CN102127522B - Human umbilical mesenchymal stem cell and preparation method thereof - Google Patents

Abstract

The invention discloses a human umbilical mesenchymal stem cell and a preparation method thereof. The human umbilical mesenchymal stem cell is prepared by carrying out aseptic treatment, mechanical shearing, Wharton jelly exposure, collagenase digestion and isolated culture on an umbilical stalk of a healthy fetus by a term abdominal delivery. After freezing and thawing, the cell activity does not decline dramatically, thereby ensuring that the human umbilical mesenchymal stem cell is an ideal seed cell for cellular therapy. The invention also relates to a primitive mesenchymal stem cell prepared by the method, and the primitive mesenchymal stem cell is confirmed as the mesenchymal stem cell by the identification of morphology, immunophenotype and invitro differentiation experiments in accordance with the mesenchymal stem cell standards established by International Society for Cellular Therapy (ISCT). The cell count, cell activity, purity, doubling time and multiplication capacity of the human umbilical mesenchymal stem cell are apparently higher than those of bone marrow-derived mesenchymal stem cell.

Description

Human umbilical cord mesenchymal stem cells and preparation method thereof

Technical field

The present invention relates to the preparation method of primary mesenchymal stem cells, relating in particular to people's umbilical cord jelly of Wharton is material prepn human umbilical cord mesenchymal stem cells and preparation method thereof.

Technical background

(Mesenchymal stem cell MSC) is one type of tissue stem cell with multidirectional differentiation potential to mescenchymal stem cell, can obtain from multiple tissue such as fetal, liver, marrow, fat.There is the possibility of height virus pollution in bone marrow derived MSC, and obvious downtrending appears in its cell quantity and amplification, differentiation capability with age.Additive method has certain difficulty as from bleeding of the umbilicus and peripheral blood, cultivating MSC, and MSC quantity in the peripheral blood of the bleeding of the umbilicus of term birth or mobilization remains in dispute.Come in to have scholar separation and Culture MSC from the rejected material umbilical cord in puerperium, and carry out biology phenotypic evaluation such as morphology, differentiation function and surface marker.Umbilical cord is to be connected in the cord structures that embryo's umbilical region and placenta are asked, outward by amnion, include the mucus reticular tissue of differentiation, in the reticular tissue except that the yolk sac and allantois of locking, Umbilical artery and umbilical vein in addition.MSC in umbilical cord source is divided into four kinds: 1. derive from jelly of Wharton (Wharton ' s jelly; WJ), it is the circumvascular Saliva Orthana appearance tissue of parcel, apart from the about 3mm of blood vessel periphery; Be rich in mucinase and glycosaminoglycan, formed inoblast hydrogel structure on every side; 2. derive from around the umbilical blood vessels; 3. derive from bleeding of the umbilicus; 4. derive under the umbilical vein blood vessel endothelium.McElreavey etc. find a kind of fibroblast-like cells with high differentiation potential from the WJ tissue of people's umbilical cord.Progenitor cell is rich in discovery WJ zones such as Sarugaser, this regional cell be called as human cord blood pipe peripheral cells (human umbilical cord perivascular cell, HUCPV).The HUCPV cell has higher fibroblast-like cells colony and forms ability, but and bone induce after proliferation and differentiation form the bone tubercle.Employings such as Romanov are cultivated the method for bone marrow MSCs and are cultivated, and are separated to the cell of similar bone MSCs from human umblilical vein endothelial and subendothelial layer.Above-mentioned research shows that umbilical cord tissue contains abundant MSCs, can become the good source of MSCs.

The method of separation and culturing human umbilical cord MSC is not quite similar at present, and separating effect is also far from each other.Separation method comprises collagenase digestion substantially, plant piece method or both combinations, umbilical vein inner membrance digestion method, and wherein the collagenase digesting fado is main with collagenase II.

People's umbilical cord MSC can be the ideal tissue engineering seed cell to multiple histocyte differentiation under suitable inductive condition.MSC induces to implant and is expected to become more widely cell therapy in section.Research shows that MSC can implant the muscle deterioration tissue and to myocyte's differentiation, also can promote the implantation of hemopoietic stem cell; MSC also can be applicable to biotechnology such as cartilaginous tissue reconstruction; Also possibly become the carrier cell that carries suicide gene or anti-cancer adenoviruses medicine in the following anticancer therapy.

Summary of the invention

The technical problem that the present invention will solve is; Provide a kind of material source to be easy to get; Human umbilical cord mesenchymal stem cells that cell doubling time is short and preparation method thereof, the mescenchymal stem cell for preparing by the inventive method has stronger self and multidirectional differentiation potential, and is easy to freezing preservation.

