CN102660497A - Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells - Google Patents
Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells Download PDFInfo
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Abstract
The invention relates to a method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells after reviving the umbilical cord, and the step comprises the following steps of preparing umbilical cord tissue freezing liquid; sterilizing and washing the umbilical cord tissues; cutting the tissues into blocks; placing the tissue blocks and the freezing liquid into a freezing tube, cold storing the tissue blocks for 0.5 hour under the temperature condition of 4 DEG C and then storing the tissue blocks for one day under the temperature condition of minus 80 DEG C, and then freezing the tissue blocks in liquefied nitrogen; and taking the umbilical cord tissues out of the liquefied nitrogen to use, thawing the umbilical cord tissues in constant-temperature water bath, utilizing mesenchymal stem cell culture base to washing the umbilical cord tissues through a drop method, and separating and increasing the mesenchymal stem cells of the revived umbilical cord tissues through a tissue attachment method. The method can effectively protect the frozen umbilical cord tissue, so that the umbilical cord tissues can be revived to use, and the method is particularly suitable for separating and increasing the mesenchymal stem cells after the umbilical cord tissue is revived.
Description
Technical field
The present invention relates to handle the method for umbilical cord tissue, be specifically related to the method that the umbilical cord flesh tissue is frozen, the back stem cell that recovers and recover separates and increases, particularly the method for separation and amplification of mesenchymal stem cells from the umbilical cord flesh tissue.
Background technology
(mesenchymal stem cells MSC) derives from growth early stage mesoderm and ectoderm to mescenchymal stem cell, has characteristics such as multidirectional differentiation potential, immunomodulatory and self-replacation, receives people's attention day by day.Mescenchymal stem cell is in vivo or under the external specific inductive condition; Can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium; Still has multidirectional differentiation potential after continuous passage cultivation and the freezing preservation; Can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes, especially very big clinical value is arranged treating aging and injuries of tissues and organs reparation.
MSC contains abundant in marrow, but aging along with the age, the stem cell number in the marrow also can significantly reduce, the also decline significantly of proliferation and differentiation ability.In addition; Marrow MSC transplants to allosome possibly cause immunoreation; And extract the stem cell process to the damaging of patient and the other problems that when gathering, runs into; All directly influenced the clinical application of marrow MSC, made that seeking marrow becomes an important problem in other alternative mescenchymal stem cells sources in addition.
Recent research shows, also contains mescenchymal stem cell in the umbilical cord tissue and can successfully separate.This tissue-derived mescenchymal stem cell has not only kept the biological characteristics of mescenchymal stem cell, and the stem cell of separating is more original, and stronger proliferation and differentiation ability is arranged.The functionally active of its immunocyte is low, has lowered the risk that triggers immunoreation and cause graft versus host disease greatly.Occult virus and infection by microorganisms and propagation probability are lower.Gatherer process is simple, and puerpera and newborn infant are not had any harm and damage.Above reason is enough to make umbilical cord mesenchymal stem cells to become the desirable surrogate of mesenchymal stem cells MSCs.
But the method for umbilical cord tissue separate stem cells and technology also are not fully matureds, and the processing of each part umbilical cord tissue with separate after cell cultures all need regular hour and personnel's consumption.Therefore with umbilical cord tissue frozen and have need in recovery carry out the isolating way of stem cell and relatively more meet cost benefit.Therefore, this area needs the method for separating of simple a, recovery frozen with umbilical cord tissue efficiently and mescenchymal stem cell and amplification, with the demand in satisfied medicine, scientific research, field such as clinical.
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 mescenchymal stem cell separates and increases after the recovery.The present invention finds to use frozen, the recovery of specific operation step and separates and amplification, can obtain mescenchymal stem cell effectively.The present invention is based on this discovery and be accomplished.
Therefore, first aspect present invention provides the method for handling umbilical cord tissue, and this method comprises followingly carries out frozen step to the umbilical cord flesh tissue that exsomatizes:
(1) preparation umbilical cord tissue frozen storing liquid: comprise rHSA and DMSO (DMSO 99.8MIN.) in the said 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 cleaning: 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 through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(3) umbilical cord tissue is handled: 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 the frozen container, then frozen container is put into the programmed cooling device; Earlier 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), frozen container is freezing in liquid nitrogen, subsequent use then again.
