CN105420187A - Kit for culturing hUC-MSC step by step and hUC-MSC obtained by adopting same - Google Patents

Abstract

The invention discloses a serum-free culture scheme for human umbilical cord mesenchymal stem cells (hUC-MSC). According to the scheme, a fractional step method is adopted to culture the hUC-MSC: first, a TME culture medium is used for culture for 3-4 hours so as to promote attachment of the hUC-MSC, and then the medium is replaced by a TMD culture medium for rapid amplification. By the adoption of the TME and TMD serum-free fractional step culture method, the problems of poor cell attachment capacity, slow amplification, easy differentiation and the like of a serum-free hUC-MSC culture method are well solved, and a foundation is laid for a long-term stable serum-free hUC-MSC culture technology.

Description

A kind of test kit for substep cultivation hUC-MSC and the hUC-MSC adopting described test kit to obtain

Technical field

The present invention relates to the research field of stem cell, novel, the efficient serum-free substep particularly relating to a kind of hUC-MSC cultivates test kit.

Background technology

Mescenchymal stem cell is prevalent in human multiple tissue and organ, and has multi-lineage potential, has and stimulates the function such as tissue regeneration, immunity moderation, have broad application prospects in cell therapy field.

Mesenchymal stem cells MSCs is widely applied clinically, and current research shows, the mescenchymal stem cell in umbilical cord source not only can become the ideal substitute of mesenchymal stem cells MSCs, and has larger application potential.Wherein, come from the human umbilical cord mesenchymal stem cells (humanUmbilicalCordmesenchymalstemcells of people's umbilical cord, hUC-MSC) the peculiar mark of multiple embryonic stem cell is expressed, there is differentiation potential large, multiplication capacity is strong, immunogenicity is low, draw materials conveniently, the restriction of amoral ethics problem, be easy to the features such as preparation of industrialization, and research confirms that hUC-MSC is at sacred disease, immunity system, endocrine system and cancer, good result for the treatment of is had in the animal model of the diseases such as heart trouble and clinical study, therefore the multipotential stem cell of most potential applicability in clinical practice is likely become.

Clinical to be applied to further by hUC-MSC, external a large amount of amplification of the most important hUC-MSC of being exactly reaches effective clinical treatment dosage, is also one of most important technology while that therefore the vitro culture of hUC-MSC becoming the most basic.FBS, mycillin are added in the many employings of existing hUC-MSC cultural method in basic medium, but non-human serum complicated component, hUC-MSC is easily broken up in long-term culturing process, and the non-danger that there is propagation xenogenesis pathogenic agent.

In addition, although existing researcher develops polytype serum substitute, but the serum substitute cultivated for hUC-MSC that existing market can be bought and the culture effect of perfect medium still undesirable, the characteristics such as the maintenance of the stability after especially cultivating for adherent, the propagation of stem cell and cell long-period all cannot produce a desired effect.

Summary of the invention

Therefore the object of the invention is the needs for this area, a kind of substratum for cultivating hUC-MSC is provided, thus obtain the hUC-MSC that adherent ability is good, propagation fast, is easily broken up.

Another object of the present invention is to provide the hUC-MSC obtained by substratum of the present invention.

Specifically, the invention provides the test kit culture scheme that a kind of novel serum-free substep cultivates hUC-MSC, this test kit at least comprises two kinds of substratum of different composition, the substep utilizing the substratum in this test kit can carry out hUC-MSC under serum-free environment is cultivated and long-term amplification cultivation, guarantees that it still keeps multipotency and stronger multiplication capacity in long-term cultivation situation simultaneously.

Technical scheme of the present invention is as follows.

On the one hand, the invention provides a kind of test kit cultivating hUC-MSC for substep, described test kit comprises separated TME substratum and TMD substratum.The equal serum-free composition of these two kinds of substratum, and cultivate hUC-MSC for substep.

Wherein, described hUC-MSC is from spontaneous labor or the isolated human umbilical cord mesenchymal stem cells of caesarean delivered healthy newborn umbilical cord tissue.Current this area has multiple known human umbilical cord mesenchymal stem cells separation method.