Realize the technical scheme of the object of the invention, adopting fetal cord is the raw material preparing mescenchymal stem cell.The preparation method is: after umbilical cord takes out from preservation liquid; Cut off bilateral ligation part, remove extravasated blood in the cord vessels, the umbilical cord immersion is contained among antibiotic Hanks ' the Balanced Salt Solution (HBSS) (1 *); Wash umbilical cord and umbilical vein inner chamber repeatedly 3 times with D-PBS; Reject blood vessel, expose jelly of Wharton, then umbilical cord is cut into 1-3mm³Put into reagent bottle after the tissue block of size, add 0.1% Digestive system, place to continue digestion 4-10h in 37 ℃ of isothermal vibration appearance, 100 eye mesh screens filter, centrifugal collecting cell.Add HBSS liquid cells washed 3 times, with DMEM/F12 culture medium solution re-suspended cell, adjustment cell density 4.8 * 10³～1 * 10⁴/ cm², being inoculated in 6 orifice plates, 37 ℃, volume(tric)fraction are 5%CO₂Cultivate in the incubator, change liquid behind the 24h, whenever changed liquid once later at a distance from 3 days, when treating that cell reaches 80% fusion, the cultivation of going down to posterity, use or freezing preservation are subsequent use immediately.

As optimization, the mescenchymal stem cell of preparation is around umbilical cord jelly of Wharton, the cord vessels, any source under bleeding of the umbilicus and the umbilical vein blood vessel endothelium or all.

The solution that is used to clean umbilical cord and blood vessel remained blood thereof is the D-PBS solution that does not contain calcium ions and magnesium ions; The preferred serum-free DMEM/F12 of the flushing used liquid of jelly of Wharton tissue block culture medium solution.

As optimization, umbilical cord is to be cut into 1-3mm after cleaning blood vessel through D PBS³Tissue block, get into next separable programming.

The preparation method who is used for primary mesenchymal stem cells; Available II Collagen Type VI enzyme digestion, IV Collagen Type VI enzyme digestion, II Collagen Type VI enzyme and pancreatin associating digestion method or II Collagen Type VI enzyme and Unidasa mixed solution digestion umbilical cord tissue; As optimization, first-selected II Collagen Type VI enzyme and Unidasa mixed solution digest.

As optimization, the Digestive system that is used to digest the umbilical cord tissue piece is the mixing solutions of II Collagen Type VI enzyme and Unidasa, and its mass and size percentage concentration is 0.1%.

As optimization, the nutrient solution that inoculating cell uses is to contain volume(tric)fraction to be the FBS of 10-20% and the DMEM/F12 of 25mmol/L Stimulina.

For mescenchymal stem cell according to method for preparing of the present invention, carry out the biology phenotypic evaluation according to the standard that " international cell therapy association " (ISCT) formulates, show following technical characterictic:

1) cell attachment growth under best culture system condition can form the CFU-F colony, and the relative homogeneous of form being the spindle cell of be arranged in parallel growth or swirl shape growth;

2) expression of adhesion molecules receptor marker thing CD29, CD44, mesenchymal cell mark CD73, CD105, stem cell labeling thing CD90; And do not express endothelial cell marker thing CD31 and hemopoietic stem cell sign CD34, yet expression of HLA-DR not;

3) external scleroblast and the cardiac-like muscle cell of being induced to differentiate into.

Above-mentioned qualification result shows the mescenchymal stem cell non-hematopoietic stem cell that is prepared by invention, and has above-mentioned technical characterictic, meets the mescenchymal stem cell product acceptance criteria.

The mescenchymal stem cell of pressing the inventive method preparation is in 5-6 days culture cycle, and cell concentration can be bred 20 times, can be rich in active mescenchymal stem cell in a large number; And can preserve for a long time and not lose its activity, and operation is simple, and through flow cytometer detect, the cell cycle detects and the vitro differentiation experimental identification; The mescenchymal stem cell purity and the cytoactive that obtain are higher; Have good multiplication potentiality, can set up cell bank fast, with low cost; Can directly be used for scientific experiment research and assisting therapy clinically, be rich in application prospect.

Description of drawings

Fig. 1 is the cellular form figure of the adherent back of primary cell to 80% fusion, and wherein, A is the 1st day cellular form; B is the 3rd day cellular form; C is the 7th day cellular form.

Fig. 2 is the immunophenotype cell content figure of the 4th generation mescenchymal stem cell flow cytometer detection.

Fig. 3 A is external evoked scleroblast and the cardiac-like muscle cell aspect graph of being divided into of the 4th generation mescenchymal stem cell--a bone sialoprotein immunohistochemical staining.