According to the method for first aspect present invention, this method also comprises the following step that the stripped flesh tissue of frozen umbilical cord is recovered:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue 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, this 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: other gets the cell cultures plate, and tissue block is tiled in the plate, and the tissue block quantity in each plate maintains 5-20 piece (for example 10-15 piece), makes be attached on the plate until tissue in the air-dry 2-50 of tissue block minute;
(7) umbilical cord tissue is cultivated: slowly add the mescenchymal stem cell substratum along the plate edge and get final product to organizing to flood; Put plate into CO
2Concentration is that 37 ℃ of incubators of 5% are cultivated, and is cultured to 3-7 days and plate is taken out from incubator when (for example 4-6 days, for example the 5th day), adds an amount of (tissue is flooded to get final product) mescenchymal stem cell substratum; When 9-11 days (for example the 10th day), the substratum in the plate is shifted out, add an amount of (tissue is flooded to get final product) fresh mescenchymal stem cell substratum, continue to cultivate; When 11-13 days (for example the 12nd day), remove all umbilical cord tissue pieces and continue and cultivate; After this (for example per 2 days) were once changed liquid entirely in every 1-3 days;
(8) passage: when the attached cell fusion rate in the plate reaches the 50-70% left and right sides, use digestive ferment (for example TrypLE Express, invitrogen product) to make attached cell break away from the plate bottom; Centrifugal, remove supernatant, add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity and carry out amplification cultivation; After this liquid was changed once in (for example per 2 days) in every 1-3 days, after fusion rate reaches 70-90%, promptly got umbilical cord mesenchymal stem cells, went down to posterity in case of necessity;
And optional following one or more steps:
(9) to step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: 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, subsequent use.
According to the method for first aspect present invention, this 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 rHSA and DMSO (DMSO 99.8MIN.) in the said 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 cleaning: 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 through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(3) umbilical cord tissue is handled: 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 the frozen container, then frozen container is put into the programmed cooling device; Earlier 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), frozen container is freezing in liquid nitrogen, subsequent use then again;
The step of (B) the stripped flesh tissue of frozen umbilical cord being recovered:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue 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) umbilical cord tissue to recovery below carries out the step that mescenchymal stem cell separates and increases:
(6) the adherent processing of umbilical cord tissue: other gets the cell cultures plate, and tissue block is tiled in the plate, and the tissue block quantity in each plate maintains 5-20 piece (for example 10-15 piece), makes be attached on the plate until tissue in the air-dry 2-50 of tissue block minute;
(7) umbilical cord tissue is cultivated: slowly add the mescenchymal stem cell substratum along the plate edge and get final product to organizing to flood; Put plate into CO
2Concentration is that 37 ℃ of incubators of 5% are cultivated, and is cultured to 3-7 days and plate is taken out from incubator when (for example 4-6 days, for example the 5th day), adds an amount of (tissue is flooded to get final product) mescenchymal stem cell substratum; When 9-11 days (for example the 10th day), the substratum in the plate is shifted out, add an amount of (tissue is flooded to get final product) fresh mescenchymal stem cell substratum, continue to cultivate; When 11-13 days (for example the 12nd day), remove all umbilical cord tissue pieces and continue and cultivate; After this (for example per 2 days) were once changed liquid entirely in every 1-3 days;
(8) passage: when the attached cell fusion rate in the plate reaches the 50-70% left and right sides, use digestive ferment (for example TrypLE Express, invitrogen product) to make attached cell break away from the plate bottom; Centrifugal, remove supernatant, add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity and carry out amplification cultivation; After this liquid was changed once in (for example per 2 days) in every 1-3 days, after fusion rate reaches 70-90%, promptly got umbilical cord mesenchymal stem cells, went down to posterity in case of necessity;
And optional
(D) following one or more step:
(9) to step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: 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, subsequent use.
According to the method for first aspect present invention, wherein said umbilical cord is the fresh in vitro tissue of umbilical cord.
According to the method for first aspect present invention, comprise rHSA and DMSO in the wherein said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains the 55-95 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains the 70-90 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains 80 weight parts in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 4-20 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 7-15 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 10 weight parts in the said umbilical cord tissue frozen storing liquid.In one embodiment, contain the rHSA of 80 weight parts of having an appointment, the DMSO of about 10 weight parts in the said umbilical cord tissue frozen storing liquid.In the above-mentioned embodiment, the summation of each component concentration is 100% in the umbilical cord tissue frozen storing liquid.The inventor finds; The umbilical cord tissue frozen storing liquid (promptly wherein the weight ratio of rHSA and DMSO is 80:10) that contains the DMSO of the rHSA of 80 weight parts of having an appointment, about 10 weight parts is preferred especially, than the prescription that changes 10% or more in the arbitrary component content that makes this umbilical cord tissue frozen storing liquid protecting umbilical cord tissue not receive to have significant advantage aspect the effect such as refrigerating process destruction.In the present invention, owing to also possibly contain those skilled in the art general other dosing solvent or solute in the frozen storing liquid, therefore above-mentioned is a kind of relative quantity with rHSA 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 said refrigeration of step (1) is in refrigerator, to preserve.
According to the method for first aspect present invention, wherein said umbilical cord is the fresh in vitro tissue of umbilical cord.
According to the method for first aspect present invention, wherein the said umbilical cord tissue of step (2) is in Biohazard Safety Equipment, to handle.
