CN103266081A - Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord - Google Patents
Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord Download PDFInfo
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- CN103266081A CN103266081A CN2013100204894A CN201310020489A CN103266081A CN 103266081 A CN103266081 A CN 103266081A CN 2013100204894 A CN2013100204894 A CN 2013100204894A CN 201310020489 A CN201310020489 A CN 201310020489A CN 103266081 A CN103266081 A CN 103266081A
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Abstract
The present invention relates to an efficient method for isolating and culturing mesenchymal stem cells from umbilical cords, i.e. a staged suspension explant culture method, comprising the following steps: (1) digesting an umbilical cord in vitro by using a tissue digestive enzyme; (2) culturing the digested cells with a mesenchymal stem cell culture medium for culturing to obtain mesenchymal stem cells; (3) culturing the tissue being not fully digested after digestion by a mesenchymal stem cell culture medium to obtain the mesenchymal stem cells. According to the present invention, the mesenchymal stem cells obtained by the method are in line with mesenchymal stem cell characteristics in surface markers, growth characteristics, multi-directional induced differentiation and other aspects. The method is simple, a lot of mesenchymal stem cells could be obtained at the same time, and the method provides a new way for obtaining mesenchymal stem cells.
Description
Technical field
The present invention relates to a kind of high efficiency method of separation and Culture mescenchymal stem cell, particularly from the method for umbilical cord separation and Culture mescenchymal stem cell.
Background technology
(mesenchymal stem cells is to derive to grow the multipotential stem cell that early stage mesoderm and an ectodermic class have height self ability and multidirectional differentiation potential MSCs) to mescenchymal stem cell.Can be under external suitable condition continuous passage cultivate, frozen, and its form of frozen front and back, phenotype and differentiation capability are not seen change.Mescenchymal stem cell is not expressed CD80, CD86, collaborative stimulation molecule such as HLA-DR
[1], externally suppress mixed lymphocyte reacion, inducing immune tolerance in the body, the graft versus host disease (GVH disease) (GVHD) that clinical confirmation causes after for prevention and treatment recessive allele hematopoietic cell transplantation has good therapeutic action; Remarkable to autoimmune disorder curing psoriasis effect simultaneously.As seed cell, be mainly used in treating histocyte that body can't repair naturally and the multiple refractory disease (for example Spinal injury, brain paralysis, amyotrophic lateral sclerosis, apoplexy, diabetes, diabetic foot, liver cirrhosis etc.) of organ damage clinically.
Mescenchymal stem cell is as the important seed cell of area researches such as preclinical medicine, clinical medicine, regenerative medicine, organizational project, and seeking convenient abundant cultivation amplification condition tissue-derived, that set up long-term stability is the required of various researchs and application.At first, the main source of MSCs is adult's marrow, but adult's bone marrow MSCs cell quantity and proliferation and differentiation potential descend with the increase at age, viral infection rate is higher, and the collection of donor MSCs palpus row bone marrow puncture, the source is restricted, and immunological rejection is less to be selected for use the cell in allosome source because having; (embryonic stem cell though ES) higher evaluation is arranged in theory, can be used for heteroplastic transplantation to embryonic stem cell, and its application is limit by ethics and law; Though the MSCs of its immunophenotype of the mescenchymal stem cell in bleeding of the umbilicus and the peripheral blood and functional characteristics and derived from bone marrow is very similar, incubation time is long, multiplication capacity is low, also the best of non-mescenchymal stem cell source.And umbilical cord belongs to the embryo and organizes outward, contains the stem cell of a large amount of multidirectional differentiation, and cell growth amplification rate is fast, and umbilical cord becomes " waste " after fetus is given birth to, so draw materials conveniently, the source is abundant, more do not have ethnics Problem, can be used as splendid mescenchymal stem cell source.And studies show that: the specific marker CD90 of the mescenchymal stem cell strongly expressed mesenchymal cell in umbilical cord source, CD73, CD105, and do not express mark CD45, CD14, CD11, CD34, the CD19 of hematopoiesis system or interior skin system cell lowly expresses or does not express major histocompatibility antigen HLA-DR
[2～4]In sum: umbilical cord is the source of mescenchymal stem cell comparative advantages
[5]
The method of separation and Culture mescenchymal stem cell has from umbilical cord tissue: the endothelium digestion method
[6], tissue block adherent method, enzyme digestion etc.Be difficult to very separating mesenchymal stem cell from umbilical cord tissue by the endothelium digestion method; Obtain time-consuming grow (about about 15 days) of primary cell by the tissue block adherent method, and be difficult to separate stem cells from umbilical cord tissue up hill and dale; Obtain the primary cell limited amount by the simple enzyme digestion method, can obtain 10 about January
9-10
10, therefore need a kind of method that can more efficiently obtain umbilical cord source mescenchymal stem cell badly.
Summary of the invention
The purpose of this invention is to provide the method that a kind of simple and fast can obtain umbilical cord source mescenchymal stem cell simultaneously in a large number, namely stage is planted piece suspension culture method.
