncretizing effect of 0.5 hour is the most abundant, has clear superiority.
According to the method for first aspect present invention, wherein the freezing flow process of the described sample of step (4) is under the temperature condition of-10 ℃ to-150 ℃ freezing 0.25-3 days.In one embodiment, described freezing be under the temperature condition of-30 ℃ to-120 ℃.In one embodiment, described freezing be under the temperature condition of-50 ℃ to-100 ℃.In one embodiment, described freezing be under the temperature condition of-80 ℃.In one embodiment, described freezing time is 0.4-2 days.In one embodiment, described freezing time is 0.8-1.5 days.In one embodiment, described freezing time is 1 day.The inventor finds; freezing 1 day is particularly preferred under the temperature condition of-80 ℃, than the freezing flow process that changes 10% or more in the arbitrary temperature that makes this freezing flow process, time protecting umbilical cord tissue not to be subjected to have significant advantage aspect freezing destroyed texts.
According to the method for first aspect present invention, wherein described the thawing of step (5) is to carry out in water bath with thermostatic control.
According to the method for first aspect present invention, comprise FBS, L-Glutamine, Gentamicin and DMEM-F12 in wherein said mescenchymal stem cell substratum.In one embodiment, the FBS that contains 10-20% in described mescenchymal stem cell substratum.In one embodiment, contain 15% the FBS of having an appointment in described mescenchymal stem cell substratum.In one embodiment, the L-Glutamine that contains 0.5-2% in described mescenchymal stem cell substratum.In one embodiment, contain 1% the L-Glutamine (L-glutaminate) of having an appointment in described mescenchymal stem cell substratum.In one embodiment, the Gentamicin (gentamicin) that contains 0.02-0.1% in described mescenchymal stem cell substratum.In one embodiment, contain 0.05% the Gentamicin of having an appointment in described mescenchymal stem cell substratum.In one embodiment, the DMEM-F12 that contains 80-90% in described mescenchymal stem cell substratum.In one embodiment, contain 84% the DMEM-F12 of having an appointment in described mescenchymal stem cell substratum.In one embodiment, contain the FBS of 15 weight parts that has an appointment, approximately L-Glutamine, approximately Gentamicin and the about DMEM-F12 of 84 weight parts of 0.05 weight part of 1 weight part in described mescenchymal stem cell substratum.The inventor finds, contain the FBS of 15 weight parts that has an appointment, approximately 1 weight part L-Glutamine, approximately 0.05 weight part Gentamicin and approximately the mescenchymal stem cell substratum of the DMEM-F12 of 84 weight parts be particularly preferred, than in the arbitrary component content that makes this substratum change 10% or more formula increase umbilical cord tissue adherent, shorten time equivalence that attached cell climbs out of from tissue and have significant advantage aspect really.
According to the method for first aspect present invention, wherein the described mescenchymal stem cell substratum of step (5) is to clean umbilical cord tissue by topical application.Topical application can clean out in-house DMSO effectively, thereby avoids the loss of cell survival rate in the recovery process.
According to the method for first aspect present invention, wherein the described filtration of step (5) is removed waste liquid by the realization of 50-200 μ m strainer, preferred 100 μ m strainers.
According to the method for first aspect present invention, wherein said culture dish is the culture dish of diameter 5-20cm, preferably the culture dish of diameter 10cm.
According to the method for first aspect present invention, wherein in step (6), the air-dry time of tissue block is 2-50 minute, for example 5-25 minute, and for example 10-15 minute.The inventor finds in the air-dry time of 10-15 minute, umbilical cord tissue is adherent to increasing, time equivalence fruit aspect that shorten that attached cell climbs out of from tissue is best, is shorter than 5 minutes or air-dry time all has significant difference when being longer than 25 minutes than air-dry time.