In test kit provided by the invention, described TME substratum comprises a-MEM, beta-mercaptoethanol and non-essential amino acid.Preferably, described TME substratum contains the a-MEM of the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume and 95-100 parts by volume, wherein, described non-essential amino aqueous acid comprises glycine, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and the Serine that concentration is respectively 8-12mM; More preferably, described TME substratum contains the a-MEM of the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume and 99 parts by volume; Further preferably, described TME substratum is made up of described a-MEM, beta-mercaptoethanol and non-essential amino aqueous acid.

In test kit provided by the invention, described TMD substratum contains a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino acid, recombination human basic fibroblast somatomedin (b-FGF) and serum substitute.

Preferably, described TMD substratum comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume, the serum substitute of 8-12 parts by volume, the a-MEM/DMEM-F12 of 85-95 parts by volume and final concentration are 5-15ng/ml, wherein, described non-essential amino aqueous acid comprises glycine, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and the Serine that concentration is respectively 8-12mM; More preferably, described TMD substratum comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume, the serum substitute of 10 parts by volume, the a-MEM/DMEM-F12 of 89 parts by volume and final concentration are 10ng/ml.Preferably, described TMD substratum is made up of described a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino aqueous acid, recombination human basic fibroblast somatomedin and serum substitute.

According to the embodiment of the application, described non-essential amino aqueous acid can adopt Gibco company article No. to be the product of 11140.

According to the embodiment of the application, described serum substitute can adopt KnockOutTMSerumReplacement (Gibco Products, article No. 10828-010).

Use test kit of the present invention can cultivate hUC-MSC step by step, cultural method comprises: first use the TME culture medium culturing hUC-MSC in described test kit, then uses the TMD substratum in described test kit to cultivate.Namely two kinds of substratum are successively adopted to cultivate;

Further, described cultural method comprises the following steps:

(1) by hUC-MSC with 0.5-4 × 10 ⁴individual cell/cm ²density be inoculated in described test kit TME substratum in cultivate 3-6 hour;

(2) discard TME substratum, PBS cleans, and be replaced by the TMD substratum in described test kit, every 3-5 days changes fresh TMD substratum;

(3) reach after 70-90% converges until cell, for subsequent use, the frozen or repeating step (1) of collecting cell and (2) Secondary Culture;

Selectively, get the hUC-MSC cell that step (3) is collected, that detects in following items is one or more: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.

Preferably, described cultural method comprises the following steps:

(1) by hUC-MSC with 2 × 10 ⁴individual cell/cm ²density be inoculated in described test kit TME substratum in cultivate 3-4 hour;

(2) discard TME substratum, PBS cleans 1 time, is replaced by the TMD substratum in 37 DEG C of described test kits of hatching in advance, within every 3 days, changes fresh TMD substratum;

(3) until cell reach 90% converge after, for subsequent use, the frozen or repeating step (1) of collecting cell and (2) Secondary Culture;

Selectively, get the hUC-MSC cell that step (3) is collected, that detects in following items is whole: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.

According to the specific embodiment of the present invention, the described substep of the serum-free for hUC-MSC cultural method comprises the following steps:

(1) TME substratum is used in six orifice plates or T75 culturing bottle, to carry out adherent culture with certain density inoculation hUC-MSC; Preferably, the inoculum density of hUC-MSC is about 2 × 10 ⁴individual cell/cm ²;

(2), after inoculating 4 hours, carefully inhale with pipettor and abandon old TME substratum, wash one time with PBS, be replaced by 37 DEG C of TMD substratum of hatching equally in advance;

(3) until cell reach 90% converge after, utilize such scheme to carry out Secondary Culture or freeze-stored cell.

Optionally, for the hUC-MSC of step (3) gained, detect following items: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.

On the other hand, the present invention also provides the hUC-MSC obtained by aforesaid method.