A first aspect of the present invention provides a kind of method of separation and Culture mescenchymal stem cell, and it comprises the steps (1), and in step (2) and the step (3) one or two:
(1) gets stripped umbilical cord, handle with the tissue digestion enzyme; Preferably, before digestion process, also comprise the step that umbilical cord is divided into segment umbilical cord tissue piece;
(2) after the digestion process of step (1) finished, obtained cell suspension placed the mescenchymal stem cell substratum to cultivate, and namely obtains mescenchymal stem cell; Preferably, in culturing process, the cell suspension of changing when changing liquid is refitted in and continues in the new culturing bottle to cultivate, and obtains mescenchymal stem cell;
Preferably, also comprise the step that cell suspension is centrifugal before the cell suspension that obtains places the mescenchymal stem cell substratum after step (1) is handled, place the mescenchymal stem cell substratum to cultivate the cell precipitation that obtains after centrifugal;
Randomly, after obtaining mescenchymal stem cell, also comprise the step that cell dissociation is gone down to posterity;
(3) after the digestion process of step (1) finished, the umbilical cord tissue of getting not complete digestion placed the mescenchymal stem cell substratum to cultivate, and can obtain mescenchymal stem cell; Randomly, after obtaining mescenchymal stem cell, remaining umbilical cord tissue can repeatedly obtain mescenchymal stem cell by repeatedly repeating the method in this step; Randomly, after obtaining mescenchymal stem cell, also comprise the step that cell dissociation is gone down to posterity.
Wherein said comprising the steps (1), and step (2) and step (3) one or two refer to step (1)+step (2), perhaps step (1)+step (3), perhaps step (1)+step (2)+step (3).
According to each described method of first aspect present invention, wherein said tissue digestion enzyme is collagenase and/or Unidasa.
According to each described method of first aspect present invention, wherein said umbilical cord is divided into before the segment umbilical cord tissue piece, also comprise the step of removing Umbilical artery.
According to each described method of first aspect present invention, before the wherein said removal Umbilical artery, also comprise the step of cleaning the umbilical cord that exsomatizes.
According to each described method of first aspect present invention, the working concentration of wherein said collagenase is the 0.05-0.2% bulking value; And/or the working concentration of wherein said Unidasa is the 0.0005-0.001% bulking value.
According to each described method of first aspect present invention, wherein said collagenase is II Collagen Type VI enzyme.
According to each described method of first aspect present invention, wherein the condition of the digestion process described in the step (1) is 37 ℃, 1-3h.
In the present invention, wherein said mescenchymal stem cell substratum is for being fit to any substratum of growth of mesenchymal stem cells, for example can be DMEM/F12+10%FBS, non-animal derived property mescenchymal stem cell substratum or mesenchymal stem cell serum-free culture medium, in one embodiment of the invention, described mescenchymal stem cell substratum is serum free medium.In a specific embodiments of the present invention, described serum free medium is that the article No. of Gibco company is the serum free medium of A10675; In another specific embodiments of the present invention, described serum free medium is that the article No. of three favourable companies is 120408 serum free medium.
According to each described method of first aspect present invention, wherein the mescenchymal stem cell substratum that cell suspension is placed described in the step (2) is cultivated, refer to treat cell attachment growth after, be cultured to the 80-90% cytogamy.
In embodiments of the invention, place the cultivation of mescenchymal stem cell substratum after 6-9 days cell suspension, the 80-90% cytogamy can obtain mescenchymal stem cell; In specific embodiments of the present invention, cultivate after 7-8 days, obtain mescenchymal stem cell.
In embodiments of the invention, change liquid after placing the mescenchymal stem cell substratum to cultivate 3-4 the cell suspension, the cell suspension of changing is refitted in continues in the new culturing bottle to cultivate, further obtain mescenchymal stem cell.
According to each described method of first aspect present invention, wherein the umbilical cord tissue of getting not complete digestion described in the step (3) places the mescenchymal stem cell substratum to cultivate, after referring to treat that tissue block has spindle cell to climb out of on every side, be cultured to the 80-90% cytogamy.
In embodiments of the invention, the umbilical cord tissue of getting not complete digestion places the mescenchymal stem cell substratum to cultivate after 6-9 days, and the 80-90% cytogamy obtains mescenchymal stem cell.In specific embodiments of the present invention, cultivate after 7-8 days, obtain mescenchymal stem cell.
According to each described method of first aspect present invention, wherein said had digestive transfer culture comprises the step of using pancreas enzyme-EDTA solution.
In embodiments of the invention, the working concentration of pancreatin is the 0.05-0.2% bulking value in the described pancreas enzyme-EDTA solution, for example is the 0.1-0.125% bulking value; In embodiments of the invention, the working concentration of EDTA is the 0.01-0.03% bulking value in the described pancreas enzyme-EDTA solution, for example is the 0.015-0.02% bulking value.
