CN104178456B - The propagating method of a kind of people induced multi-potent stem cell and application - Google Patents

Abstract

The propagating method of a kind of people induced multi-potent stem cell and application, this method solve tradition cloning block propagating method and make cell be prone to differentiation, and transfection efficiency is low, it is difficult to the problem carrying out genetic modification.The method is unicellular passing on and cultural method, comprises the steps: that adding ROCK inhibitor with digestive enzyme fully digests induced multi-potent stem cell clone for unicellular；Use blood counting chamber or cell counter counting；According to the density of 20,000 30000 cells/square cm, cell is spread on the coated culture plate of ECM；Use the culture medium adding ROCK inhibitor that induced multi-potent stem cell is placed in 37 DEG C, 8 15%CO₂Under the conditions of cultivate；After passing on for 20 generations, the versatility of induced multi-potent stem cell does not changes, including versatility gene expression, and internal and vitro differentiation potential etc..

Description

The propagating method of a kind of people induced multi-potent stem cell and application

Technical field

The present invention relates to the propagating method of people induced multi-potent stem cell, be specifically related to people induced multi-potent stem cell slender Born of the same parents are passed on and culture technique.

Background technology

2006, Kyoto Univ Japan Shinya Yamanaka was in the upper rate of world-renowned scholarly journal " cell " First report the research of induced multi-potent stem cell.They turn Oct3/4, Sox2, c-Myc and Klf4 these four Record factor gene is cloned into viral vector, then introduces l cell, and discovery can induce it to convert, It is successfully obtained the pluripotent stem cell being similar to embryonic stem cell (embryonic stem cell, ES cell), name For induced multi-potent stem cell (iPSCs, induced pluripotent stem cell).Produce iPSCs form, Gene and protein expression, epigenetic modification state, cell multiplication capability, embryoid and deformity tumor generative capacity, The aspects such as differentiation capability are all similar to embryonic stem cell, thus affirmed iPSCs versatility.Demonstrate,prove according to this Daylight can be undifferentiated state by reprogramming technology reverse in the mature cell of terminal differentiation, and reaches to recover The purpose of differentiation versatility potential.

Shinya Yamanaka research group utilizes identical technology, above-mentioned 4 same transcription factor is imported In human dermal fibroblasts, also have successfully been obtained iPSCs.Primary human fibroblast sample synovial cell The most also can be reconstructed into as iPSCs with being derived from the fibroblastic cell line of neonate.This kind of iPSCs is at cell Form, multiplication capacity, surface antigen marker, gene expression profile, pluripotent stem cell specific gene apparent The aspects such as hereditism's state, telomerase activation are similar to hESCs, and when cultivating in vitro and in Mice Body Teratoma all can be divided into different cell types (Takahashi K and the Yamanaka S etc. of 3 germinal layers in being formed People, utilizes specificity factor induction adult fibroblasts to pluripotent stem cell.Cell 2007；131:861-872). Meanwhile, winconsin university J.A Thomson research group also reports and successfully induces fetal fibroblast Be converted into the mankind iPSCs with hESCs basic feature, except that they use slow virus as carrier, And in 14 candidate genes, have selected 4 genes such as Oct4, Sox2, Nanog, Lin28 carry out transduceing (Yu Monarch's English et al., becomes pluripotent stem cell system from the somatic induction of people, science, and 2007；318:1917-1920). This is referred to as, by educational circles, the human relations that the important breakthrough of bioscience " milestone " is expected to help scientist to walk around clone technology Reason, morals dispute, open gate for medical application.

The research nucleus reprogramming that appears as of iPSCs provides new thinking, at stem-cell research field, table See genetics research field and field of biomedical research all causes strong repercussion, be not only because it Importance in terms of basic research, more regenerative medicine bring new hope.At actual application aspect, iPSCs Preparation method relatively easy and stable, it is not necessary to use sexual cell or destroy body early embryo.This is technically The most all than additive method the most advantageously.The foundation of iPSCs has furthered stem cell further and clinical disease is controlled The distance treated, has huge diving in terms of cell replacement treatment and pathogenetic research, new medicament screen It is being worth.Additionally, iPSCs presents the most day by day in the effect of the aspect such as nervous system disease, cardiovascular disease, iPSCs The most successfully it is divided into neuronal cell, neurogliocyte, cardiovascular cell and primordial germ thin Born of the same parents etc., because having the genomic constitution identical with patient self, so the immunologic rejection of body self will not be caused anti- Should.

At present cultivation and the propagating method of iPSCs is not quite similar, culture medium have serum and serum replacement point, pass There are enzyme digestion and machinery to pass on method for method, use corresponding cultivation and propagating method according to experiment demand.Logical The normal mode of passing on is by after clone use Dispase or collagenase preliminary treatment, makes clone become the loosest, Pass in the way of then clone excision becomes cloning block on paving to trophoblastic cell, condition of culture one used As be 37 DEG C, 5%CO₂.The defect of the method is that cell is prone to differentiation, it is difficult to expand on a large scale.And utilize list The propagating method of cell would generally affect the versatility of cell, and cell caryogram is had an impact by long-term cultivation.

Summary of the invention

For the problems referred to above of prior art, the invention provides a kind of people induced multi-potent stem cell (hiPSCs) The unicellular method passing on and cultivating, the method can expand rapidly hiPSCs, makes cell keep preferably raw Long status, overcome hiPSCs be prone to differentiation, be difficult to expand on a large scale, frozen and inefficient problem of recovering, It is prone to carry out consideration convey dye and single cell clone, brings great convenience for operations such as genetic modifications.

