patents.google.com

CN102191229A - Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) - Google Patents

️Wed Sep 21 2011

Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) Download PDF

Info

Publication number

CN102191229A

CN102191229A CN 201010125235 CN201010125235A CN102191229A CN 102191229 A CN102191229 A CN 102191229A CN 201010125235 CN201010125235 CN 201010125235 CN 201010125235 A CN201010125235 A CN 201010125235A CN 102191229 A CN102191229 A CN 102191229A Authority

China

Prior art keywords

msc

umbilical cord

cell

tissue

rapidly

Prior art date

2010-03-16

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Pending

Application number

CN 201010125235

Other languages

Chinese (zh)

Inventor

侯亚义

刘柳

李鹏飞

赵晓寅

赵叶琳

王亚平

Original Assignee

侯亚义

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2010-03-16

Filing date

2010-03-16

Publication date

2011-09-21

2010-03-16 Application filed by 侯亚义 filed Critical 侯亚义

2010-03-16 Priority to CN 201010125235 priority Critical patent/CN102191229A/en

2011-09-21 Publication of CN102191229A publication Critical patent/CN102191229A/en

Status Pending legal-status Critical Current

Images

Landscapes

Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention belongs to the field of biological medicines and discloses a method for rapidly and effectively acquiring an umbilical cord mesenchymal stem cell (MSC). The method is characterized in that the umbilical cord MSC is rapidly and effectively acquired by taking the umbilical cord of a newly born fetus as a resource and is rapidly identified by using an optimized umbilical cord tissue digestive enzyme formula and a digestive cell adherence mode. The method is developed by improvement based on the traditional MSC, the time for acquiring the MSC is shortened to the half of the traditional method, the yield is improved by one time, and various surface characteristics and functions of the MSC are not influenced. The invention provides the method for rapidly and effectively acquiring the MSC for the scientific researches and clinical application of the umbilical cord MSC.

Description

A kind of method that effectively obtains umbilical cord mesenchymal stem cells (MSC) fast

Technical field:

The present invention is a kind of method that effectively obtains umbilical cord MSC fast, specifically utilizes cell culture technology and rational tissue management technique to improve and obtains umbilical cord MSC yield and speed, and the method for Rapid identification.

Background technology

Mescenchymal stem cell is to belong to a mesoblastic class multipotential stem cell, mainly is present between reticular tissue and organ in the matter.MSC has powerful multiplication capacity and multidirectional differentiation potential, in suitable body or can be divided into various kinds of cell such as hematopoietic cell, myocyte, liver cell, scleroblast, chondrocyte, stroma cell under the external environment.MSC also has immunoloregulation function except the general biology characteristics with stem cell simultaneously.It is convenient relatively that this cell also has the source in addition, is easy to separation, cultivation, amplification and purifying, still has the stem cell characteristic after the amplification of repeatedly going down to posterity, and do not have the characteristic of immunological rejection.Therefore MSC has become focus in the stem-cell research in recent years, has application promise in clinical practice in fields such as Immunological diseases treatment, hematopoietic stem cell transplantation, organizational project, genetically engineereds.

MSCs can suppress lymphocytic propagation, has very strong immunoloregulation function.Experiment confirm in the body, MSC are transplanted the survival time that can obviously prolong cutify and are reduced recessive allele hematopoietic stem cell transplantation GVHD, tens of routine Patients with SLE that we have also successfully utilized the MSC transplantation treatment.Think that at present this MSC secretes various immune modulatory molecules, with contacting of immunocyte, immunity of organism is relevant thereby reduced immunogenicity, induced chimaera form the inducing immune tolerance regulation and control.

The present MSC that studies show that can also treat ischemic disease.MSC and peripheral hematopoietic stem cells combined transplantation can promote the hematopoiesis of patient with breast cancer and high-risk patients with acute myeloid leukemia to recover, and not relevant negative interaction has shown the ability that good short hematopoiesis recovers.This result of treatment and the MSC of MSC cell secrete various Hemopoietic factors and generate bone marrow matrix and help the amplification in vitro, hematopoietic stem cell transplantation of hemopoietic stem cell after go back to the nest and implant, can shorten that to transplant the time that the back hemopoietic function of bone marrow recovers relevant.Find all in the research of rat brain obstruction of artery and myocardial infarction model that simultaneously the MSC cell has the effect that improves illness.

Simultaneously MSC also has the multidirectional differentiation potential of stem cell as a kind of stem cell, and MSC can treat that congenital bone forming is bad, metachromatic leukodystrophy (MLD) and hurley syndrome, in parkinsonism, senile dementia, spinal cord injury, the treatment of burn also has report.