According to each described method of first aspect present invention, wherein said had digestive transfer culture method comprises:
(i) use the physiological saline washed cell;
(ii) add an amount of pancreas enzyme-EDTA solution, treat that solution is paved to inhale behind the Tissue Culture Flask bottom surface to abandon pancreas enzyme-EDTA solution;
(iii) blow and beat cell with stoste, make single cell suspension, centrifugal back counting is inoculated in the culturing bottle to be cultivated.
A second aspect of the present invention relates to the mescenchymal stem cell for preparing according to each described method of first aspect present invention.
According to each mescenchymal stem cell of second aspect present invention, it expresses CD90, CD105, CD73, does not express CD31, CD14, CD34, CD45, CD80, CD86, CD40, CD40L and HLA-DR.
According to each mescenchymal stem cell of second aspect present invention, it can be divided into scleroblast, adipocyte and/or chondrocyte.
A third aspect of the present invention relates to collagenase and/or Unidasa for the preparation of the purposes of mescenchymal stem cell.
Below the present invention is described in detail.
In the present invention, the umbilical cord of described umbilical cord for exsomatizing, it derives from people or Mammals.
In the present invention, described cellular segregation cultural method: for stage is planted piece suspension culture method.
In the present invention, the mescenchymal stem cell for preparing from umbilical cord according to the inventive method is also referred to as umbilical cord source mescenchymal stem cell.
In the present invention, umbilical cord is divided into the digestion that segment is beneficial to the tissue digestion enzyme, for example is cut into the segment of 0.5-2cm.
In the present invention, after umbilical cord tissue and digestive ferment mixing, for abundant reaction, can be placed in the single cell suspension preparing instrument.
In the present invention, the part that the treatment process of cell is not described in detail is carried out according to the ordinary method of this area.
In the present invention, after to the umbilical cord tissue digestion process, can first natural subsidence tissue block for some time, 8-10min for example, and then shift the upper strata suspension, be beneficial to collect more cell.Simultaneously, the upper strata cell suspension can carry out centrifugal, centrifugal condition and for example can abandon supernatant for 2000～2500rpm, 15～20 ℃, centrifugal 20～30min with after physiological saline or the dilution of other damping fluid again, gets cell precipitation.Before cell precipitation being placed the cultivation of mescenchymal stem cell substratum, can earlier cell precipitation be washed with physiological saline or other damping fluid, namely by centrifugal, resuspended method washed cell.The cell density that cell precipitation is inoculated in the substratum is about 10
5Individual cell/cm
2, place CO
2Cultivate in the incubator.Change liquid after cultivating 48h-72h, every day is the observation of cell form under inverted microscope, is cultured to 80-90% and merges, and namely obtains the MSCs primary cell; The cell suspension of changing in the time of will changing liquid simultaneously places new culturing bottle to cultivate again, and method is the same.Two portions cell is added together, and this is the gained P0 of first part cell.
In the present invention, after to the umbilical cord tissue digestion process, indigested umbilical cord tissue piece is inoculated in before the culture dish, can namely use the resuspended tissue block of physiological saline earlier with the physiological saline washing, and centrifugal, centrifugal condition for example is 1500-2000rpm, 15-20 ℃, and 5-10min.When being inoculated in culture dish, can keep a determining deviation between the tissue block, to make things convenient for climbing out of and digestion process of cell, spacing for example can be 0.3-0.5cm.Tissue block suitably can be dried during inoculation, for example dry 15-30min, slowly add the mescenchymal stem cell substratum then.There is the spindle shape cell to climb out of around can seeing tissue block behind the general 2-3d, changes liquid behind the 3-4d, treat that tissue block peripheral cell density gets final product had digestive transfer culture when reaching the 80-90% fusion.Can adopt during digestion and disperse digestion (be about to the digestion around tissue block of an amount of digestion drop and climbed out of cell), keep the residue tissue block and continue to cultivate, cell all climbs out of in tissue block so repeatedly.The P0 cell that this obtains for second section.
In the present invention, when being paved with 80-90%, cell is used for going down to posterity cultivation usually.Inhale earlier when going down to posterity and abandon old liquid, can wash twice with physiological saline, add an amount of pancreas enzyme-EDTA solution, for example be 0.1% pancreatin-0.02%EDTA solution, rock and make the bottom surface pave the back to inhale and to abandon Digestive system, static 5-8min blows and beats cell gently with old liquid then again, makes single cell suspension, centrifugal, abandon supernatant, centrifugal condition for example can be 1000-1500rpm, 8-10min.The re-suspended cell precipitation, counting.Press 4000-6000cell/cm
2Be inoculated in the culturing bottle, cultivate in incubator, cultivation continued to go down to posterity after 3-4 days.
In the present invention, the mescenchymal stem cell for preparing by the inventive method, identify from cellular form, growth curve, cell cycle, surface markers and multidirectional differentiation capability aspect, all meet the mescenchymal stem cell characteristic, show that the present invention successfully separates to have obtained mescenchymal stem cell from umbilical cord.