According to the method for first aspect present invention, wherein the step of the said tiling of step (2) is that umbilical cord tissue is tiled in the culture dish, and said culture dish diameter is the culture dish of 5-20cm, and preferred petridish 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, sodium salt that wherein said PBS damping fluid is a 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, the concentration of phosphate radical is 0.01-0.5M in the said PBS damping fluid, preferred 0.02-0.1M.In hereinafter test of the present invention, used PBS damping fluid is a sodium phosphate salt, and wherein the concentration of phosphate radical is 0.025M, and pH is 6.5.Need to prove that the inventor finds that 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; Carry out umbilical cord tissue then and be cut into tissue block, said culture dish diameter is the culture dish of 5-20cm, and preferred petridish diameter is the culture dish of 10cm.
According to the method for first aspect present invention, wherein in the step (3), the size of tissue block is about the 0.2-2.5 cubic centimetre, preferably is about the 0.5-1.5 cubic centimetre, and preferred about 1 cubic centimetre cube is block.
According to the method for first aspect present invention, wherein in the step (3), the tissue block size is at the 0.5-1.5 cubic centimetre, and preferred 0.5-1.0 cubic centimetre is very preferred in the time of particularly big or small about 1 cubic centimetre.Although the intended tissue fragment helps the realization of the inventive method for a short time; Yet the inventor finds in test under 0.2 cubic centimetre, 0.5 cubic centimetre, 1 cubic centimetre three kinds of states; They to cold storage effect, resuscitation effect, the adherent effect of increase umbilical cord tissue of improving umbilical cord tissue, shorten the effect aspect basically identicals such as time that attached cell climbs out of from tissue, and volume greater than after 1.5 cubic centimetres to cold storage effect, resuscitation effect, the adherent effect of increase umbilical cord tissue of improving umbilical cord tissue, shorten attached cell and disadvantageous effect arranged from the effects such as time that tissue climbs out of.
According to the method for first aspect present invention, wherein the said 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 said frozen container of step (4) is frozen pipe.
According to the method for first aspect present invention, wherein the described programmed cooling device of step (4) is the programmed cooling box.
Method according to first aspect present invention; Wherein the density of the described tissue block of step (4) in frozen container is the upper limit to guarantee that arbitrary tissue block all has enough DMSO that protection is provided; Said density is meant the tissue block number of individuals in the unit space, preferred 0.1-0.8/cm
3, preferred 0.5/cm
3
According to the method for first aspect present invention, wherein described deepfreeze of step (4) and freezing are put into the refrigerator realization with programmed cooling device (for example programmed cooling box).
According to the method for first aspect present invention, wherein the described sample of step (4) refrigeration flow process is under 1 ℃ to 7 ℃ temperature condition deepfreeze 0.2-2 hour.In one embodiment, said deepfreeze is under 2 ℃ to 6 ℃ temperature condition.In one embodiment, said deepfreeze is under 3 ℃ to 5 ℃ temperature condition.In one embodiment, said deepfreeze is under 5 ℃ temperature condition.In one embodiment, the said deepfreeze time is 0.3-1.5 hour.In one embodiment, the said deepfreeze time is 0.4-1 hour.In one embodiment, the said deepfreeze time is 0.5 hour.The inventor finds that deepfreeze 0.5 hour was preferred especially under 4 ℃ temperature condition, 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 0.5 hour syncretizing effect fullest of deepfreeze under 4 ℃ the temperature condition 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-10 ℃ to-150 ℃ temperature condition freezing 0.25-3 days.In one embodiment, said freezing be under-30 ℃ to-120 ℃ temperature condition.In one embodiment, said freezing be under-50 ℃ to-100 ℃ temperature condition.In one embodiment, said freezing be under-80 ℃ temperature condition.In one embodiment, said freezing time is 0.4-2 days.In one embodiment, said freezing time is 0.8-1.5 days.In one embodiment, said freezing time is 1 day.The inventor finds; Freezing 1 day is preferred especially under-80 ℃ temperature condition, 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 receive to have significant advantage aspect the effect such as refrigerating process destruction.
According to the method for first aspect present invention, wherein said the thawing of step (5) is in water bath with thermostatic control, to carry out.
According to the method for first aspect present invention, comprise FBS, L-Glutamine, Gentamicin and DMEM-F12 in the wherein said mescenchymal stem cell substratum.In one embodiment, the FBS that contains 10-20% in the said mescenchymal stem cell substratum.In one embodiment, contain 15% the FBS of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the L-Glutamine that contains 0.5-2% in the said mescenchymal stem cell substratum.In one embodiment, contain 1% the L-Glutamine (L-glutaminate) of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the Gentamicin (qingfengmeisu qiong) that contains 0.02-0.1% in the said mescenchymal stem cell substratum.In one embodiment, contain 0.05% the Gentamicin of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the DMEM-F12 that contains 80-90% in the said mescenchymal stem cell substratum.In one embodiment, contain 84% the DMEM-F12 of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, contain the Gentamicin of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part and the DMEM-F12 of about 84 weight parts in the said mescenchymal stem cell substratum.The inventor finds; The mescenchymal stem cell substratum of DMEM-F12 that contains Gentamicin and about 84 weight parts of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part is preferred especially, than the arbitrary component content in making this substratum change prescription 10% or more adherent at the increase umbilical cord tissue, shorten and have significant advantage aspect the effects such as time that attached cell climbs out of from tissue.