MSC can be used as the good carrier that carries foreign gene in addition, the people has been arranged cytokine, imports in the MSC cell as IL23, EPO etc., enters acquisition long-time stably express in back in the body.Someone imports the gene of IFN β in the MSC, finds that the microenvironment of tumour makes MSC implant easily, and the MSC that enters in the tumour suppresses tumor growth by secretion IFN β cytokine, has shown that MSC has the tumor biotherapy function of possibility.These results have pointed out MSC wide range of therapeutic function, and present countries in the world are all dropped into a large amount of manpower, material resources and financial resources and carried out extensive research.But above-mentioned treatment plan all needs a large amount of MSC to transplant, and therefore sets up stable MSC resources bank fast and effectively and has very important meaning.

It is generally acknowledged that the content of MSCs in myeloid tissue is the abundantest, but along with wearing out of age, the MSCs number significantly reduces in the autologous bone marrow, the proliferation and differentiation ability fails significantly, and there is a potentially dangerous of virus infection, the collection difficulty of while donor MSC, the research of other alternative MSCs " resources bank " is brought into schedule beyond the feasible searching marrow.At present, isolate MSC the tissue beyond marrow such as fat, embryo, amniotic fluid, placenta, Cord blood and umbilical cord.And in these tissues, umbilical cord belongs to Biohazard Waste, the easiest acquisition, and to its research and use and not relate to ethnics Problem, and have and stablize a large amount of sources, therefore have the value of research.Present studies show that, umbilical cord mesenchymal stem cells is present in umbilical cord jelly of Wharton and the blood vessel surrounding tissue, cultivate the inoblast sample that obtains at this position, express alpha-smooth muscle actin and multiple MSCs marker (CD29, CD44, CD51, CD105, SH2, SH3), but do not express endothelium or the special antigens c D34 of white corpuscle, CD45.Simultaneously add U-18496 in the umbilical cord mesenchymal stem cells substratum, MSCs breaks up to the myocardial cell, changes culture condition and also can point out umbilical cord MSC cell to have identical phenotype and differentiation potential with marrow MSC cell to adipocyte and osteocyte differentiation.

The method that is used for the separating mesenchymal stem cell at present mainly contains 4 kinds, i.e. fluidic cell separating method, magnetic bead partition method, density gradient centrifugation and adherent sieve method.Convection type cell sorting method and magnetic bead partition method need not select for use a plurality of surface markers to detect because of finding the surface marker that the mescenchymal stem cell specificity is very strong, investigator so far as yet; In addition, these two kinds of active influences of method pair cell are big, requirement for experiment condition is higher, and are unfavorable for the separation sorting of cell, so not widespread use as yet at present.Density gradient centrifugation is owing to need accurate instrument and suitable parting liquid, and pair cell also has certain influence, and is not suitable for large-scale production application.And adherent sieve method, the cellular form homogeneous that is obtained, rate of propagation is fast, and growth conditions is stable, and easy and simple to handle, and cost is low, is expected to become practical production method.Pertinent literature has been reported the acquisition methods of umbilical cord MSC cell, and it is relatively low to obtain efficient, and speed is slower.We have improved the traditional endotheliocyte digestion method and the prescription of digestive ferment, adopt and partly organize adherent method can obtain umbilical cord MSC cell in a large number fast.And very high acquisition efficient and yield arranged.Present method is simple, more helps being used for production practice.

Summary of the invention:

The purpose of this invention is to provide a kind of pass through to optimize umbilical cord tissue digestive ferment prescription and the adherent mode of peptic cell, effectively utilize the method for the umbilical cord of newborn fetus fast for resource acquisition umbilical cord MSC and quick MSC evaluation, specific as follows:

1. clip cuts open the palace and produces the nearly placenta end of healthy puerpera umbilical cord 20-30cm, places the PBS of aseptic precooling to preserve.Umbilical cord is confirmed to use after the safety through strict pathogen detection.

2. in the Bechtop, glass culture dish is put in the umbilical cord taking-up, peeled off Umbilical artery and umbilical vein, the umbilical cord tissue of peeling off blood vessel is put in the DMEM/F12 substratum is cut into 2mm ³About tissue block.