The beneficial effect of the invention
The mescenchymal stem cell in a kind of people's umbilical cord of the present invention source separates, efficient amplification method, operation is simple, and: obtain more primary cell (1) within a short period of time, enlarged the deposit of seed cell, for efficient amplification provides radix, and amplification back cell identifies according to the minimum standard that international cell therapy association (ISCT) proposes, and namely mescenchymal stem cell must satisfy three and just can be considered to mescenchymal stem cell.At first cell must be adherent growth; Secondly mescenchymal stem cell must be expressed the specific marker of mesenchymal cells such as CD90, CD105CD73, and does not express the mark of hematopoiesis system or interior skin system cell: CD45, CD14, CD19, CD34 and major histocompatibility antigen HLA-DR; Again, must have the multidirectional differentiation potential that is divided into bone, cartilage, adipocyte.(2) in embodiments of the invention, the tissue digestion enzyme does not use pancreatin, has avoided the use of serum in the substratum.(3) plant piece suspension culture method for postdigestive umbilical cord tissue employing, improved the output of primary cell, also more undigested tissue block adherent method has shortened the time simultaneously.(4) go down to posterity to inhale in the process and abandon pancreatin, reduce the pancreatin activity by dilution method and reach the effect that stops pancreatin, avoided the use of serum in the substratum.
By this law, about 8 days, can obtain 3～4 * 10
7Primary cell is treated can obtain 5～7 * 10 about 14 days
7Primary cell can increase about January and obtain 1～5 * 10
11The MSCs cell.The umbilical cord MSCs that present method obtains can satisfy clinical and needs experimental study in external a large amount of amplifications.By to its biological characteristics as from surface marker, growth characteristic, the multidirectional aspect such as differentiation of inducing umbilical cord MSCs being identified and systematic research, confirm that the cell that repeatedly climbs out of from cell suspension and tissue block all can be at subculture in vitro separately, and the character indifference.This method is simple to operate, repeatable strong, builds the storehouse for cell technological method comparatively reliably is provided.
Description of drawings
Fig. 1 is the cellular form of single cell suspension amplification back and digestion tissue block adherent amplification back umbilical cord source MSCs;
Wherein scheme A and be the 8th day growth figure of digestion back tissue block, the arrow indication is tissue block in the diagram, and hair tonic goes out cell around the tissue block, is divergent shape, and nearly tissue block cell is tight, and outside cell is loose; Figure B is the 8th day growth figure of cell suspension, and the cell growing way is than homogeneous, and most of cell becomes fusiformis; Figure C for the tissue block cell dissociation reach the 10th generation cell, the form homogeneous is whirlpool shape growth; Figure D be cell suspension digestion reach the 10th generation cell, the form homogeneous is the growth of whirlpool shape.
Fig. 2 is the cell growth curve of the MSCs in umbilical cord source;
Wherein scheme E and be cell suspension be expanded to the 5th generation umbilical cord source mescenchymal stem cell growth curve, figure F be tissue block be expanded to the 5th generation umbilical cord source mescenchymal stem cell growth curve.
Fig. 3 is the MSCs in the umbilical cord source differentiated result of inducing to scleroblast, adipocyte and chondrocyte;
Wherein scheme K and be cell suspension be expanded to the 5th generation umbilical cord source mescenchymal stem cell differentiated result, K1 is to osteoblast differentiation, K2 is that K3 breaks up to the chondrocyte to the adipocyte differentiation;
Wherein scheme M and be tissue block be expanded to the 5th generation umbilical cord source mescenchymal stem cell differentiated result, M1 is to osteoblast differentiation, M2 is that M3 breaks up to the chondrocyte to the adipocyte differentiation.
Fig. 4 is the cell cycle analysis figure of the MSCs in umbilical cord source;
Wherein G be cell suspension increase the 5th generation mescenchymal stem cell periodogram, figure H be tissue block be expanded to the 5th generation mescenchymal stem cell periodogram; G0/G1 phase cell all accounts for more than 80%.
Fig. 5 for cell suspension be expanded to the 5th generation umbilical cord source mescenchymal stem cell the flow cytometer showed figure of surface marker.
Fig. 6 for tissue block be expanded to the 5th generation umbilical cord source mescenchymal stem cell flow cytometer showed figure;
The umbilical cord source equal high expression level of property mescenchymal stem cell of Fig. 5 and Fig. 6 or the mark of strongly expressed mescenchymal stem cell: CD90, CD105, CD73; And do not express CD31, CD14, CD34, CD45, CD80, CD86, CD40, CD40L and HLA-DR.