According to the method for first aspect present invention, wherein the said mescenchymal stem cell substratum of step (5) is to clean umbilical cord tissue through 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 said filtration of step (5) is removed waste liquid and is realized preferred 100 μ m strainers through 50-200 μ m strainer.
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 the about 10cm of diameter.
According to the method for first aspect present invention, wherein in the 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 10-15 minute air-dry time; Umbilical cord tissue is adherent, to shorten attached cell be best from the effect aspects such as time that tissue climbs out of to increasing, and is shorter than 5 minutes or air-dry time all has significant difference when being longer than 25 minutes than air-dry time.
According to the method for first aspect present invention, wherein in the step (7), the inventor finds; When being cultured to the 5th day plate is taken out from incubator, add an amount of mescenchymal stem cell substratum, continue to cultivate; In the time of the 10th day, the substratum in the plate is shifted out, add the mescenchymal stem cell substratum of proper amount of fresh, continue to cultivate; In the time of the 12nd day, remove all umbilical cord tissue pieces and continue and cultivate, after this per 2 days once change liquid entirely is preferred especially.This changes liquid and has effectively shortened attached cell with the setting of organizing clean-up time and reach the time of specifying fusion rate.
According to the method for first aspect present invention, in step (9), it is to utilize the trypan blue staining to count the number of frozen front and back viable cell that said cytoactive detects.
According to the method for first aspect present invention, in step (9), said cell contamination detects and utilizes small amounts of cells to cultivate, and detects cell and whether receives the pollution of fungi and bacterium.In one embodiment; Whether it is to utilize the etiology method that said cell contamination detects, detect cell and receive and be selected from following one or multinomial infection: hepatitis B two double, 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 said 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 said HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (10), said 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), said umbilical cord mesenchymal stem cells is present in the cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises DMEM-F12, DMSO 99.8MIN. and rHSA.In one embodiment, this cells frozen storing liquid comprises about 65 parts DMEM-F12, about 10 parts DMSO 99.8MIN., about 15 parts rHSA.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% DMSO 99.8MIN..
According to the method for first aspect present invention, this method may further comprise the steps:
(1) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO of 10 weight parts (DMSO 99.8MIN., dimethyl sulfoxide) in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(2) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(3) umbilical cord tissue is handled: 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 1cm
3Square shape;
(4) umbilical cord tissue is cold deposits: under 4 ℃ low temperature environment; Tissue block and frozen storing liquid are added in the frozen pipe; Then frozen pipe is put into the programmed cooling box, first deepfreeze 0.5 hour under 4 ℃ temperature condition, under the temperature condition again-80 ℃ freezing 1 day; Frozen pipe is freezing in liquid nitrogen, subsequent use then.
Further, can comprise and the matching used method for resuscitation of frozen method:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue is taken out from liquid nitrogen; Being placed on thaws in the 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 recovery back mescenchymal stem cell 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 be attached on the plate until tissue in the air-dry 10-15 of tissue block minute;
(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) along the plate edge and get final product to organizing to flood; Plate is placed CO
2Concentration 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; The tenth day with the media transfer in the plate, add the fresh mescenchymal stem cell substratum of 15ml; Remove all umbilical cord tissue pieces at the 12 day and continue to cultivate, once changed liquid in after this per two days entirely;
(8) passage: the attached cell fusion rate when the plate the inside reaches about 60%; Digestive ferment capable of using (TrypLE Express) breaks away from the plate bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid in per two days once after fusion rate reaches 80%, promptly get, go down to posterity again in case of necessity.
Further, can be directed against above step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type.
Further, the umbilical cord mesenchymal stem cells after can above step (8) gained being gone down to posterity is frozen in liquid nitrogen, subsequent use.
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 multidirectional differentiation potential that carry out cell are identified; And pair cell carries out molecular genetics diagnosis, preserves all related datas of cell, sets up the DB of umbilical cord stem cell and carries out related with freeze-stored cell.Therefore among the present invention in one aspect; The method of setting up umbilical cord stem cell DB 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 will going down to posterity is the frozen and relevant fetus information of record in liquid nitrogen, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell, and pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of umbilical cord stem cell and carry out related with freeze-stored cell.
In addition, in first aspect of the present invention, the method that the back stem cell that provides frozen to the umbilical cord flesh tissue, recovers and recovered 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, it obtains according to the said method of the arbitrary embodiment of first aspect present invention.
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, said 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%.
Be further described in the face of the present invention down.The document that the present invention quoted, and the document of being quoted in the document, their full content is incorporated this paper by reference into.
In the present invention; In arbitrary technical scheme of the arbitrary aspect of the present invention; Its arbitrary technical characterictic is equally applicable to arbitrary embodiment of arbitrary aspect of the present invention, as long as they can not cause contradiction, and this being useful in each other in case of necessity can be done suitable modification.