3. the enzyme that conventional umbilical cord tissue digestion is used influences primary cell state if want with organizing complete digestion often to need the long time as the mixed solution of collagenase or collagenase and pancreatin.And if digestion time is shorter, can not will organize complete digestion, then can influence separation efficiency.And than higher, organizing that bulk does not shred usually can not complete digestion to the pulverizing of tissue block or the requirement that shreds for this digestion method.Simultaneously, use collagenase or collagenase to become thick with the tissue of the mixed solution digestion of pancreatin more, need a large amount of damping fluid dilutions, repeatedly flushing just can be obtained corresponding cell, has more strengthened the difficulty that the MSC cell obtains.Therefore, we have disposed a kind of enzyme that is specifically designed to umbilical cord tissue digestion: CDH digestive ferment, this enzyme can digest umbilical cord tissue in the short period of time fully, and be lower to the requirement that tissue block is pulverized, and Digestive system is thickness not, the centrifugal MSC cell that obtains of easier flushing.

4. after the conventional umbilical cord tissue digestion, can filter, obtain single cell suspension by 100 eye mesh screens, it is adherent to be inoculated in Tissue Culture Flask then, and this method MSC obtains comparatively fast, but the MSC cell that obtains is generally less, and because the intermediate treatment process is too many, cell state is affected.Also have bibliographical information to obtain MSC by the method for tissue block adherent, but this method is too high to the tissue block processing requirements, the most tissues piece can not be adjacent to cell bottle wall, and generally needs for 3 weeks just can obtain the MSC cell.Observation by us is found, after tissue digests by digestive ferment, the MSC cell is general agglomerating or stick to not complete digestion tissue, and the MSC cell that major part digested out after Digestive system was filtered by 100 eye mesh screens can not pass through screen cloth because of agglomerating, and efficient is obtained in influence.We have invented following method to this: the postdigestive tissue juice of CDH digestive ferment need not to pass through screen cloth, most of individual cells or MSC cell mass all can be adherent, even the completely little block organization of not digestion is arranged, after passing through the CDH digestive ferment, also become and very easily be adjacent to cell bottle wall, can obtain the MSC cell fast.This method combines umbilical cord tissue digestion and obtains unicellular and organize the two-fold advantage of adherent method, has effectively improved yield and the speed of MSC.

5. concrete operating process is as follows: tissue that shreds and CDH mixed enzyme solution add in the 50ml centrifuge tube by 1: 1 mixed, place 37 ℃ of shaking tables, and the 200rpm vibration is about digestion 3h.Postdigestive organize PBS washing three times after, be resuspended in the DMEM/F12 substratum that contains 12% foetal calf serum, the T-25 culturing bottle is gone in the shop.

6. after cultivating 2 days, discard not adherent cell and tissue, add the fresh DMEM/F12 substratum that contains 10% foetal calf serum, change liquid every other day.When cell grows to 80% abundance (about 8 days), cell dissociation first three the generation ratio of cell in 1: 1 is inoculated in the new T-25 culturing bottle.Cell dissociation all went down to posterity by 1: 3 later on.Can obtain first-generation MSC, about 2 * 10 on the 8th day ⁶Individual cell, but and the corresponding phenotype of streaming detection MSC.

Description of drawings:

The MSC cellular form in Fig. 1, umbilical cord source: (A) MSC that adopts present method to obtain cultivates the cellular form after 3 days, and the attached cell that the hair tonic of tissue block place goes out is divergence form shuttle shape.The single attached cell of part is also arranged and begin the division increment.(B) adopt traditional digestion process mode to cultivate after 3 days and have only a few cell adherent, the part sample can't obtain cell.(C) first-generation MSC that adopts present method to obtain cultivates the form after 8 days, and the cell abundance reaches 80%, becomes the shuttle type fibroblast-like cells of the relative homogeneous of form, the growth of whirlpool shape.(D) adopt traditional digestion process mode to cultivate that a few cell begins increment after 8 days, reach that (E F) does not change with present method MSC that obtains and the 15th generation MSC form that adopts traditional digestion process mode to cultivate but can not reach 80% cell abundance.

Fig. 2, the quick phenotype of the MSC cell streaming of obtaining detect: the third generation MSC cell dissociation that will cultivate in T-25 plastic culture bottle obtains single cell suspension, adopt machine testing on the mono-clonal fluorescence antibody difference mark, carry out data analysis with CellQuest software: the positive phenotype CD105 of the criterion of MSC cell, CD73, CD90, CD44, CD29, HLA-ABC positive rate are not less than 95%; Negative CD34, CD14, CD11b, CD19, HLA-DR, CD31 positive rate are not higher than 2%.