Embodiment
Be described in detail below in conjunction with the embodiment of the present invention of embodiment, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Collection of specimens: the umbilical cord sample of following examples is guaranteed aseptic all in the obstetrics and gynecology hospital delivery room or the Operation theatre collection.Transportation is stored in the aseptic wide-necked bottle, preserves liquid for containing 20u/mL heparin, 1% albuminous physiological saline, 4 ℃ of preservations.(annotating: should control in 12h from collect specimen to the time of handling sample)
Embodiment 1: separation and the cultivation of the mescenchymal stem cell in people's umbilical cord source
(1) the umbilical cord sample of gathering with normal saline flushing is removed residual blood.Length, blunt separation arteries weighed, measured, all the other umbilical cord tissues are cut into the 0.5-1cm segment, novel organized processing instrument (the full-automatic tissue processor that Ni company produced in beautiful day, model: gentleMACSTM) are put in.
(2) add II Collagen Type VI enzyme (sigma company, C6885), final concentration is 0.1%, (sigma company, H4274), final concentration is 0.0005%, in 37 ℃ of digestion 60min, stops digestion then to add Unidasa.
(3) natural subsidence 8min shifts the supernatant cell suspension to centrifuge tube, with 2 times of volume physiological saline dilutions, and 2000rpm, 15 ℃, centrifugal 20min obtains cell precipitation; To not digest simultaneously completely tissue block with the physiological saline dilution after, 2000rpm, 15 ℃, centrifugal 5min abandons supernatant, obtains stand-by not digestion tissue block completely.
(4) cell precipitation that step (3) is obtained is resuspended in the physiological saline, 1500rpm, centrifugal 10min, abandon supernatant, with throw out be resuspended in mesenchymal stem cell serum-free culture medium (Gibco company, article No. is: A10675), obtain cell suspension, according to 10
5Individual cell/cm
2Be inoculated in the culturing bottle, place 37 ℃, 5%CO
2, humidity is cultivated greater than 95% incubator.Change liquid after 3-4 days,, digest with 0.125% pancreatin+0.015%EDTA, according to 6000 cell/cm 85% the time to about when cytogamy to 8d
2Inoculation is gone down to posterity.What obtain in this way is first part's umbilical cord source mescenchymal stem cell.Simultaneously, in order further to improve cell yield, the original cell suspension of changing when changing liquid with 3-4 days is refitted in and continues in the new culturing bottle to cultivate, and cultural method is the same, and the cell that obtains is integrated with first part's umbilical cord source mescenchymal stem cell.
(5) the centrifugal stand-by tissue block completely of not digesting that obtains of step (3) is inoculated in culture dish according to the spacing of 0.5cm, place in the Bechtop, slowly drip mesenchymal stem cell serum-free culture medium behind the 15min, action softly avoids tissue block floating, change liquid after cultivating 4d, beginning to go down to posterity for the first time to 8d obtains the P0 cell, digests with 0.125% pancreatin+0.02%EDTA, according to 6000 cell/cm
2Inoculation is gone down to posterity; Tissue block continues to cultivate in the culture dish, repeats digestion step during to 11d, goes down to posterity; Repeat digestion step during to 15d, go down to posterity.What obtain in this way is second section umbilical cord source mescenchymal stem cell.
Can from umbilical cord tissue, obtain a large amount of mescenchymal stem cells according to present method, can obtain 3.26 * 10 when reaching for the 5th generation
11Mescenchymal stem cell.
Embodiment 2: separation and the cultivation of the mescenchymal stem cell in people's umbilical cord source
(1) the umbilical cord sample of gathering with normal saline flushing is removed residual blood.Weigh, measure length, blunt separation arteries, all the other umbilical cord tissues are cut into the 0.5-1cm segment, be put in the novel organized processing instrument.
(2) add II Collagen Type VI enzyme, final concentration is 0.05%, adds Unidasa, and final concentration is 0.001%, in 37 ℃ of digestion 90min, stops digestion then.
(3) natural subsidence 10min shifts the supernatant cell suspension to centrifuge tube, with 5 times of volume physiological saline dilutions, and 2500rpm, 15 ℃, centrifugal 15min obtains cell precipitation; To not digest simultaneously completely tissue block with the physiological saline dilution after, 2000rpm, 15 ℃, centrifugal 5min abandons supernatant, obtains stand-by not digestion tissue block completely.
(4) cell precipitation that step (3) is obtained is resuspended in the physiological saline, 1500rpm, centrifugal 10min, abandon supernatant, with throw out be resuspended in mesenchymal stem cell serum-free culture medium (three favourable companies, article No. is: 120408), obtain cell suspension, according to 10
5Individual cell/cm
2Be inoculated in the culturing bottle, place 37 ℃, 5%CO
2, humidity is cultivated greater than 95% incubator, changes liquid after 3-4 days, to 8d when the cytogamy to 85%, digest with 0.1% pancreatin+0.02%EDTA, according to 7000 cell/cm
2Inoculation is gone down to posterity.What obtain in this way is first part's umbilical cord source mescenchymal stem cell.Simultaneously, in order further to improve cell yield, the original cell suspension of changing when changing liquid with 3-4 days is refitted in and continues in the new culturing bottle to cultivate, and cultural method is the same, and the cell that obtains is integrated with first part's umbilical cord source mescenchymal stem cell.