In the present invention, term " umbilical cord mesenchymal stem cells " is meant the mescenchymal stem cell that derives from umbilical cord.Therefore in the present invention, particularly relate in the linguistic context of the present invention, term " umbilical cord mesenchymal stem cells " can exchange with " umbilical cord stem cell ", " stem cell ", " mescenchymal stem cell " and use, and indicates only if having clearly in addition.
In the present invention, term " PBS damping fluid " perhaps " PBS " be meant phosphate buffered saline buffer.Generality prescription and compound method and their general aspects that those skilled in the art know the PBS that under situation of the present invention, uses be pH value or pH scope for example.
In the present invention, term " umbilical cord " is meant neonatal umbilical cord, is meant the umbilical cord within 4 hours postpartum especially.
Mescenchymal stem cell (mesenchymal stem cell; MSC) for example human mescenchymal stem cell is separated from marrow the earliest; Derive from mesoblastic one type of tissue stem cell with multidirectional differentiation potential and self ability; Has ability (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991 in vivo with under the external specified conditions to multiple adult cytodifferentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte, myocyte, liver cells; 9:641-650.Pittenger MF; Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999; 284:143-147).Up-to-date research shows that mescenchymal stem cell has immunomodulatory and hematopoiesis support effect, 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 make up; Important carrier cell in the gene therapy; And, in HSCT and organ transplantation, be with a wide range of applications because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, people successfully from multiple tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood separation and Culture go out mescenchymal stem cell.
The mescenchymal stem cell of being reported at present is mainly derived from marrow, adopts density gradient centrifugation to obtain.Though separation method is easy, donor is got marrow need experience a relatively more painful operation, and has very high infection chance in the process of drawing materials and after drawing materials; Because the content of MSC is extremely rare among the human bone marrow, per 10
5～10
6Approximately have only 1 in the individual mononuclearcell, and along with the increase at age, the quantity of mescenchymal stem cell, propagation and differentiation capability descend significantly all in the marrow, make it in research with use in the especially clinical application and be restricted.Originate from the umbilical cord of embryonic development period extraembryonic mesoderm and form, contain a large amount of mesenchyme compositions by a matter, blood vessel and nurse cell.
Up-to-date research shows and contains abundant stem cell in the umbilical cord, and 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 many shortcomings are arranged still, for example purity is not enough and/or quantity is not high, and then demonstrates the expectation that these methods still can not satisfy people.The for example invention of CN 101270349A (one 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 (one 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 (one 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 further improved remaining aspect the purity of extract and/or the recovery.
The invention discloses a kind of umbilical cord tissue is frozen, recovery and recovery back stem cell separates and the method for amplification, and this method capable of using is preserved umbilical cord mesenchymal stem cells and is set up the umbilical cord stem cell bank.Contriver of the present invention is summing up on the basis of frozen in the past, recovery umbilical cord tissue; Utilize umbilical cord tissue frozen storing liquid protection refrigerated umbilical cord tissue; Utilize topical application to clean the recovery umbilical cord tissue,, successfully from the umbilical cord tissue of recovery, separate obtaining a large amount of mescenchymal stem cells in conjunction with the adherent culture method.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 is inmature than adult stem cell in the umbilical cord; Content is abundant; Be with a wide range of applications clinically; We use conventional cell cryopreservation 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.
Because contain abundant hemopoietic stem cell in the 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 of treatment means.Same umbilical cord mesenchymal stem cells is as a kind of more importantly stem cell resource; We use conventional cell cryopreservation method it to be chilled in-196 degrees centigrade medium-term and long-term preservation of profound hypothermia liquid nitrogen; Set up the umbilical cord stem cell bank, for stem-cell therapy is in the future preserved seed.
According to the method for the invention, the prescription of umbilical cord tissue frozen storing liquid and sample refrigerated flow process can successfully and effectively be protected in the refrigerated process umbilical cord tissue.According to the method for the invention, utilize topical application to clean out, avoided the loss of cell survival rate in the recovery process at the DMSO of tissue the inside.According to the method for the invention, wherein the shape of umbilical cord tissue piece, size, quantity and air-dry time all are that the factor of adherent effect is organized in influence, thereby directly influence 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.According to the method for the invention, wherein the mescenchymal stem cell culture medium prescription can successfully and effectively carry out amplification in vitro to umbilical cord mesenchymal stem cells.According to the method for the invention, wherein changing liquid has shortened attached cell with the setting of organizing clean-up time and has reached 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 the ability to cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.Comparison with existing method: MSC mainly adopts modus operandi extraction donor marrow or perfusion method to separate umbilical cord at present, and adherent culture obtains.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, the cell composition is inmature, and wide material sources conveniently are easy to get, and therefore method of the present invention will have broad application prospect in the clinical application of stem cell.