Fig. 3 B is that external evoked scleroblast and the cardiac-like muscle cell aspect graph of being divided into of the 4th generation mescenchymal stem cell--RT-PCR detects the expression electrophorogram of myocardium specific gene,swimming lane 1,2-α-muscle rhabdomyosarcoma filamentous actin;Swimming lane 3,4-cardiac muscle sarcoplasmic reticulum calcium atpase; 5,6-people's cardiac muscle transcription factor GATA4;Swimming lane 7,8-α-myoglobulin heavy chain;Swimming lane 9,10 people cardiac muscle transcription factor NKX2.5;Swimming lane 11, the 12-Troponin I;Swimming lane 13,14-confidential reference items GADPH.

Embodiment

Below in conjunction with accompanying drawing and specific embodiment the present invention is further described, but the present invention is not limited to following embodiment.

Embodiment 1: the preparation of mesenchymal stem cells of human umbilical cord and placenta

Pluripara's informed consent is gathered mature dose of palace and is produced healthy fetal cord or placenta.The puerpera need do detections such as antibody of AIDS virus, hepatitis B virus antibody, antibody of HCV, syphilis helicoid antibody, gpt, mycoplasma before umbilical cord or the placenta collection, all qualifiedly can gather after the security guaranteeing.

1, umbilical cord or placenta are after operating table takes off, and immersion contains in antibiotic 0.9%saline water 4 ℃ of preservations;

2, in the clean platform of behaviour, take out umbilical cord or placenta, wash clean surperficial residual blood with D-PBS;

3, umbilical cord or placenta are cut into 1-3mm³Put into the blue lid of 200mL reagent bottle after the tissue block of size, add the Digestive system of mass and size percentage concentration 0.1%, place to continuedigestion 4～10h in 37 ℃ of constant-temperature shaking appearance;

4,100 eye mesh screens filter collecting cell;

5, add D-Hank ' s liquid cells washed 3 times, use to contain the DMEM/F12 re-suspended cell of volume(tric)fraction as 10-20% foetal calf serum and Stimulina, the adjustment cell density is (4.8 * 10³～1 * 10⁴)/cm², be inoculated in 6 orifice plates, in 37 ℃, volume(tric)fraction 5% CO₂Cultivate in the incubator, change liquid behind the 24-72h, Fig. 1.

Embodiment 2: the cell characteristic of mesenchymal stem cells of human umbilical cord and placenta

1, immunophenotype detects CD29, CD44, and, CD73, CD90, CDl05, CD31, CD34, HLA-DR (seeing table 1).4-7 is inoculated in for the mescenchymal stem cell in human umbilical cord and placenta source rolls in the bottle, treat that cell reaches to use the 10ml trysinization when 80-90% merges, change in the 50ml centrifuge tube cell suspension and centrifugal 3 minutes with 1000rpm.After centrifugal, discard supernatant,PBS washing 2 times is divided into everypipe 1 * 10⁶Cell, first pipe is blank (only containing mescenchymal stem cell), is used to regulate the voltage of flow cytometer, second pipe adds homotype control antibodies (PE-MOUSE IgGl/FITC-MOUSE IgG1/APC-Mouse IgG1 κ) 10 μ l; Add othercorresponding antibodies 10 μ l in all the other pipes, mixing, 4 ℃ of lucifuges were hatched 30 minutes; PBS washing 1 time is abandoned supernatant after centrifugal, and it is resuspended to add 500 μ l PBS; Mixing, promptly be available on the machine (flow cytometer FACSAria, BD company) detected.The cellular immunization phenotype is: CD29, CD44, CD73, CD90, the positive mark of CD105, CD31, CD34, the negative mark of HLA-DR (seeing table 1,2).

The surface marker of table 1 preferred detection mesenchymal stem cells of human umbilical cord and placenta of the present invention

The detected result of table 2 mesenchymal stem cells of human umbilical cord and placenta surface marker

2, the skeletonization of cell and become myocardium differentiation potential and differentiation back is identified

The 4-6 of digestion amplification in vitro is for mescenchymal stem cell, with 5 * 10⁴TCS is inoculated in 6 orifice plates, and every hole adds 2ml DMEM/F12 nutrient solution.When cell reaches the 60%-80% fusion, change corresponding inductive differentiation medium.