(5) the centrifugal stand-by tissue block completely of not digesting that obtains of step (3) is inoculated in culture dish according to the spacing of 0.3cm, place in the Bechtop, slowly drip mesenchymal stem cell serum-free culture medium behind the 30min, action softly avoids tissue block floating, change liquid after cultivating 4d, beginning to go down to posterity for the first time to 8d obtains the P0 cell, and 0.1% pancreatin+0.015%EDTA digestion is according to 7000 cell/cm
2Inoculation is gone down to posterity; Tissue block continues to cultivate in the ware, repeats digestion step during to 11d, goes down to posterity; Repeat digestion step during to 15d, go down to posterity.What obtain in this way is second section umbilical cord source mescenchymal stem cell.
Can from umbilical cord tissue, obtain a large amount of mescenchymal stem cells according to this law, obtain 1.25 * 10 during the 5th generation
11Mescenchymal stem cell.
Embodiment 3: separation and the cultivation of the mescenchymal stem cell in people's umbilical cord source
(1) the umbilical cord sample of gathering with normal saline flushing is removed residual blood.Weigh, measure length, blunt separation arteries, all the other umbilical cord tissues are cut into the 0.5-1cm segment, be put in the novel organized processing instrument.
(2) add II Collagen Type VI enzyme, final concentration is 0.2%, adds Unidasa, and final concentration is 0.001%, in 37 ℃ of digestion 60min, stops digestion then.
(3) natural subsidence 10min shifts the supernatant cell suspension to centrifuge tube, with 5 times of physiological saline dilutions, and 2500rpm, 15 ℃, centrifugal 25min obtains cell precipitation; To not digest simultaneously completely tissue block with the physiological saline dilution after, 2000rpm, 15 ℃, centrifugal 5min abandons supernatant, obtains stand-by not digestion tissue block completely.
(4) cell precipitation that step (3) is obtained is resuspended in the physiological saline, 1500rpm, centrifugal 10min, abandon supernatant, with throw out be resuspended in mesenchymal stem cell serum-free culture medium (three favourable companies, article No. is: 120408), obtain cell suspension, according to 10
5Individual cell/cm
2Be inoculated in the culturing bottle, place 37 ℃, 5%CO
2, humidity is cultivated greater than 95% incubator, changes liquid after 3-4 days, to 9d when the cytogamy to 90%, 0.1% pancreatin+0.02%EDTA digests, according to 8000 cell/cm
2Inoculation is gone down to posterity.What obtain in this way is first part's umbilical cord source mescenchymal stem cell.Simultaneously, in order further to improve cell yield, the original cell suspension of changing when changing liquid with 3-4 days is refitted in and continues in the new culturing bottle to cultivate, and cultural method is the same, and the cell that obtains is integrated with first part's umbilical cord source mescenchymal stem cell.
(5) the centrifugal stand-by tissue block completely of not digesting that obtains of step (3) is inoculated in culture dish according to the spacing of 0.3cm, place in the Bechtop, slowly drip mesenchymal stem cell serum-free culture medium behind the 30min, action softly avoids tissue block floating, change liquid after cultivating 4d, beginning to go down to posterity for the first time to 8d obtains the P0 cell, and 0.1% pancreatin+0.015%EDTA digestion is according to 8000 cell/cm
2Inoculation is gone down to posterity; Tissue block continues to cultivate in the ware, repeats digestion step during to 11d, goes down to posterity; Repeat digestion step during to 14d, go down to posterity.What obtain in this way is second section umbilical cord source mescenchymal stem cell.
Can from umbilical cord tissue, obtain a large amount of mescenchymal stem cells according to present method, can obtain 2.1 * 10 when reaching for the 5th generation
11Mescenchymal stem cell.
Embodiment 4: the morphological observation of umbilical cord source mescenchymal stem cell
Adopt first part's umbilical cord mesenchymal stem cells primary cell that embodiment 1-3 method separates more than adherent in 24～48 hours, cultivated 3～4 days, observe visible attached cell under the inverted microscope and be fusiformis, polygon, increased the spindle cell that becomes the relative homogeneous of form in 8～9 days, to 80～90% fusion attached cell layers; Second section digestion back tissue block many about 4 days to the periphery spindle cell that dissociates, grew up to the monolayer cell layer centered by tissue block around the tissue block in 7～8 days, middle intensive, the edge is loose.The cell that dual mode obtains is in 15 generations of external continuous biography, and cellular form does not have obvious change (Fig. 1).
The morphological observation result of umbilical cord source mescenchymal stem cell shows: cell has the adherent characteristic of mescenchymal stem cell, is fusiformis.