Method according to first aspect present invention, wherein in step (7), the inventor finds, when being cultured to the 5th day, plate is taken out from incubator, adds appropriate mescenchymal stem cell substratum, continue to cultivate, in the time of the 10th day, the substratum in plate is shifted out, add the mescenchymal stem cell substratum of proper amount of fresh, continue to cultivate, removed all umbilical cord tissue pieces and continue in the time of the 12nd day and cultivate, after this every 2 days once change liquid entirely is particularly preferred.This changes liquid and organizes the setting of clean-up time effectively to shorten attached cell and reaches the time of specifying fusion rate.
According to the method for first aspect present invention, in step (9), it is to utilize trypan blue staining to count the number of frozen front and back viable cell that described cytoactive detects.
According to the method for first aspect present invention, in step (9), described cell contamination detects and utilizes a small amount of cell cultures, detects the pollution whether cell is subject to fungus and bacterium.In one embodiment, it is to utilize the etiology method that described cell contamination detects, and detects cell and whether is subject to being selected from following one or more infection: Hepatitis B virus, the third liver, virus of AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method for first aspect present invention, in step (9), it is the method for utilizing molecular genetics that described inherited disease detects, and detects freeze-stored cell and whether has inherited disease.
According to the method for first aspect present invention, in step (9), it is to detect cell HLA-ABC/DR phenotype that described HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (10), described umbilical cord mesenchymal stem cells is frozen in liquid nitrogen through the programmed cooling process.
According to the method for first aspect present invention, in step (10), described umbilical cord mesenchymal stem cells is present in cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises DMEM-F12, dimethyl sulfoxide (DMSO) and human serum albumin.In one embodiment, this cells frozen storing liquid comprises approximately the DMEM-F12 of 65 parts, approximately dimethyl sulfoxide (DMSO), the about human serum albumin of 15 parts of 10 parts.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO).
According to the method for first aspect present invention, the method comprises the following steps:
(1) preparation umbilical cord tissue frozen storing liquid: the DMSO (dimethyl sulfoxide (DMSO) that comprises human serum albumin and 10 weight parts of 80 weight parts in described umbilical cord tissue frozen storing liquid, dimethyl sulfoxide), the cold liquid storage for preparing is placed on 4 ℃ of Refrigerator stores until use;
(2) sterilization and clean: in Biohazard Safety Equipment, with alcohol, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle;
(3) umbilical cord tissue is processed: the umbilical cord tissue that step (2) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into big or small 1cm3Square shape;
(4) umbilical cord tissue is cold deposits: under the low temperature environment of 4 ℃, tissue block and frozen storing liquid are added in cryopreservation tube, then cryopreservation tube is put into program temperature reduction box, first deepfreeze 0.5 hour under the temperature condition of 4 ℃, under the temperature condition of again-80 ℃ freezing 1 day, then cryopreservation tube is freezing in liquid nitrogen, standby.
Further, can comprise and the matching used method for resuscitation of cryopreservation methods:
(5) freezing and storing umbilical tissue recovery: the freezing umbilical cord tissue of step (4) is taken out from liquid nitrogen, being placed on thaws in water bath with thermostatic control begins to melt to half frozen storing liquid, utilize mescenchymal stem cell substratum (it for example comprises 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and clean umbilical cord tissue, utilize the 100um strainer that waste liquid is removed.
Further, can comprise that the rear mescenchymal stem cell of recovery separates and the method for amplification:
(6) the adherent processing of umbilical cord tissue: the freezing and storing umbilical tissue of recovery is tiled in another 10cm cell cultures plate, and the tissue block quantity in each plate maintains the 10-15 piece, makes the air-dry 10-15 of tissue block minute until tissue is attached on plate;
(7) umbilical cord tissue is cultivated: slowly add mescenchymal stem cell substratum (it for example comprises 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to flood to tissue along the plate edge and get final product; Plate is placed in CO2Concentration is that 37 ℃ of incubators of 5% are cultivated, and when being cultured to the 5th day, plate is taken out from incubator, adds 5ml mescenchymal stem cell substratum; With the media transfer in plate, added the fresh mescenchymal stem cell substratum of 15ml at the tenth day; Removed all umbilical cord tissue pieces at the 12 day and continue to cultivate, once entirely changing liquid in after this every two days;
(8) passage: the attached cell fusion rate inside plate reaches 60% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is broken away from plate bottom, remove supernatant liquor after centrifugal, and add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid once in every two days until after fusion rate reaches 80%, and get final product, go down to posterity again in case of necessity.