2.1 osteogenic induction differentiation

Inductive differentiation medium is the DEXAMETHASONE BP98 that contains 100nmol/L, the sodium of 10mmol/L, the DMEM/F12 nutrient solution of 50 μ mol/L vitamins Cs and 10%FBS; Whenever changed liquid 1 time at a distance from 2 days; Cultivated PBS washedcell 1 time, fixing 10 minutes of 70% ethanol 21 days; Row bone sialoprotein immunohistochemical staining is identified the nodular generation of calcium.Microscopically is observed, the expression that is positive of the cell after inducing.

Induce differentiation 2.2 become cardiac muscle

Inductive differentiation medium is to contain the U-18496 of 10 μ mol/L and the DMEM/F12 nutrient solution of 10%FBS, adds induced liquid after 24 hours, is replaced by the DMEM/F12 nutrient solution of 10%FBS immediately, whenever changes liquid 1 time at a distance from 2 days later, cultivates for 8 weeks.Extract the RNA of cell, carry out RT-PCR and detect people cardiac muscle transcription factor GATA4 and NKX2.5, myocardium sarcoplasmic reticulum calcium atpase, Troponin I, the expression of α-muscle rhabdomyosarcoma filamentous actin.The result shows that these genes all have expression to a certain degree.Shown in Fig. 3 A, 3B, RT-PCR detects confirmation, does not express above-mentioned myocardial cell's mark before people's umbilical cord MSCs induces, and myocardial cell's mark all has expression to a certain degree after 5-Aza induces.

The mescenchymal stem cell of aforesaid method amplification is in 5-6 days culture cycle, and cell concentration can be bred 20 times, can be rich in active umbilical cord and placenta mesenchymal stem cell in a large number; And can preserve for a long time and do not lose its activity, and operation is simple, and detect and the vitro differentiation experimental identification through flow cytometer; The mescenchymal stem cell purity and the cytoactive that obtain are higher; Have good multiplication potentiality, can set up cell bank fast, with low cost; Can directly be used for scientific experiment research and assisting therapy clinically, be rich in application prospect.

In sum, content of the present invention is not confined in the above embodiments, and the knowledgeable people in the same area can propose other embodiment easily within technical director's thought of the present invention, but this embodiment is included within the scope of the present invention.

Claims (8)

1. a preparing human umbilical mesenchymal stem cells is characterized in that, it is to adopt following process method preparation: after preserving liquid taking-up umbilical cord; Cut off bilateral ligation part, remove extravasated blood in the cord vessels, the umbilical cord immersion is contained among the antibiotic Hanks'Balanced Salt Solution; Wash umbilical cord and umbilical vein inner chamber repeatedly 3 times with D-PBS; Reject blood vessel, expose jelly of Wharton, then umbilical cord is cut into 1-3mm³Put into reagent bottle after the tissue block of size, adding mass and size percentage concentration is 0.1% Digestive system, places to continue digestion 4～10h in 37 ℃ of isothermal vibration appearance, and 100 eye mesh screens filter, centrifugal collecting cell; Add HBSS liquid cells washed 3 times, with DMEM/F12 culture medium solution re-suspended cell, adjustment cell density 4.8 * 10³～1 * 10⁴/ cm², being inoculated in 6 orifice plates, 37 ℃, volume(tric)fraction are 5%CO₂Cultivate in the incubator, change liquid behind the 24h, whenever changed liquid once later at a distance from 3 days, when treating that cell reaches 80% fusion, the cultivation of going down to posterity, use or freezing preservation are subsequent use immediately.

2. preparing human umbilical mesenchymal stem cells according to claim 1; It is characterized in that; Said umbilical cord adopts the mature palace of cuing open to produce healthy fetal cord, and the puerpera has done antibody of AIDS virus, hepatitis B virus antibody, antibody of HCV, syphilis helicoid antibody, gpt, detection of mycoplasma before gathering.

3. preparing human umbilical mesenchymal stem cells according to claim 2 is characterized in that, the umbilical cord after the collection places 4 ℃ of preservations, handles within the 6h.

4. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, said D-PBS solution is calcium ions and mg ion not.

5. preparing human umbilical mesenchymal stem cells according to claim 1; It is characterized in that, add a kind of in II Collagen Type VI enzyme, type, II Collagen Type VI enzyme and pancreatin mixed solution or II Collagen Type VI enzyme and the Unidasa mixed solution in the said Digestive system.

6. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, the time of digestion umbilical cord tissue piece is 6h.

7. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, said DMEM/F12 culture medium solution is a serum-free DMEM/F12 culture medium solution.

8. preparing human umbilical mesenchymal stem cells according to claim 1 is characterized in that, said DMEM/F12 culture medium solution is to contain the FBS that volume(tric)fraction is 10-20% and the DMEM/F12 culture medium solution of 25mmol/L Stimulina.

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