Preferably, described hUC-MSC has following characteristics:

(1) adhering to plastic culture vessel becomes fusiformis swirl shape to grow;

(2) the positive ratio expressing CD29, CD44, CD73, CD90, CD105 and HLA-ABC is greater than 99.0%; The positive ratio expressing CD45, CD34 and HLA-DR is less than 1.0%;

(3) externally scleroblast and stearoblast is induced to differentiate into;

(4) viable cell detected ratios reaches more than 99%;

(5) in typical " S type " growth curve characteristic;

(6) versatility genetic expression, described versatility gene be selected from SSEA-4, OCT-4, NANOG and SOX-2 one or more.

Another aspect, the present invention is also provided in the TME substratum and/or TMD substratum that adopt in above-mentioned cultural method.

TME substratum in test kit described in the present invention and TMD substratum are serum-free component, and form distinct, to avoid in culturing cell culturing process because serum batch difference causes the situation of cell growth process instability, also eliminate the possibility propagating the danger of xenogenesis pathogenic agent.

In addition, utilize two kinds of substratum in test kit to carry out substep to cultivate, TME culture medium culturing is first used to promote that hUC-MSC is adherent, then be replaced by TMD substratum and carry out rapid amplifying, solve cell attachment ability in conventional serum-free culture well, breed shortcoming slowly, and in long-term cultivation process, cell still can keep good multiplication capacity and multi-lineage potential, and the vitro culture for zooblast provides a kind of solution efficiently.

Further, operation is simple for test kit of the present invention, shortens the original cuiture time.

Through cells were tested by flow cytometry, through viability examination, differentiation capability qualification and versatility genetic analysis, the mescenchymal stem cell motility rate obtained by the inventive method is high, and purity is high, differentiation capability is strong, and the cell bank of foundation can be directly used in scientific research and clinical assisting therapy.

Accompanying drawing explanation

Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:

Fig. 1 uses the figure cultivating hUC-MSC containing blood serum medium, and wherein Figure 1A is the cellular form of inoculation after 2 hours, and Figure 1B is the cellular form of inoculation after 24 hours, and Fig. 1 C is the cellular form of inoculation after 48 hours.

Fig. 2 is the cytological map in substratum composition screening process, wherein Fig. 2 A is that high density beta-mercaptoethanol substratum inoculates 4 hours later cell forms at cell, Fig. 2 B is lower concentration serum substitute culture medium inoculated 48 hours later cell form, Fig. 2 C is high density serum substitute culture medium inoculated 24 hours later cell form, Fig. 2 D is lower concentration bFGF culture medium inoculated 24 hours later cell form, and Fig. 2 E is high density bFGF culture medium culturing cell cellular form after going down to posterity.

Fig. 3 is the figure of the TME culture medium culturing hUC-MSC used in test kit, and wherein Fig. 3 A is the cellular form of inoculation after 2 hours, and Fig. 3 B is the cellular form of inoculation after 24 hours, and Fig. 3 C is the cellular form of inoculation after 48 hours.

Fig. 4 is the figure of the TMD culture medium culturing hUC-MSC used in test kit, and wherein Fig. 4 A is the cellular form of inoculation after 2 hours, and Fig. 4 B is the cellular form of inoculation after 24 hours, and Fig. 4 C is the cellular form of inoculation after 48 hours.

Fig. 5 is the figure utilizing test kit of the present invention substep to cultivate hUC-MSC, wherein Fig. 5 A uses the cellular form of TME culture medium inoculated after 2 hours in test kit, Fig. 5 B uses the TME culture medium inoculated in test kit to be replaced by the cellular form that TMD substratum continues to be cultured to 24 hours after 4 hours, and Fig. 5 C uses the TME culture medium inoculated in test kit to be replaced by the cellular form that TMD substratum continues to be cultured to 48 hours after 4 hours.

Fig. 6 (6A to 6I) is the result of hUC-MSC through flow cytometry analysis cell surface molecule of carrying out serum-free substep cultivation acquisition adopted in test kit of the present invention, and the positive ratio showing described hUC-MSC expression CD29, CD44, CD73, CD90, CD105, HLA-ABC is greater than 99%; The positive ratio expressing CD45, CD34, HLA-DR is less than 1%.