Further, can be for above step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type.
Further, the umbilical cord mesenchymal stem cells after above step (8) gained can being gone down to posterity is frozen in liquid nitrogen, standby.
Further, can be with the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out the molecular genetics diagnosis, preserve all related datas of cell, set up the database of umbilical cord stem cell and carry out related with freeze-stored cell.Therefore in the present invention in one aspect, the method of setting up umbilical cord stem cell database is provided, it comprises that first aspect present invention is separated and the step of amplification umbilical cord mesenchymal stem cells, and following steps: the cell after going down to posterity is the frozen and relevant fetus information of record in liquid nitrogen, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out the molecular genetics diagnosis, preserve all related datas of cell, set up the database of umbilical cord stem cell and carry out related with freeze-stored cell.
In addition, in a first aspect of the present invention, the method that after providing frozen to the umbilical cord flesh tissue, recover and having recovered, stem cell separates and increases.Therefore second aspect present invention provides a kind of umbilical cord mesenchymal stem cells.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, the described method of its embodiment arbitrary according to first aspect present invention obtains.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, its cell purity is greater than 90%, for example greater than 95%.In one embodiment, described umbilical cord mesenchymal stem cells is after going down to posterity more than 3 generations, and cell purity is greater than 90%, for example greater than 95%.
Below the present invention is further illustrated.The document that the present invention quotes, and the document of quoting in the document, their full content is incorporated this paper by reference into.
In the present invention, in arbitrary technical scheme of either side of the present invention, its arbitrary technical characterictic is equally applicable to arbitrary embodiment of either side of the present invention, as long as they can not cause contradiction, and this mutually being useful in case of necessity can be done suitable modification.
In the present invention, term " umbilical cord mesenchymal stem cells " refers to derive from the mescenchymal stem cell of umbilical cord.Therefore in the present invention, particularly relate in linguistic context of the present invention, term " umbilical cord mesenchymal stem cells " can with " umbilical cord stem cell ", " stem cell ", " mescenchymal stem cell " Alternate, indicate unless separately have clearly.
In the present invention, term " PBS damping fluid " or " PBS " refer to phosphate buffered saline buffer.Those skilled in the art know the generality formula of the PBS that uses and compound method and their general aspects for example pH value or pH scope under situation of the present invention.
In the present invention, term " umbilical cord " refers to neonatal umbilical cord, refers to especially the umbilical cord within 4 hours postpartum.
mescenchymal stem cell (mesenchymal stem cell, MSC) for example the mankind's mescenchymal stem cell is separated from marrow the earliest, derive from the tissue stem cell that a mesoblastic class has multi-lineage potential and self-renewal capacity, have to scleroblast in vivo with under external specified conditions, the chondrocyte, adipocyte, endotheliocyte, neurocyte, the myocyte, the ability of the multiple adult cytodifferentiation such as liver cell (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991, 9:641-650.Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999, 284:143-147).The up-to-date mescenchymal stem cell that studies show that has immunomodulatory and Hematopoiesis Support affect, and is easy to foreign gene importing expression.Therefore the seed cell of mescenchymal stem cell during still tissue-engineered bone, cartilage and cardiac muscle build, important carrier cell in gene therapy, and because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, be with a wide range of applications in hematopoietic stem cell transplantation and organ transplantation.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, people successfully from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood separation and Culture go out mescenchymal stem cell.