Embodiment
Can further describe the present invention through following embodiment, 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 TP that are used in the test.Though for realizing that employed many materials of the object of the invention and working method 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 back stem cell that recovers and recover separates and increases
The frozen method of umbilical cord tissue may further comprise the steps:
(1) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO of 10 weight parts (DMSO 99.8MIN., dimethyl sulfoxide) in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(2) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(3) umbilical cord tissue is handled: 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 1cm
3Square shape;
(4) umbilical cord tissue is cold deposits: under 4 ℃ low temperature environment; Tissue block and frozen storing liquid are added in the frozen pipe; Then frozen pipe is put into the programmed cooling box, first deepfreeze 0.5 hour under 4 ℃ temperature condition, under the temperature condition again-80 ℃ freezing 1 day; Frozen pipe is freezing in liquid nitrogen, subsequent use then.
May further comprise the steps with the matching used method for resuscitation of frozen method:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue is taken out from liquid nitrogen; Being placed on thaws in the 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.
Recovery back mescenchymal stem cell separates and the method for amplification may further comprise the 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 be attached on the plate until tissue in the air-dry 10-15 of tissue block minute;
(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) along the plate edge and get final product to organizing to flood; Plate is placed CO
2Concentration 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; The tenth day with the media transfer in the plate, add the fresh mescenchymal stem cell substratum of 15ml; Remove all umbilical cord tissue pieces at the 12 day and continue to cultivate, once changed liquid in after this per two days entirely;
(8) passage: the attached cell fusion rate when the plate the inside reaches about 60%; Digestive ferment capable of using (TrypLE Express) breaks away from the plate bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid and once after fusion rate reaches 80%, go down to posterity again in per 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%, be passaged to the T25 culturing bottle after, reach 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; Comprise the rHSA of 80 weight parts and the DMSO (DMSO 99.8MIN. of 10 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 the refrigerated process, is embodied in the isolating mescenchymal stem cell counting in freezing and storing umbilical tissue recovery back and obviously descends.
In the test of above step; Sample refrigerated flow process is a deepfreeze 0.5 hour under 4 ℃ temperature condition; Under the temperature condition again-80 ℃ freezing 1 day, the arbitrary temp of this freezing flow process was departing from said temperature 60% when above, and the time is departing from 80% when above; Umbilical cord tissue is not effectively protected in the refrigerating process, is embodied in the isolating mescenchymal stem cell counting in freezing and storing umbilical tissue recovery back 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, during wherein any three kinds of fixed ratio, alternative ratio does not all reach 75% the 17th day fusion rate after departing from said ratio 10% when above, being passaged to the T25 culturing bottle.For example in the mescenchymal stem cell substratum that uses, comprise 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12; And when 12 weight part FBS, 13.5 weight part FBS, 16.5 weight part FBS or 18 weight part FBS; Under four kinds of situation; After being passaged to the T25 culturing bottle, the 17th day fusion rate all between 55-75%.
In the test of above step, the 14th back of changing the liquid time and be cell cultures is for once changing liquid in per 2 days entirely, and the liquid time of changing is arbitrarily wherein being departed from above-mentioned time 80% when above, and attached cell did not reach fusion rate 80% in 20 days.For example change the liquid time and be and changed liquid entirely for the first time in the 16th day of cell cultures, or once changed liquid entirely for per 4 days backward, under two kinds of situation, attached cell reached 80% at 25-30 days.
Embodiment 2, the method that umbilical cord tissue is frozen, the back stem cell that recovers and recover separates and increases
The method of reference implementation 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 back stem cell that recovers and recover separates and increases
The method of reference implementation 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 back stem cell that recovers and recover separates and increases
The method of reference implementation 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 back stem cell that recovers and recover separates and increases
The method of reference implementation 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 of each acquisition of embodiment 1-5 is digested, get 1 * 10 after the digestion
6Cell joins in the 1ml cells frozen storing liquid (containing 65 parts of DMEM-F12+10 part DMSO 99.8MIN.s+15 parts of rHSAs), and it is frozen to enter into liquid nitrogen container at last through programmed cooling.
The biological characteristics of embodiment 7, umbilical cord MSC is identified
1, cell growth and morphology characteristics thereof
Through the separation and Culture of embodiment 1 and embodiment 6, the cultivation of umbilical cord mononuclearcell can obviously be seen the fusiformis attached cell at microscopically after 72 hours, can form the turbine-like cell clone about 10 days, can form the adherent layer of about 80% fusions after the had digestive transfer culture.In the culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, is prone to be passaged to 5-15 generation by trysinization, and its form and growth characteristic also do not have obvious change.
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 dynamic observes the variation of cell surface marker in the culturing process.The digestion collecting cell gets 8 * 10 behind the counting
6Individual 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 cell, and 1% Paraformaldehyde 96 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, dynamic observes the cell in the 3rd, 6,9,12,15 generations, does not have obviously to change.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), the lasting feminine gender of HLA-DR (MHC-II quasi-molecule); CD29 and CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (being 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
Cell grows to about 80% when merging, digestion collecting cell about 1 * 10
6Individual, PBS washes once, and the ethanol of adding 70% is fixed, and 4 ℃ to be measured.During detection, the centrifugal ethanol that goes is washed once with PBS more earlier, adds RNase I 500u, 37 ℃ of reaction 30min, and PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, last machine testing cell DNA content.