Fig. 7 is cell viability, the growth characteristics analytical results of Vi-Cell cell viability analyser to the hUC-MSC obtained, wherein Fig. 7 A is the diameter distribution profile of hUC-MSC, Fig. 7 B is the growth curve of hUC-MSC, Fig. 7 C is the real-time activity analysis of hUC-MSC, result shows that the activity of described hUC-MSC is more than 99.7%, cell dia is distributed in about 13 μm, and has the multiplication characteristic of latent period, increased logarithmic phase, plateau.

Fig. 8 is that the hUC-MSC of acquisition is to scleroblast and osteoblastic Induction of committed differentiation result, the scarlet compound that the calcium tubercle generation color reaction that wherein Fig. 8 A shows sodium alizarinsulfonate and osteogenetic process produces, it is painted that Fig. 8 B shows the fat bubble specificity of oil red O to stearoblast.

Fig. 9 is the hUC-MSC versatility differential protein that immuning fluorescent dyeing analysis obtains, and from left to right, is followed successively by SSEA-4, SOX-2, OCT-4, NANOG from top to bottom.

Embodiment

Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.

Unreceipted actual conditions, carry out according to the normal condition in field belonging to the present invention or the suggestion condition of instrument reagent supplier; For dated commercial source, be the conventional products that can commercially availablely buy.

embodiment 1routine has blood serum medium to cultivate hUC-MSC

Tested substratum: the α-MEM of 89 parts by volume, 10% foetal calf serum (FBS), 100U/ml penicillin, 100U/ml Streptomycin sulphate, the beta-mercaptoethanol of 0.1 parts by volume, the b-FGF of 10ng/ml, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume.

In Biohazard Safety Equipment, get the hUC-MSC in the 3rd generation being located away from spontaneous labor neonatal umbilical cord China general formula glue tissue, with 2 × 10 ⁴individual cell/cm ²density is inoculated in T75 Tissue Culture Flask, adds the tested substratum of 15mL, moves into CO ₂concentration is in 37 DEG C of constant incubators of 5%.Inoculate the adherent situation of observation of cell after 2 hours, a large amount of cell attachment of hUC-MSC, has feeler to stretch out; Observe after 48 hrs, hUC-MSC reaches 90% and converges; Cell feeler is unfolded, bright.

Cellular form is see Fig. 1.But, in cell cultivation process, introduce serum and can cause and propagate xenogenesis pathogenic agent risk, and cell growth process also can be caused unstable because of serum batch difference.

embodiment 2conventional commercial serum free medium cultivates hUC-MSC

Reference example 1 method, with same cell source, equal densities inoculation, adds the commercially available serum free medium of 15mL (match industry Products, article No. HUXUC-90061) culturing cell.Inoculate observation of cell after 2 hours adherent, cell becomes clear, how rounded, and feeler is for stretching; At inoculation observation of cell after 24 hours, hUC-MSC becomes clear under the microscope, and feeler extends, and increases not obvious; In inoculation after 48 hours, cell confluency rate reaches about 50%; Inoculate observation of cell after 72 hours, hUC-MSC cell becomes clear, and reaches more than 90% and converges, collected by trypsinisation cell cryopreservation.

Selectively, cell reach 100% converge after, continue cultivate after, cell is curling from culturing bottle edge to come off.It can thus be appreciated that when lacking serum composition, cell easily comes off, and is difficult to maintain good adhered state.

embodimentthe screening of 3 substratum compositions

(1) screening of TME substratum composition

Tested substratum: 0.01, the beta-mercaptoethanol of 0.02,0.05,0.1,0.15,0.2,0.3 or 0.5 parts by volume, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the a-MEM of 99 parts by volume.

Reference example 1 method, with same cell source, equal densities inoculation, adds the tested culture medium culturing cell of 15mL.The adherent situation of observation of cell.