The mescenchymal stem cell of reporting at present is mainly derived from marrow, adopts density gradient centrifugation to obtain.Although separation method is easy, donor is got marrow need to experience a more painful operation, and has very high infection chance in the process of drawing materials and after drawing materials; Because the content of MSC in the human bone marrow is extremely rare, every 105～106Approximately only have 1 in individual mononuclearcell, and along with the increase at age, in marrow, the quantity of mescenchymal stem cell, propagation and differentiation capability descend significantly all, it is restricted in research with in using especially clinical application.The umbilical cord that originates from the embryonic development period extraembryonic mesoderm is comprised of a matter, blood vessel and nurse cell, contains a large amount of mesenchyme compositions.
Up-to-date studies show that contains abundant stem cell in umbilical cord, separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application from umbilical cord.
Thereby existing separate stem cells is set up the method for stem cell bank, shortcomings is arranged still, for example purity is not enough and/or quantity is not high, and then demonstrates these methods and still can not satisfy people's expectation.The for example invention of CN 101270349A (Chinese patent application numbers 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separates and the amplification in vitro cultural method "; The invention of CN 101693884A (Chinese patent application numbers 200910117522.9, open day on April 14th, 2010) disclosed being entitled as method of separating and extracting stem cells " a kind of from placenta, umbilical cord or fatty tissue "; CN 102146359A (Chinese patent application numbers 201110005964.1, open day on August 10th, 2011) disclosed being entitled as " extracted the method for primary mesenchymal stem cells and serum-free amplification " from placenta invention.These methods are remaining to be further improved aspect the purity of extract and/or the rate of recovery.
The invention discloses a kind of umbilical cord tissue is frozen, method of rear stem cell separation and amplification of recovering and recover, and can utilize this method preservation umbilical cord mesenchymal stem cells and set up the umbilical cord stem cell bank.The present inventor is summing up on the basis of frozen, recovery umbilical cord tissue in the past; utilize the freezing umbilical cord tissue of umbilical cord tissue frozen storing liquid protection; utilize topical application to clean the recovery umbilical cord tissue, in conjunction with stationary culture, successfully separate obtaining a large amount of mescenchymal stem cells from the umbilical cord tissue of recovery.The mescenchymal stem cell purity that the inventive method obtains is high, quantity is many, has the biological characteristics identical with mesenchymal stem cells MSCs, can be to differentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte.Because stem cell in umbilical cord is inmature than adult stem cell, rich content, be with a wide range of applications clinically, we use conventional cell freezing method that mescenchymal stem cell is frozen as bleeding of the umbilicus, set up the umbilical cord stem cell bank, for further investigation and the clinical treatment of stem cell lay the foundation later on.
Owing to containing abundant hemopoietic stem cell in bleeding of the umbilicus, people set up unbilical blood bank these important Biological resources of umbilical hemopoietic stem cell are stored, for multiple disease in the blood system and disease of immune system provide a kind for the treatment of means.Same umbilical cord mesenchymal stem cells is as a kind of more importantly stem cell resource, we use conventional cell freezing method it to be chilled in the medium-term and long-term preservation of profound hypothermia liquid nitrogen of-196 degrees centigrade, set up the umbilical cord stem cell bank, for stem-cell therapy is in the future preserved seed.
The method according to this invention, the freezing flow process of the formula of umbilical cord tissue frozen storing liquid and sample can successfully and effectively be protected in freezing process umbilical cord tissue.The method according to this invention utilizes topical application that the DMSO inside tissue is cleaned out, and has avoided the loss of cell survival rate in the recovery process.The method according to this invention, wherein the shape of umbilical cord tissue piece, size, quantity and air-dry time are all that the factor of adherent effect is organized in impact, thereby directly affect attached cell from time that tissue climbs out of.The present invention of experimental data proof can effectively increase the adherent effect of umbilical cord tissue, shortens attached cell from time that tissue climbs out of.The method according to this invention, wherein the mescenchymal stem cell culture medium prescription can successfully and effectively carry out amplification in vitro to umbilical cord mesenchymal stem cells.The method according to this invention is wherein changed liquid and is organized the setting of clean-up time to shorten attached cell and reaches the time of specifying fusion rate.