Fig. 3, (0.25% pancreatin+0.02%EDTA) is digested to single cell suspension, by 1 * 10 with cell dissociation buffer with attached cell ⁴Cells/well is seeded in 24 orifice plates, every 3 holes of digestion in 24 hours, and collecting cell, and expect blue living cell counting with 0.4% tire, the drafting growth curve.The increment of third generation cell is very fast, and at logarithmic phase, cell doubling time all is about 36 hours.The every biography generation of cell, cell count increases by 2 times approximately.Report basically identical with pertinent literature and patent.Obtained first-generation MSC, about 2 * 10 on the 8th day ⁶Individual cell estimates that every 20cm umbilical cord can obtain 7 * 10 after one month ¹⁰The MSC cell yield.

Embodiment:

The objective of the invention is to utilize cell culture technology and rational tissue management technique to improve and obtain umbilical cord MSC yield and speed, and the method for Rapid identification.The contriver has announced a kind of umbilical cord tissue digestive ferment prescription and adherent mode of peptic cell of optimization, and quick MSC authentication method.With regard to specific embodiment concrete utilization of the present invention is described below.

Example 1 umbilical cord MSC cell obtains fast

1. clip cuts open the palace and produces nearly placenta end umbilical cord 20-30cm in puerpera's operation, places the PBS of aseptic precooling to preserve.

2. umbilical cord confirms that through strict pathogen detection (microbial pathogenes such as treponema pallidum, hiv virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, mycoplasma detect) use the back safely.Get rid of the umbilical cord sample of factors such as premature labor, premature rupture of fetal membrane, chorioamnionitis, systemic lupus erythematous, ephritis, diabetes simultaneously, guarantee that the MSC that obtains is normal.

3. in the Bechtop, glass culture dish is put in the umbilical cord taking-up, be cut into the long segment of about 5cm, clean with PBS.After Cord blood cleaned fully, peel off Umbilical artery and umbilical vein, and the umbilical cord tissue that will peel off blood vessel is put in the DMEM/F12 substratum and is cut into 2mm with surgical scissors ³About tissue block.

4. dispose CDH digestive ferment (II Collagen Type VI enzyme 250U/ml, neutral protease 100U/ml, Unidasa 10U/ml), 37 degree are dissolved in the DMEM/F12 substratum, and the filter degerming of filtering 0.22 μ m is standby.Digestive ferment is preferably now with the current, spends the shelf time above 1

week

5. the tissue that shreds and CDH mixed enzyme solution by 1: 1 volume ratio mix with the 50ml centrifuge tube in, place 37 ℃ of shaking tables, 200rpm vibration is about digestion 3h (treat tissue block digests substantially get final product, the residual MSC that do not influence of the tissue block that minority is less obtains efficient).

6. with 4 ℃ in postdigestive tissue juice, the centrifugal 5min of 300g abandons supernatant, abandons supernatant, will precipitate with PBS to be resuspended in the 50ml fresh culture, and 4 ℃, the centrifugal 5min of 300g abandons clearly, and PBS washes twice, notes cell and tissue precipitation not being discarded.Last abandons supernatant all over after centrifugal, precipitation is resuspended in the DMEM/F12 substratum that contains 12% foetal calf serum, and the T-25 culturing bottle is gone in the shop.The shop can not be filtered with 100 eye mesh screens before going into the Tissue Culture Flask culturing bottle, filters the acquisition efficient that can influence the MSC cell.(precipitation of the umbilical cord digestion gained of about 5cm is laid in 1 T-25 culturing bottle)

7. after cultivating 2 days, discard not adherent cell and tissue, add the fresh DMEM/F12 substratum that contains 10% foetal calf serum, change liquid every other day.The MSC cell can grow along adherent tissue block or adherent cell.When cell grows to 80% abundance (about 8 days), (0.25% pancreatin+0.02%EDTA), in first three generation, be inoculated in the ratio of cell in 1: 1 in the new T-25 culturing bottle cell dissociation buffer, to guarantee growth velocity.Cell dissociation all went down to posterity by 1: 3 later on.

8. every 20cm umbilical cord obtained first-generation MSC, about 2 * 10 on the 8th day ⁶Individual cell can obtain 7 * 10 after one month ¹⁰The MSC cell yield.

Example 2 umbilical cord MSC cell Rapid identification:

Morphological observation: how adherent in 2 days by primary cell and tissue block that present method obtains, cultivate the attached cell that promptly can be observed in 4-6 days to go out from the tissue block hair tonic, be divergence form shuttle shape.Also can be observed simultaneously the single attached cell of part and begin the division increment.After 1 time go down to posterity (8 days), the cell increment is very fast, becomes the shuttle type fibroblast-like cells of the relative homogeneous of form, the growth of whirlpool shape.Considerable change does not take place in 15 cellular fories of continuous passage.According to the report of document and other patents, obtaining first-generation MSC cell needed for 2 weeks, and success ratio is not high.Cell count that present method obtains and acquisition time and success ratio all obviously are better than according to other documents and patent.