Result: in the medium respectively containing in two concentration groups of 0.01 and 0.02 parts by volume beta-mercaptoethanol, cell attachment speed is comparatively slow, and in inoculation after 4 hours, still have part cell not adherent, after about 8 hours, cell is substantially all adherent; In the medium respectively containing in four concentration groups of 0.05,0.1,0.15 and 0.2 parts by volume beta-mercaptoethanol, completely adherent at inoculation 4 hours later cell, cell becomes clear, and puts out the feelers; In the medium respectively containing in two concentration groups of 0.3 and 0.5 parts by volume beta-mercaptoethanol, in inoculation after 4 hours, cell is also adherent, but part cell state is deteriorated, and occurs differentiation early symptom (see Fig. 2 A).

(2) screening of TMD substratum composition

TME substratum: the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the a-MEM of 99 parts by volume.

Tested substratum: the beta-mercaptoethanol of 0.1 parts by volume, recombination human basic fibroblast somatomedin (the b-FGF of 10ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), 1, the KnockoutFBS serum substitute (10828-028, Gibco company) of 2,5,8,10,12,15 or 20 parts by volume, the a-MEM of 89 parts by volume.

Reference example 1 method, with same cell source, equal densities inoculation, adds 15mLTME culture medium culturing cell.Inoculate observation of cell after 2 hours adherent, continue to cultivate, inoculation about 4 hours later cell are namely completely adherent, change the tested substratum of 15mL.Observation of cell growing state.

Result: in the medium respectively containing in three concentration groups of 1,2,5 parts by volume serum substitutes, cell proliferation is slow, at inoculation observation of cell after 24 hours, the cell aggregation of hUC-MSC part, cell is flat, index difference, degree of converging reaches about 20%, inoculates observation of cell after 48 hours, and hUC-MSC cell becomes clear, reach about 60% converge after, substantially stop breed (see Fig. 2 B); In the medium respectively containing in three concentration groups of 8,10,12 parts by volume serum substitutes, cell growth state is good, at inoculation observation of cell after 24 hours, hUC-MSC is that fusiformis swirl shape is assembled, and range of extension is high, and cell becomes clear, degree of converging reaches 40-60%, inoculate observation of cell after 48 hours, hUC-MSC cell becomes clear, and reaches more than 90% and converges; In the medium respectively containing in two concentration groups of 15,20 parts by volume serum substitutes, occur the situation identical with low concentration group, Growth of Cells is slow, cell flattening, clear-cut (see Fig. 2 C).

(3) screening of TMD substratum composition

TME substratum: the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the a-MEM of 99 parts by volume.

Tested substratum: the beta-mercaptoethanol of 0.1 parts by volume, 1,2,5,8,10,12,15,18 or the recombination human basic fibroblast somatomedin (b-FGF of 20ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), the KnockoutFBS serum substitute (10828-028, Gibco company) of 10 parts by volume, the a-MEM of 89 parts by volume.

Reference example 1 method, with same cell source, equal densities inoculation, adds 15mLTME culture medium culturing cell.Inoculate observation of cell after 2 hours adherent, continue to cultivate, inoculation about 4 hours later cell are namely completely adherent, change the tested substratum of 15mL.Observation of cell growing state.

Result: in the medium respectively containing 1, in two concentration groups of 2ng/mlbFGF, cell proliferation is slow, and cell state is poor, presents under-nutrition state (see Fig. 2 D); In the medium respectively containing 5,8,10,12, in the concentration group of 15ng/mlbFGF, cell normal growth, brightness is high, grows; In the medium respectively containing 18, in the concentration group of 20ng/mlbFGF, cell proliferation is good, bright, but in repeatedly succeeding generations, cell easily breaks up, and cell can become bulk to collect, or feeler elongated (see Fig. 2 E).

embodiment 4utilize the TME culture medium culturing hUC-MSC in test kit

TME substratum: the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the a-MEM of 99 parts by volume.

In Biohazard Safety Equipment, get the hUC-MSC in the 3rd generation being located away from spontaneous labor neonatal umbilical cord China general formula glue tissue, with 2 × 10 ⁴individual cell/cm ²density is inoculated in T75 Tissue Culture Flask, adds 15mLTME substratum, moves into CO ₂concentration is in 37 DEG C of constant incubators of 5%.To inoculate after 2 hours that observation of cell is adherent to put out the feelers, shifting out incubator 24 hours and 48 hours and observe, in order, but there is a large amount of floating dead cell in cell attachment.Breed not obvious; After inoculation the 3rd day, change fresh ME substratum and continue to cultivate, cell came off death gradually at the bottom of bottle, breeds not obvious.