The present invention is simple to operate; convenient and practical; can effectively protect frozen umbilical cord tissue; avoid cell survival rate loss in the recovery process; can obtain a large amount of mescenchymal stem cells; the differentiation performance is good, has to the ability of the cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.With now methodical comparison: at present MSC mainly adopts modus operandi to extract donor marrow or perfusion method separates umbilical cord, the adherent culture acquisition.It is few that this method is got cell quantity, and donor is being got the possibility that infection is all arranged after marrow is got in the marrow neutralization.The present invention's success separates the higher mescenchymal stem cell of a large amount of purity of acquisition in umbilical cord, and uses this method to set up the umbilical cord stem cell bank and lay in this stem cell that has application prospect.This method is simple and easy to do, and because umbilical cord is the same with bleeding of the umbilicus, Cell Component is inmatureer, and wide material sources conveniently are easy to get, and therefore method of the present invention will have prospect widely in the clinical application of stem cell.
Embodiment
Can conduct further description the present invention by the following examples, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although for to realize that many materials and working method that the object of the invention is used are well known in the art, the present invention still does detailed as far as possible description at this.
Embodiment 1, the method that umbilical cord tissue is frozen, the rear stem cell that recovers and recover separates and increases
The umbilical cord tissue cryopreservation methods comprises the following steps:
(1) preparation umbilical cord tissue frozen storing liquid: the DMSO (dimethyl sulfoxide (DMSO) that comprises human serum albumin and 10 weight parts of 80 weight parts in described umbilical cord tissue frozen storing liquid, dimethyl sulfoxide), the cold liquid storage for preparing is placed on 4 ℃ of Refrigerator stores until use;
(2) sterilization and clean: in Biohazard Safety Equipment, with alcohol, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle;
(3) umbilical cord tissue is processed: the umbilical cord tissue that step (2) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into big or small 1cm3Square shape;
(4) umbilical cord tissue is cold deposits: under the low temperature environment of 4 ℃, tissue block and frozen storing liquid are added in cryopreservation tube, then cryopreservation tube is put into program temperature reduction box, first deepfreeze 0.5 hour under the temperature condition of 4 ℃, under the temperature condition of again-80 ℃ freezing 1 day, then cryopreservation tube is freezing in liquid nitrogen, standby.
Comprise the following steps with the matching used method for resuscitation of cryopreservation methods:
(5) freezing and storing umbilical tissue recovery: the freezing umbilical cord tissue of step (4) is taken out from liquid nitrogen, being placed on thaws in water bath with thermostatic control begins to melt to half frozen storing liquid, utilize mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and clean umbilical cord tissue, utilize the 100um strainer that waste liquid is removed.
After recovery, the method for mescenchymal stem cell separation and amplification comprises the following steps:
(6) the adherent processing of umbilical cord tissue: the freezing and storing umbilical tissue of recovery is tiled in another 10cm cell cultures plate, and the tissue block quantity in each plate maintains the 10-15 piece, makes the air-dry 10-15 of tissue block minute until tissue is attached on plate;
(7) umbilical cord tissue is cultivated: slowly add mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to flood to tissue along the plate edge and get final product; Plate is placed in CO2Concentration is that 37 ℃ of incubators of 5% are cultivated, and when being cultured to the 5th day, plate is taken out from incubator, adds 5ml mescenchymal stem cell substratum; With the media transfer in plate, added the fresh mescenchymal stem cell substratum of 15ml at the tenth day; Removed all umbilical cord tissue pieces at the 12 day and continue to cultivate, once entirely changing liquid in after this every two days;
(8) passage: the attached cell fusion rate inside plate reaches 60% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is broken away from plate bottom, remove supernatant liquor after centrifugal, and add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid once until fusion rate goes down to posterity after reaching 80% again in every two days.