Embodiment 5: the mensuration of umbilical cord source growth of mesenchymal stem cells curve
To digest with 0.125% pancreatin+0.02%EDTA according to the separation of embodiment 1-3 method, the 5th generation MSCs attached cell that obtains of going down to posterity, by 2 * 10
4Cells/well is seeded in 24 orifice plates, every 3 holes of 24h digestion, and collecting cell, and expect blue living cell counting with 0.4% platform, get the mean value of 5 experimental results, drafting growth curve (see figure 2).
As can be seen, cell proliferation is very fast, and at logarithmic phase, cell doubling time all is about 30h.
The measurement result of umbilical cord source growth of mesenchymal stem cells curve shows: the mescenchymal stem cell that obtains by this method has Normocellular growth characteristics.
Embodiment 6: the external multidirectional differentiation capability of umbilical cord source mescenchymal stem cell detects
1, to osteoblast differentiation
Will according to embodiment 1-3 method separate go down to posterity obtain exponential phase of growth cell by every hole 4 * 10
4Density be inoculated in six orifice plates, place 37 ℃, 5%CO
2, humidity is cultivated greater than 95% incubator.Behind the 24h, add the osteogenic induction system, inductor is to contain 10% foetal calf serum (FBS), 0.1 μ mol/L dexamethasone, 50 μ mol/L xitix, (Hyclone SH30022.01B), changed liquid in every 3-4 days to the HG-DMEM substratum of 10mmol/L β-phospho-glycerol.Detect with alkaline phosphatase (ALP) dyeing after cultivating for 2 weeks, osteogenic induction 2 all experimental group ALP dyeing visible cells are hyacinthine (seeing K1 among Fig. 3, M1).
2, to adipocyte differentiation will according to embodiment 1-3 method separate go down to posterity obtain exponential phase of growth cell by every hole 4 * 10
4Density be inoculated in six orifice plates, place 37 ℃, 5%CO
2, humidity is cultivated greater than 95% incubator.Behind the 24h, add into fat and induce system, inductor is the HG-DMEM substratum that contains 10%FBS, 1 μ mol/L dexamethasone, 0.5mmol/L1-methyl-3-isobutyl--xanthine (IBMX), 50 μ mol/L xitix.Changed liquid in every 3-4 days.Become fat to induce 2 weeks back employing Oil Red O dyeing to detect fat granule, fat granule forms (seeing K2 among Fig. 3, M2) in the visible cell.
3, will separate cell exponential phase of growth that obtains that goes down to posterity according to embodiment 1-3 method to chondrocyte's differentiation and use complete culture solution resuspended, by every pipe 4 * 10
5 Place 15 milliliters of plastic centrifuge tubes of the sharp end, 1200rpm, 10 minutes are centrifugal, unscrew the centrifuge tube lid, just standing on 37 ℃, 5%CO2, humidity is cultivated greater than 95% incubator.Behind the 24h, add into the chondrocyte induction system, inductor is 0.1 μ mol/L dexamethasone, 50 μ mol/L xitix, 1mmol/L Sodium.alpha.-ketopropionate, 10ng/mL transforming growth factor-beta 3(TGF-β 3), 1 * ITS+1(Sigma) HG-DMEM substratum.Changed liquid in every 3-4 days.Become the dyeing of 4 week of chondrocyte induction back employing alcian blue to detect (seeing K3 among Fig. 3, M3).
The result that the external multidirectional differentiation capability of umbilical cord source mescenchymal stem cell detects shows that to have to three be the ability of differentiation to umbilical cord source mescenchymal stem cell external.
Embodiment 7: the The cell cycle of umbilical cord source mescenchymal stem cell
To separate cell dissociation exponential phase of growth that goes down to posterity and obtain according to embodiment 1-3 method, add 4 ℃ of fixing and permeable membrane 24h of cold 70% ethanol, hatch 30min for the RNase A37 of 10 μ g/mL ℃, add 50 μ g/mL propidium iodides again, 4 ℃ of lucifuges are hatched 30min, flow cytometer (BD) detects, ModiFIT software analysis result.The flow cytometry analysis Cell cycles showed, the cell above 80% is in the G0/G1 phase (Fig. 4).
The The cell cycle result of umbilical cord source mescenchymal stem cell shows: it is many that the mescenchymal stem cell that obtains by this method is in static or original relatively cell, reflected that it has high differentiation capability.
Embodiment 8: the immunophenotype analysis of umbilical cord source mescenchymal stem cell
To separate cell routine exponential phase of growth that obtains that goes down to posterity according to embodiment 1-3 method and digest the back by 1 * 10
6The packing of/pipe is gone in the streaming pipe, adds the streaming antibody of respective markers in every pipe, mixing.4 ℃ of lucifuges were hatched 30 minutes, the antibody on flush away is unmarked.Subsequently cell precipitation is resuspended among the PBS.(FACA Calibur type USA) detects flow cytometer.Each fluorescent-labeled antibody all available from BD company (Beckman, USA).The surface marker of institute's mark has: CD31, CD14, CD90, CD105, CD73, CD34, CD45, CD80, CD86, CD40, CD40L and HLA-DR.Wherein Fig. 5 for cell suspension be expanded to the 5th generation umbilical cord source mescenchymal stem cell the flow cytometer showed figure of surface marker, Fig. 6 for tissue block be expanded to the 5th generation umbilical cord source mescenchymal stem cell flow cytometer showed figure; The mark of both equal high expression levels or strongly expressed mescenchymal stem cell: CD90, CD105, CD73; And do not express CD31, CD14, CD34, CD45, CD80, CD86, CD40, CD40L and HLA-DR.