Cellular form is see Fig. 3.

embodiment 5utilize the TMD culture medium culturing hUC-MSC in test kit

TMD substratum: the beta-mercaptoethanol of 0.1 parts by volume, recombination human basic fibroblast somatomedin (the b-FGF of 10ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), the KnockoutFBS serum substitute (10828-028, Gibco company) of 10 parts by volume, the a-MEM of 89 parts by volume

Reference example 4 method, with same cell source, equal densities inoculation, adds 15mLTMD culture medium culturing cell.Equally in inoculation adherent situation of observation of cell after 2 hours, hUC-MSC still has a large amount of cells float not adherent; Observation of cell after 24 hours, cell attachment is uneven, localized clusters; Observe after 48 hrs, hUC-MSC cell attachment more big area is assembled; After inoculation the 3rd day, change fresh TMD substratum and continue to cultivate, part cell came off changing in liquid process, and small part cell be local ageing death, after being cultured to 96 hours, and cell confluency degree about 90%.

Cellular form is see Fig. 4.

embodiment 6the serum-free substep utilizing test kit of the present invention to carry out hUC-MSC is cultivated

TME substratum: the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid (11140, Gibco company) of 1 parts by volume, the a-MEM of 99 parts by volume;

TMD substratum: the beta-mercaptoethanol of 0.1 parts by volume, recombination human basic fibroblast somatomedin (the b-FGF of 10ng/ml, Peprotech company), the non-essential amino aqueous acid (11140 of 1 parts by volume, Gibco company), the KnockoutFBS serum substitute (10828-028, Gibco company) of 10 parts by volume, the a-MEM of 89 parts by volume.

Reference example 4 method, with same cell source, equal densities inoculation, adds 15mLTME culture medium culturing cell.Inoculate observation of cell after 2 hours adherent, continue to cultivate, inoculation about 4 hours later cell are namely completely adherent, change fresh TMD substratum; At inoculation observation of cell after 24 hours, hUC-MSC is that fusiformis swirl shape is assembled, and range of extension is high, and cell becomes clear, and degree of converging reaches 40-60%; Inoculate observation of cell after 48 hours, hUC-MSC cell becomes clear, and reaches more than 90% and converges, collected by trypsinisation cell cryopreservation.

Selectively, cell reach 100% converge after, after continuing to cultivate, cell is not rolled and is come off, and remains long-time well adherent.

Cellular form is see Fig. 5.

Embodiment 6 is known compared with embodiment 1, the present invention takes TME substratum and TMD substratum to carry out the cultivation of serum-free substep, achieve the identical result having serum free culture system with routine, but turn avoid simultaneously and cause propagation xenogenesis pathogenic agent risk at culturing cell owing to introducing serum, it also avoid in culturing process because serum batch difference causes the situation of cell growth process instability.

embodiment 7the surface marker of flow cytometry analysis hUC-MSC

Example 6 cultivate the 3rd generation cell, after Growth of Cells to 90% converges, 2mL0.125% trysinization, then centrifugal 6 minutes of 1200rpm at 4 DEG C, abandons supernatant collecting cell, and PBS cleaning twice, by cell often pipe 1 × 10 ⁵individually be transferred to streaming pipe, add 5 μ LCD34-PE, CD45-FITC, CD29-FITC, CD44-PE, CD73-PE, CD105-PE, CD90-FITC, HLA-ABC-FITC, HLA-DR-PE, IgG1-PE (Isotype control) and IgG1-FITC (Isotype control) antibody respectively, at mixing 4 DEG C, lucifuge hatches 30 minutes, PBS cleaning once, centrifugally remove supernatant, add the resuspended mixing of 500 μ LPBS damping fluid, upper machine testing (flow cytometer XL, Beckman company), each sample collection 1 × 10 ⁴individual cell.