In the test of above step, the umbilical cord tissue of recovery began to have attached cell to climb out of at the 10th day that cultivates, and was cultured to the 17th day cell confluency and reached 60%, after being passaged to the T25 culturing bottle, reached 90% the 22nd day fusion rate.After 3 generations went down to posterity, cell purity was greater than 95%.
In the test of above step; DMSO (the dimethyl sulfoxide (DMSO) that comprises human serum albumin and 10 weight parts of 80 weight parts in the umbilical cord tissue frozen storing liquid; dimethyl sulfoxide); in two kinds of components of this frozen storing liquid; any one ratio is departing from said ratio 20% when above; frozen storing liquid can not effectively be protected umbilical cord tissue in freezing process, is embodied in the mescenchymal stem cell counting that separates after the recovery of freezing and storing umbilical tissue and obviously descends.
In the test of above step; the freezing flow process of sample is deepfreeze 0.5 hour under the temperature condition of 4 ℃; under the temperature condition of again-80 ℃ freezing 1 day; the arbitrary temp of this freezing flow process is departing from said temperature 60% when above; and the time departing from 80% when above; in refrigerating process, umbilical cord tissue is not effectively protected, and is embodied in the mescenchymal stem cell counting that separates after the recovery of freezing and storing umbilical tissue and obviously descends.
In the test of above step, comprise 15 weight part FBS, 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12 in the mescenchymal stem cell substratum.In four kinds of components of this substratum, any three kinds of ratios wherein are fixedly the time, and alternative ratio after being passaged to the T25 culturing bottle, does not all reach 75% the 17th day fusion rate departing from said ratio 10% when above.For example comprise 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12 in the mescenchymal stem cell substratum that uses, and when 12 weight part FBS, 13.5 weight part FBS, 16.5 weight part FBS or 18 weight part FBS, in four kinds of situations, after being passaged to the T25 culturing bottle, the 17th day fusion rate all between 55-75%.
In the test of above step, change the liquid time and be cell cultures the 14th after for once entirely changing liquid in every 2 days, the liquid time of changing is arbitrarily wherein being departed from the above-mentioned time 80% when above, attached cell did not reach fusion rate 80% in 20 days.For example change the liquid time and be and entirely changed for the first time liquid in the 16th day of cell cultures, or once entirely changed liquid for every 4 days backward, in two kinds of situations, attached cell reached 80% at 25-30 days.
Embodiment 2, the method that umbilical cord tissue is frozen, the rear stem cell that recovers and recover separates and increases
The method of reference example 1 is carried out.The umbilical cord tissue of recovery began to have attached cell to climb out of at the 7th day that cultivates, and was cultured to the 13rd day cell confluency and reached 60%.After being passaged to the T25 culturing bottle, reach 90% the 19th day fusion rate.After 3 generations went down to posterity, cell purity was greater than 90%.
Embodiment 3, the method that umbilical cord tissue is frozen, the rear stem cell that recovers and recover separates and increases
The method of reference example 1 is carried out.The umbilical cord tissue of recovery began to have attached cell to climb out of at the 8th day that cultivates, and was cultured to the 15th day cell confluency and reached 60%.After being passaged to the T25 culturing bottle, reach 90% the 18th day fusion rate.After 3 generations went down to posterity, cell purity was greater than 90%.
Embodiment 4, the method that umbilical cord tissue is frozen, the rear stem cell that recovers and recover separates and increases
The method of reference example 1 is carried out.The umbilical cord tissue of recovery began to have attached cell to climb out of at the 7th day that cultivates, and was cultured to the 14th day cell confluency and reached 60%.After being passaged to the T25 culturing bottle, reach 90% the 17th day fusion rate.
Embodiment 5, the method that umbilical cord tissue is frozen, the rear stem cell that recovers and recover separates and increases
The method of reference example 1 is carried out.The umbilical cord tissue of recovery began to have attached cell to climb out of at the 7th day that cultivates, and was cultured to the 13rd day cell confluency and reached 60%.After being passaged to the T25 culturing bottle, reach 90% the 20th day fusion rate.