Through measure the 3rd generation and the 6th generation cell dna content, cell cycle analysis, G
0/ G
1Phase, S phase and G
2M phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25%.The result shows that the cell of vitro culture has typical stem cells hyperplasia characteristics, promptly has only few cell to be in the active propagation phase (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 processed cell suspension (every hole inoculation 0.5ml in 2 * 104/ml), 24 orifice plates, 37 ℃, 5%CO with the LG-DMEM substratum of 10%FBS
2, cultivate under the saturated humidity.Get 3 multiple holes every day, trypan blue dyeing back living cell counting number, calculating mean value was observed 7 days continuously.With the incubation time is transverse axis, and 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 used time (h), N: cell count by No.
Cytometric result draws cell growth curve through every day, calculates the doubling time.Can find out that by cell growth curve cell was in exponential phase of growth at 2-4 days.According to formula calculate the 5th generation cell doubling time of exponential phase of growth in 18-30 hour scope.
5, the evaluation of the multidirectional differentiation potential of umbilical cord MSC
(1) osteogenic induction
Above MSC of 3 generations is by 1 * 10
5Six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO
2, under the saturated humidity, cultivate 24h in the MSC substratum after, use instead and contain 10% through the DMEM-HG of screening FBS and add DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO
2, under saturated humidity, cultivate, amount was changed liquid in per 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; Adding DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM cultivated for 1 week; Cellular form takes place significantly to change, and becomes polygon by fusiform inoblast appearance, is similar to neuronal cell appearance; 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 the cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating for 4 weeks, visible obviously calcification tubercle.The alkaline phosphatase staining of 2 whens week is strong positive reaction, reaches more than 95%, and in addition the inductive control group is then most of not 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 sedimentary calcium in the bone tubercle, induces the visible a large amount of black bone tubercle of group, tangible three-dimensional arrangement is arranged, and control group does not all have positive reaction at any time.
(2) become fat to induce
Above MSC of 3 generations is by 1 * 10
5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO
2, under the saturated humidity, in the MSC substratum, cultivate 24h after, use instead and contain 10% high sugared DMEM, and add DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml through screening FBS, be put in 37 ℃, 5%CO
2, cultivate under the saturated humidity, amount was changed liquid in per 3 days half, and in 2 weeks of coinduction, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS; Adding DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml cultivated 3 days; Form promptly takes place and changes in cell, is shunk gradually by fusiform inoblast appearance to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7 days, there have small fat to ooze under the mirror in the visible cell to be existing, and along with the prolongation of incubation time, fat drips and increases gradually and merges, and when cultivating for 2 weeks, the agglomerating fat of visible fusion drips and is 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 generations above cell, according to every pipe 2 * 10
5The cell branch installs to the 15ml polypropylene centrifuge tube; Low-speed centrifugal makes cell in test tube, form micelle, in containing the DMEM-HG of 2.5%FBS, adds Regular Insulin, Transferrins,iron complexes, each 6.25 μ g/ml of Sodium Selenite, 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%CO
2, cultivate under the saturated humidity, amount was changed liquid, 2 weeks of cultured continuously in per 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 does not have indigo plant and dyes.
Through the detection of above a series of data targets, demonstrate and use the MSC that the inventive method separation obtains, have ability to scleroblast, adipocyte, chondrocyte's differentiation, confirm that the MSC that the inventive method obtains has the stem cell characteristic.
The foundation of embodiment 8, umbilical cord stem cell bank
1, the detection of cytoactive
Utilize the trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize small amounts of cells to cultivate, detect cell and whether receive the pollution of fungi and bacterium.Utilize the etiology method, detect cell whether receive hepatitis B two double, third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST and infect.
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 in cell source
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of umbilical cord stem cell DB
After preserving normal umbilical cord stem cell, set up the DB of umbilical cord stem cell, comprising the first five items data, and foundation and freeze-stored cell is related.