Result is see Fig. 6.

embodiment 8cell viability instrument analyzes cell viability, the growth characteristics of hUC-MSC

Example 6 cultivate the 3rd generation cell be inoculated in T25 culturing bottle, reach after 95%-100% converges until cell, 0.125% trysinization, collecting cell is with 1 × 10 ⁵individual/hole density is inoculated in two 6 orifice plates.Until all adherent and some growth of cell after 10 hours, collect two porocytes and add 500 μ LPBS and make cell suspension, upper machine analysis (cell viability analyser Vi-CellXR, Beckman company).After this every 12 hours sampling analysis, draw growth curve.

Result is see Fig. 7, and show that hUC-MSC is active more than 99.7%, cell dia is distributed at 9-15 μm, and has the multiplication characteristic of latent period, increased logarithmic phase, plateau.

embodiment 9the qualification of hUC-MSC multi-lineage potential

1) Osteoinductive differentiation

The 3rd generation hUC-MSC that Example 6 is cultivated is with 3 × 10 ⁴individual cell/cm ²be seeded to 6 porocyte culture plates, after 24 hours, freshly prepared people UCMSC Osteoinductive differentiation substratum (HUXUC-90021 is added in every hole, match industry product) 2mL, after this within every 3 days, fresh Osteoblast Differentiation inducing culture is changed, after 2 weeks, paraformaldehyde is fixed, Alizarin red staining 3-5min.

Result, see Fig. 8 A, shows the hUC-MSC that obtains in the process of the present invention at osteogenic induction after two weeks, the calcium tubercle generation scarlet color reaction of sodium alizarinsulfonate and osteogenetic process.

2) adipogenic induction differentiation

The 3rd generation hUC-MSC that Example 6 is cultivated is with 2 × 10 ⁴individual cell/cm ²be seeded to 6 porocyte culture plates, until cell reach 100% converge after, every hole is added adipogenic induction division culture medium A liquid (HUXUC-90031, match industry product) and is started induction, change adipogenic induction division culture medium B liquid after 3 days into and carry out maintenance 24 hours, so circulate.When fat ooze now more but less time, maintain 7 days with adipogenic induction liquid B, induction terminate after 4% paraformaldehyde fix, oil red O stain.

Result is see Fig. 8 B, and show the hUC-MSC that obtains in the process of the present invention at adipogenic induction after two weeks, oil red O is obviously painted to stearoblast.

embodiment 10immuning fluorescent dyeing analysis hUC-MSC specific proteins

The 5th generation hUC-MSC that Example 6 is cultivated is with 5 × 10 ³the density of individual cell per well is inoculated in 24 porocyte culture plates, treat that Growth of Cells to 30% ~ 50% converges, with 0.25%TritonX-100 punching process 20 minutes after fixing 15 minutes with 4% paraformaldehyde, lowlenthal serum adds the mouse-anti people primary antibodie (anti-SOX2 antibody, anti-OCT4 antibody, anti-NANOG antibody and anti-NANOG antibody) of having diluted after closing, lucifuge of spending the night at 4 DEG C is hatched; The goat against murine two adding FITC mark afterwards resists, and at room temperature lucifuge hatches 2 hours; Then contaminate core with DAPI/PI, hatch 20min in room temperature lucifuge, fluorescence microscopy Microscopic observation.

Result, see Fig. 9, shows that the hUC-MSC that substep is cultivated in the process of the present invention expresses SOX-2, OCT-4, NANOG and SSEA-4 specific proteins.

Specific description of embodiments of the present invention does not above limit the present invention, and those skilled in the art can make various change or distortion according to the present invention, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.

Claims (9)

1. cultivate the test kit of hUC-MSC for substep for one kind, it is characterized in that, described test kit comprises separated TME substratum and TMD substratum, wherein said TME substratum contains a-MEM, beta-mercaptoethanol and non-essential amino acid, and described TMD substratum contains a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino acid, recombination human basic fibroblast somatomedin (b-FGF) and serum substitute.

2. test kit according to claim 1, is characterized in that, described hUC-MSC is from spontaneous labor or the isolated human umbilical cord mesenchymal stem cells of caesarean delivered healthy newborn umbilical cord tissue.