The cultivation and frozen of going down to posterity of embodiment 6, umbilical cord MSC
The cell that embodiment 1-5 any one is obtained digests, and gets 1 * 10 after digestion6Cell joins in 1ml cells frozen storing liquid (containing 65 parts of DMEM-F12+10 part dimethyl sulfoxide (DMSO)+15 parts of human serum albumins), enters at last liquid nitrogen container through programmed cooling frozen.
The Identification of Biological Characteristics of embodiment 7, umbilical cord MSC
1, Growth of Cells and Morphological Characteristics thereof
By the separation and Culture of embodiment 1 and embodiment 6, the umbilical cord mononuclearcell was cultivated after 72 hours can obviously see the fusiformis attached cell at microscopically, can form the turbine-like cell clone about 10 days, can form the adherent layer that merge 80% left and right after had digestive transfer culture.In culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, easily by trysinization, is passaged to 5-15 generation, and its form and growth characteristic are also without obviously changing.
2, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, the Flow cytometry cell surface marker is dynamically observed the variation of cell surface marker in culturing process.The digestion collecting cell gets 8 * 10 after counting6Individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixing cell; Add CD45, CD105, HLA-ABC, HLA-DR, each 10 μ l of UEA-1 antibody of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC mark of PE mark, and establish a pipe and be blank; Under 4 ℃, lucifuge reaction 30min; PBS washes once, the centrifugal 10min of 1500rpm; Directly the cell of mark is abandoned supernatant, adds 200 μ l PBS piping and druming mixing cells, and 1% paraformaldehyde of 200 μ l is fixed, put 4 ℃ to be measured, the upflowing cell instrument detects in 3 days.
Flow cytometer detects the surface marker of cell, dynamically observes the cell in the 3rd, 6,9,12,15 generations, without obviously changing.not expressing the hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), HLA-DR (MHC-II quasi-molecule) continues negative, CD29 and the CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (is SH-3,4), CD105 (being SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I quasi-molecule) and UEA-1 (surface marker of endotheliocyte) are continuously the positive.After going down to posterity more than 3 generations, the cellular constituent homogeneous, purity is more than 95%.
3, the cell cycle of Flow cytometry umbilical cord MSC
When cell grows to 80% left and right and merges, digestion collecting cell approximately 1 * 106Individual, PBS washes once, adds 70% ethanol to fix, and 4 ℃ to be measured.During detection, the first centrifugal ethanol that goes, then wash once with PBS, add RNase I 500u, 37 ℃ of reaction 30min, PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, upper machine testing cell DNA content.
After measured the 3rd generation and the 6th generation cell DNA content, cell cycle analysis, G0/ G1Phase, S phase and G2M phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09%, and 2.54%, 3.25%.Result shows that the cell of vitro culture has typical stem cells hyperplasia characteristics, namely only has a few cell to be in active proliferation period (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).
4, the drafting of umbilical cord MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, the digestion counting is made cell suspension (every hole inoculation 0.5ml in 2 * 104/ml), 24 orifice plates, 37 ℃, 5%CO with the LG-DMEM substratum of 10%FBS2, cultivate under saturated humidity.Get 3 multiple holes every day, living cell counting number after Trypan Blue, calculating mean value, Continuous Observation 7 days.Take incubation time as transverse axis, cell count is the longitudinal axis, draws cell growth curve.Calculate cell in the doubling time of logarithmic phase with the Patterson formula, i.e. Td=Tlg2/Lg (Nt/No), Td: the doubling time (h), T: cell increases to Nt time used (h), N: cell count by No.
By Cytometric result drafting every day cell growth curve, calculate the doubling time.Can be found out by cell growth curve, cell was in exponential phase of growth at 2-4 days.According to formula calculate the 5th generation cell in doubling time of exponential phase of growth in 18-30 hour scope.