The immunophenotype analytical results of umbilical cord source mescenchymal stem cell shows: the cell that obtains by this method be mescenchymal stem cell meet that ISCT proposes about mescenchymal stem cell in the standard aspect the molecular marker.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Reference:
[1]Lu LL，Liu YJ，Yang SG，et al．Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials[J].Haematologica，2006，91(8):1017-1026．
[2]WeissML,Medicetty S,Bledsoe AR,et al.Human umbilical cord matrix stem cells:preliminary characterization and effect of transplantation in a rodent model of Parkinson′s disease[J].Stem Cells,2006,24(3):781-792.
[3]Conconi MT,Burra P,DiLiddo R,et al.CD105(+)cells fromWharton’s jelly show in vitro and in vivo myogenic differentiative potential[J].Int J MolMed,2006,18(6):1089–1096
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Claims (12)
1. the method for a separation and Culture mescenchymal stem cell, it comprises the steps (1), and in step (2) and the step (3) one or two:
(1) gets stripped umbilical cord and carry out digestion process with the tissue digestion enzyme; Preferably, before digestion process, also comprise the step that umbilical cord is divided into segment umbilical cord tissue piece;
(2) after the digestion process of step (1) finished, obtained cell suspension placed the mescenchymal stem cell substratum to cultivate, and obtains mescenchymal stem cell; Preferably, in culturing process, the cell suspension of changing when changing liquid is refitted in and continues in the new culturing bottle to cultivate, and obtains mescenchymal stem cell;
Preferably, also comprise the step that cell suspension is centrifugal before the cell suspension that obtains places the mescenchymal stem cell substratum after step (1) is handled, place the mescenchymal stem cell substratum to cultivate the cell precipitation that obtains after centrifugal;
Randomly, after obtaining mescenchymal stem cell, also comprise the step that cell dissociation is gone down to posterity;
(3) after the digestion process of step (1) finished, the umbilical cord tissue of getting not complete digestion placed the mescenchymal stem cell substratum to cultivate, and can obtain mescenchymal stem cell; Randomly, after obtaining mescenchymal stem cell, remaining umbilical cord tissue can repeatedly obtain mescenchymal stem cell by repeatedly repeating the method in this step; Randomly, after obtaining mescenchymal stem cell, also comprise the step that cell dissociation is gone down to posterity.
2. the process of claim 1 wherein that described tissue digestion enzyme is collagenase and/or Unidasa.
3. the process of claim 1 wherein described umbilical cord to be divided into before the segment umbilical cord tissue piece, also comprise the step of removing Umbilical artery; Randomly, before removing Umbilical artery, also comprise the step of cleaning the umbilical cord that exsomatizes.
4. the method for claim 2, the working concentration of wherein said collagenase is the 0.05-0.2% bulking value; And/or the working concentration of wherein said Unidasa is the 0.0005-0.001% bulking value.
5. the process of claim 1 wherein that the condition of the digestion process described in the step (1) is 37 ℃, 1-3h.
6. the process of claim 1 wherein that described mescenchymal stem cell substratum is serum free medium.
7. the process of claim 1 wherein that the mescenchymal stem cell substratum that cell suspension is placed described in the step (2) cultivates, refer to treat the cell attachment growth after, be cultured to the 80-90% cytogamy.
8. the process of claim 1 wherein that the umbilical cord tissue of getting not complete digestion described in the step (3) places the mescenchymal stem cell substratum to cultivate, refer to treat have spindle cell to climb out of around the tissue block after, be cultured to the 80-90% cytogamy.
9. the process of claim 1 wherein that described had digestive transfer culture comprises the step of using pancreas enzyme-EDTA solution; Preferably, described had digestive transfer culture method comprises:
(i) use the physiological saline washed cell;
(ii) add an amount of pancreas enzyme-EDTA solution, treat that solution is paved to inhale behind the Tissue Culture Flask bottom surface to abandon pancreas enzyme-EDTA solution;
(iii) blow and beat cell with stoste, make single cell suspension, be inoculated in the culturing bottle behind the counting and cultivate.
10. the mescenchymal stem cell for preparing according to each described method of claim 1-9.
11. the mescenchymal stem cell of claim 10, it expresses CD90, CD105, CD73, does not express CD31, CD14, CD34, CD45, CD80, CD86, CD40, CD40L and HLA-DR.
12. collagenase and/or Unidasa are for the preparation of the purposes of mescenchymal stem cell.
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