Streaming detects:

How the MSCs that separates, increases identifies, is present question at issue.MSCs can express kinds of surface antigen but be not special, but by checking that a plurality of surface markers can distinguish MSC and other cells, pertinent literature also has report.Flow cytometry is a kind of fast accurate detection means simultaneously, can determine to obtain the phenotype of cell very fast and effectively.Avoid traditional passing through to detect MSC cytodifferentiation ability and identified long shortcoming consuming time.

The third generation MSC cell that to cultivate in T-25 plastic culture bottle obtains single cell suspension with cell dissociation buffer digestion, and cell counting.The MSC cell is resuspended to 1 * 107 cell count/milliliter, and the single cell suspension packing is gone in the streaming pipe, every

pipe

100 μ l.The streaming fluorescence antibody that in each streaming pipe, adds respective markers, mixing.4 degree lucifuges were hatched 30 minutes.Each streaming pipe adds the antibody of 500 μ l PBS flush awaies on unmarked, and 4 ℃, the centrifugal 10min of 300g abandons supernatant, abandons supernatant, triplicate.Subsequently with cell precipitation resuspended with 100 μ l PBS in.Flow cytometer detects corresponding phenotype, and by the corresponding software analysis.The positive phenotype CD105 of the criterion of MSC cell, CD73, CD90, CD44, CD29, HLA-ABC positive rate are not less than 95%; Negative CD34, CD14, CD11b, CD19, HLA-DR, CD31 positive rate are not higher than 2%

The mensuration of example 3 umbilical cord MSC cell yields and cell growth curve.

The MSC cell that obtains (after the digestion of 0.25% pancreatin+0.02%EDTA), is expected blue living cell counting with 0.4% tire, added up first-generation cell yield with cell dissociation buffer the first-generation the 8th day.With third generation cell with cell dissociation buffer (after the digestion of 0.25% pancreatin+0.02%EDTA), be seeded in 24 orifice plates by 1 * 104 cells/well, every 3 holes of digestion in 24 hours, collecting cell, and expect blue living cell counting, the drafting growth curve with 0.4% tire.First three is slower for the cell growth, therefore need go down to posterity with 1: 1 ratio inoculation, and it is very fast that the 4th generation began the cell increment, and at logarithmic phase, cell doubling time all is about 36 hours.The every biography generation of cell, cell count increases by 2 times approximately.Report basically identical with pertinent literature and patent.Obtained first-generation MSC on the 8th day, about 2 * 106 cells estimate that every 20cm umbilical cord can obtain 7 * 1010MSC cell yield after one month.

Claims (2)

1. enzyme that is specifically designed to umbilical cord tissue digestion: CDH digestive ferment, this enzyme in the short period of time can be fully with umbilical cord tissue digestion, and be lower to the requirement that tissue block is pulverized, and Digestive system thickness not, the centrifugal MSC cell that obtains of easier flushing.The feature of this Digestive system is to contain II Collagen Type VI enzyme 250U/ml, neutral protease 100U/ml, and Unidasa 10U/ml, 37 degree are dissolved in the DMEM/F12 substratum, and the filter degerming of filtering 0.22 μ m is standby.Digestive ferment is preferably now with the current, spends the shelf time above 1 week 4.

2. method that obtains fast effectively the umbilical cord hemopoietic stem cell, it is characterized by utilization claim 1 described special-purpose umbilical cord tissue digestive ferment prescription, the mode of taking umbilical cord tissue to train is altogether effectively utilized the method for the umbilical cord of newborn fetus for resource acquisition umbilical cord MSC and quick MSC evaluation fast.

CN 201010125235 2010-03-16 2010-03-16 Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC) Pending CN102191229A (en)

Priority Applications (1)

Application Number	Priority Date	Filing Date	Title
CN 201010125235 CN102191229A (en)	2010-03-16	2010-03-16	Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC)

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
CN 201010125235 CN102191229A (en)	2010-03-16	2010-03-16	Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC)

Publications (1)

Publication Number	Publication Date
CN102191229A true CN102191229A (en)	2011-09-21

Family

ID=44600103

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
CN 201010125235 Pending CN102191229A (en)	2010-03-16	2010-03-16	Method for rapidly and effectively acquiring umbilical cord mesenchymal stem cell (MSC)