5, the evaluation of umbilical cord MSC multi-lineage potential
(1) osteogenic induction
3 above MSC of generation are by 1 * 105Inoculation six orifice plates in/hole are put in 37 ℃, 5%CO2, under saturated humidity, after cultivating 24h in the MSC substratum, use instead and contain 10% through the DMEM-HG of screening FBS and add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO2, cultivate under saturated humidity, amount was changed liquid in every 3 days half, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that the bone tubercle forms.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM to cultivate for 1 week, cellular form occurs significantly to change, become polygon by fusiform inoblast sample, be similar to the neuronal cell sample, it is outstanding that the long filament shape appears in the cell periphery, and can extend towards periphery.Continue to cultivate 2 weeks above after, calcified plaque appears in cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating for 4 weeks, visible obviously calcification tubercle.During 2 week, alkaline phosphatase staining is strong positive reaction, reaches more than 95%, and the control group of not induced is most of negative, only is shown as the weak positive less than 5%, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black with the calcium that deposits in the bone tubercle, induces the visible a large amount of black bone tubercle of group, obvious three-dimensional arrangement is arranged, and control group does not all have positive reaction at any time.
(2) Adipogenic induction
3 above MSC of generation are by 1 * 105/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO2, under saturated humidity, cultivate 24h in the MSC substratum after, use instead and contain 10% DMEM in high glucose through screening FBS, and add dexamethasone 1 μ M, INDOMETHACIN 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml, be put in 37 ℃, 5%CO2, cultivate under saturated humidity, amount was changed liquid in every 3 days half, and in 2 weeks of coinduction, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml to cultivate 3 days, form namely occurs and changes in cell, is shunk gradually by fusiform inoblast sample to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7 days has small fat to ooze existing in visible cell under mirror, along with the prolongation of incubation time, fat drips and increases gradually and merge, to cultivating 2 when all, as seen merges agglomerating fat and drips and be full of whole cell.The fat that produces in the oil red O stain visible cell is dyed redness by specificity.
(3) become chondrocyte induction
3 generation above cell, according to every pipe 2 * 105Cell divides and installs to the 15ml polypropylene centrifuge tube, low-speed centrifugal makes cell form micelle in test tube, add Regular Insulin, Transferrins,iron complexes, each 6.25 μ g/ml of Sodium Selenite in containing the DMEM-HG of 2.5%FBS, BSA 1.25 μ g/ml, Sodium.alpha.-ketopropionate 1mM/L, xitix phosphoric acid 37.5 μ g/ml, TGF-β 150ng/ml is put in 37 ℃, 5%CO2, cultivate under saturated humidity, amount was changed liquid, 2 weeks of cultured continuously in every 3 days half.
After inducing for 2 weeks, the cell micelle is broken up smear, the visible II Collagen Type VI of alcian blue (Alcian blue) dyeing forms extracellular matrix and is blue, and control group dyes without indigo plant.
By the detection of above a series of data targets, demonstrate and use the MSC that the inventive method separation obtains, have to the ability of scleroblast, adipocyte, Chondrocyte Differentiation, the MSC that the proved inventive method obtains has the stem cell characteristic.
The foundation of embodiment 8, umbilical cord stem cell bank
1, the detection of cytoactive
Utilize trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize a small amount of cell cultures, detect the pollution whether cell is subject to fungus and bacterium.Utilize the etiology method, detect whether cell is subject to Hepatitis B virus, the third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infects.
3, the detection of inherited disease
Utilize the method for molecular genetics, detect freeze-stored cell and whether have inherited disease.
4, HLA-ABC/DR joins type
Detect cell HLA-ABC/DR phenotype, and place on record.
5, the investigation of cell derived
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of umbilical cord stem cell database
After preserving normal umbilical cord stem cell, set up the database of umbilical cord stem cell, comprising the first five items data, and foundation and freeze-stored cell is related.