3. test kit according to claim 1 and 2, it is characterized in that, described TME substratum contains the a-MEM of the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume and 95-100 parts by volume, wherein, described non-essential amino aqueous acid comprises glycine, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and the Serine that concentration is respectively 8-12mM;

Preferably, described TME substratum contains the a-MEM of the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume and 99 parts by volume;

Preferably, described TME substratum is made up of described a-MEM, beta-mercaptoethanol and non-essential amino aqueous acid.

4. test kit according to any one of claim 1 to 3, it is characterized in that, described TMD substratum comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.05-0.2 parts by volume, the non-essential amino aqueous acid of 0.5-2 parts by volume, the serum substitute of 8-12 parts by volume, the a-MEM/DMEM-F12 of 85-95 parts by volume and final concentration are 5-15ng/ml, wherein, described non-essential amino aqueous acid comprises glycine, L-Ala, L-Tianmen acid amides, L-Aspartic acid, L-glutamic acid, proline(Pro) and the Serine that concentration is respectively 8-12mM;

Preferably, described TMD substratum comprises the recombination human basic fibroblast somatomedin that the beta-mercaptoethanol of 0.1 parts by volume, the non-essential amino aqueous acid of 1 parts by volume, the serum substitute of 10 parts by volume, the a-MEM/DMEM-F12 of 89 parts by volume and final concentration are 10ng/ml;

Preferably, described TMD substratum is made up of described a-MEM/DMEM-F12, beta-mercaptoethanol, non-essential amino aqueous acid, recombination human basic fibroblast somatomedin and serum substitute.

5. use the test kit substep according to any one of claim 1 to 4 to cultivate the method for hUC-MSC, it is characterized in that, described method comprises: first use the TME culture medium culturing hUC-MSC in described test kit, then uses the TMD substratum in described test kit to cultivate.

6. method according to claim 5, is characterized in that, said method comprising the steps of:

(1) by hUC-MSC with 0.5-4 × 10 ⁴individual cell/cm ²density be inoculated in described test kit TME substratum in cultivate 3-6 hour; Cell carries out adherent significantly in this step;

(2) discard TME substratum, PBS cleans, and be replaced by the TMD substratum in described test kit, every 3-5 days changes fresh TMD substratum; Cell increases in this step significantly;

(3) reach after 70-90% converges until cell, for subsequent use, the frozen or repeating step (1) of collecting cell and (2) Secondary Culture;

Selectively, get the hUC-MSC cell that step (3) is collected, that detects in following items is one or more: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.

7. the method according to claim 5 or 6, is characterized in that, said method comprising the steps of:

(1) by hUC-MSC with 2 × 10 ⁴individual cell/cm ²density be inoculated in described test kit TME substratum in cultivate 3-4 hour;

(2) discard TME substratum, PBS cleans 1 time, is replaced by the TMD substratum in 37 DEG C of described test kits of hatching in advance, within every 3 days, changes fresh TMD substratum;

(3) until cell reach 90% converge after, for subsequent use, the frozen or repeating step (1) of collecting cell and (2) Secondary Culture;

Selectively, get the hUC-MSC cell that step (3) is collected, that detects in following items is whole: differentiation capability, cytoactive, cell purity, cell contamination and multiplication characteristic.

8. by the hUC-MSC of the method acquisition according to any one of claim 5 to 7.

9. hUC-MSC according to claim 8, is characterized in that, described hUC-MSC has following characteristics:

(1) adhering to plastic culture vessel becomes fusiformis swirl shape to grow;

(2) the positive ratio expressing CD29, CD44, CD73, CD90, CD105 and HLA-ABC is greater than 99.0%; The positive ratio expressing CD45, CD34 and HLA-DR is less than 1.0%;

(3) externally scleroblast and stearoblast is induced to differentiate into;

(4) viable cell detected ratios reaches more than 99%;

(5) in typical " S type " growth curve characteristic;

(6) versatility genetic expression, described versatility gene be selected from SSEA-4, OCT-4, NANOG and SOX-2 one or more.