Claims (10)
1. handle the method for umbilical cord tissue, this 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 rHSA and DMSO (DMSO 99.8MIN.) in the said 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 cleaning: 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 through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(3) umbilical cord tissue is handled: 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 the frozen container, then frozen container is put into the programmed cooling device; Earlier 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), frozen container is freezing in liquid nitrogen, subsequent use then again;
The step of (B) the stripped flesh tissue of frozen umbilical cord being recovered:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue 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) umbilical cord tissue to recovery below carries out the step that mescenchymal stem cell separates and increases:
(6) the adherent processing of umbilical cord tissue: other gets the cell cultures plate, and tissue block is tiled in the plate, and the tissue block quantity in each plate maintains 5-20 piece (for example 10-15 piece), makes be attached on the plate until tissue in the air-dry 2-50 of tissue block minute;
(7) umbilical cord tissue is cultivated: slowly add the mescenchymal stem cell substratum along the plate edge and get final product to organizing to flood; Put plate into CO
2Concentration is that 37 ℃ of incubators of 5% are cultivated, and is cultured to 3-7 days and plate is taken out from incubator when (for example 4-6 days, for example the 5th day), adds an amount of (tissue is flooded to get final product) mescenchymal stem cell substratum; When 9-11 days (for example the 10th day), the substratum in the plate is shifted out, add an amount of (tissue is flooded to get final product) fresh mescenchymal stem cell substratum, continue to cultivate; When 11-13 days (for example the 12nd day), remove all umbilical cord tissue pieces and continue and cultivate; After this (for example per 2 days) were once changed liquid entirely in every 1-3 days;
(8) passage: when the attached cell fusion rate in the plate reaches the 50-70% left and right sides, use digestive ferment (for example TrypLE Express, invitrogen product) to make attached cell break away from the plate bottom; Centrifugal, remove supernatant, add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity and carry out amplification cultivation; After this liquid was changed once in (for example per 2 days) in every 1-3 days, after fusion rate reaches 70-90%, promptly got umbilical cord mesenchymal stem cells, went down to posterity in case of necessity;
And optional
(D) following one or more step:
(9) to step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: 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, subsequent use.
2. comprise the rHSA of 80 weight parts and the DMSO of 10 weight parts according to the process of claim 1 wherein in the said umbilical cord tissue frozen storing liquid.
3. according to the process of claim 1 wherein in the step (3), the tissue that shreds is the about 0.2-2.5 cubic centimetre of size.
4. according to the process of claim 1 wherein in the step (5), what utilize that the mescenchymal stem cell substratum cleans that umbilical cord tissue adopts is topical application.
5. comprise FBS, L-Glutamine, Gentamicin and DMEM-F12 according to the process of claim 1 wherein in the said mescenchymal stem cell substratum.
6. according to the process of claim 1 wherein:
The FBS that contains 10-20% in the said mescenchymal stem cell substratum;
The L-Glutamine that contains 0.5-2% in the said mescenchymal stem cell substratum;
The Gentamicin that contains 0.02-0.1% in the said mescenchymal stem cell substratum; And/or
The DMEM-F12 that contains 80-90% in the said mescenchymal stem cell substratum.
7. contain the Gentamicin of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part and the DMEM-F12 of about 84 weight parts according to the process of claim 1 wherein in the said mescenchymal stem cell substratum.
8. according to the method for claim 1, this method may further comprise the steps:
(1) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO of 10 weight parts (DMSO 99.8MIN., dimethyl sulfoxide) in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(2) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(3) umbilical cord tissue is handled: the umbilical cord tissue that step (2) is obtained is transferred in another 10cm cell cultures plate, umbilical cord tissue is cut into the square shape of big or small 1cm3;
(4) umbilical cord tissue is cold deposits: under 4 ℃ low temperature environment; Tissue block and frozen storing liquid are added in the frozen pipe; Then frozen pipe is put into the programmed cooling box, first deepfreeze 0.5 hour under 4 ℃ temperature condition, under the temperature condition again-80 ℃ freezing 1 day; Frozen pipe is freezing in liquid nitrogen, subsequent use then;
Said frozen method can also be selected the step of recovery matching used with it:
(5) freezing and storing umbilical tissue recovery: step (4) refrigerated umbilical cord tissue is taken out from liquid nitrogen; Being placed on thaws in the water bath with thermostatic control begins to melt to half frozen storing liquid; Utilize mescenchymal stem cell substratum (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;
Said frozen method can also be selected to recover, and the back mescenchymal stem cell separates and the step of 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 be attached on the plate until tissue in the air-dry 10-15 of tissue block minute;
(7) umbilical cord tissue is cultivated: slowly add mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) along the plate edge and get final product to organizing to flood; It is that 37 ℃ of incubators of 5% are cultivated that plate is placed CO2 concentration, when being cultured to the 5th day plate is taken out from incubator, adds 5ml mescenchymal stem cell substratum; The tenth day with the media transfer in the plate, add the fresh mescenchymal stem cell substratum of 15ml; Remove all umbilical cord tissue pieces at the 12 day and continue to cultivate, once changed liquid in after this per two days entirely;
(8) passage: the attached cell fusion rate when the plate the inside reaches about 60%; Digestive ferment capable of using (TrypLE Express) breaks away from the plate bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid in per two days once after fusion rate reaches 80%, promptly get, go down to posterity again in case of necessity;
Randomly,
(9) to above step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type; And/or
(10) umbilical cord mesenchymal stem cells after above step (8) gained is gone down to posterity is frozen in liquid nitrogen, subsequent use.
9. set up the method for umbilical cord stem cell DB; It comprises the step of each said method of claim 1-8; And following steps: the cell after will going down to posterity is the frozen and relevant fetus information of record in liquid nitrogen, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell, and pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of umbilical cord stem cell and carry out related with freeze-stored cell.
10. umbilical cord mesenchymal stem cells, it obtains according to each said method of claim 1-8.
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