For achieving the above object, the present invention takes techniques below scheme:

The propagating method of a kind of people induced multi-potent stem cell, the method comprises the steps:

A. before passage, 1-2h spreads extracellular matrix to porous plate；

B. take out from incubator and converge the cell that rate 75%-85% needs pass on, use DMEM/F12 culture medium Wash once, add Accutase digestive enzyme and be placed in 37 DEG C, the CO of volume fraction 8-15%₂Incubator digests；Carefully Born of the same parents are the most digested when getting off, and add mTeSR1 culture medium and terminate digestion；

C. it is centrifuged, abandons supernatant, use the mTeSR1 culture medium re-suspended cell adding ROCK inhibitor, gently Uniformly numeration is played in featheriness；

D. the density of 20000-30000 cells/square cm is pressed by cultivation the most extracellular matrix coated for cell paving On plate, be placed in 37 DEG C, volume fraction be the CO of 8-15%₂Incubator is cultivated；

E. second day observation of cell density, adherent situation, i.e. removing ROCK inhibitor if all going well, renewing Fresh mTeSR1 culture medium is cultivated, and later every day changes liquid, again reaches 75%-85% both can pass to converging rate Generation or frozen.

Propagating method as above, it is preferable that the extracellular matrix in described step A is ECM, MatrigelLDEV-Free hESC Qualified, Vitronectin (VTN-N) or CELLstart^TM CTS^TM。

Propagating method as above, it is preferable that described step B needs the cell passed on make a living long be in right Cell during the number phase；It is highly preferred that add Y27632 as digestive enzyme using Accutase, digestion time is 7min, The most both it is ensured of unicellular passing on, excessively will not affect cell growth state because of digestion again.

Propagating method as above, the described ROCK inhibitor apoptosis under unicellular capable of inhibiting cell, Thus improve cell survival rate, preferably Y27632, HA-100, Blebbistatin or Thiazovivin, more It is preferably Y27632.

Propagating method as above, it is preferable that with trypan blue, cell is counted in described step C, for Ensure the accuracy of counting, adjust cell counting when cell density is each big lattice cell number 30-50.

The propagating method of a kind of people induced multi-potent stem cell, the method uses method as above, except described Accutase digestive enzyme replaces with pancreatin.

The propagating method of a kind of people induced multi-potent stem cell, the method uses method as above, except described MTeSR1 culture medium (StemCell, Cat#05850) replaces with TeSR2 (StemCell, Cat#05860), Essential8^TMMedium (Life, Cat#A14666SA), or NutriStem (BI, Cat#05-100-1A/B).

Propagating method as above is applied to hiPSCs cell and expands on a large scale.

Propagating method as above is applied to high-efficiency transfection hiPSCs and exogenous gene imports.

Propagating method as above is applied to obtain the clone in individual cells source, with purifying cells or screening tool There is the cell colony of specific markers.

Propagating method as above is applied to obtain the homogenic type cell strain of mutant allele.

Propagating method as above is applied to obtain the single cell suspension for genetic modification.

The beneficial effects of the present invention is, hiPSCs cell is processed into unicellular form by the method for the present invention, and It is placed in high CO₂Cultivate under conditions of concentration.It has the beneficial effect that the following aspects:

1) make the most homogeneous shakedown of cell that passes on in culture plate, inoculum density be 20000-30000 cell/ Square centimeter, the dilutest if cell-tocell current ratio is less, be unfavorable for growth, the closeest if cell grow relatively Hurry up, if it is the most aging to pass on cell not in time, thus cause differentiation；

2) hiPSCs can be made to expand on a large scale, be suitable to the industrialized production of such cell；

3) cell after frozen can obtain more than survival rate after 70% recovery；

4) efficiency making hiPSCs single cell clone improves 3-5 times, it is easy to carries out genetic modification screening and has identical The clone of genotype；

5), after passing on more than 20 generations, the hiPSCs cell using the method to pass on still has human embryo stem cell phase Like feature, and caryogram does not the most change.

Accompanying drawing explanation

Figure 1A is that embodiment 1 cultivates second day microphotograph.

Figure 1B is that embodiment 1 cultivates the 3rd day microphotograph.

Fig. 1 C is that embodiment 1 cultivates the 4th day microphotograph.

Fig. 1 D is that embodiment 1 cultivates the 5th day microphotograph.

Fig. 2 A is embodiment 3 cloning cluster suspension culture microphotograph.

Fig. 2 B is the expression that embodiment 3 utilizes qPCR three germinal layer labels of detection.

Fig. 2 C is embodiment 3 teratoma photo.

Fig. 3 is embodiment 4 karyotyping figure.

Fig. 4 is that embodiment 5GFP expresses microphotograph.

Detailed description of the invention

Embodiment 1hiPSCs is unicellular to pass on

1) before passage, 1-2h spreads ECM to 6 orifice plate.

2) from incubator, taking-up converges the cell about rate 75%-85%, washes one by DMEM/F12 culture medium Secondary, add Accutase digestive enzyme and be placed in 37 DEG C, the CO of volume fraction 12%₂Digesting in incubator, cell is basic Digested when getting off, add mTeSR1 and terminate digestion.

3) 200g is centrifuged 3min, abandons supernatant, the mTeSR1 re-suspended cell containing Y27632, blows and beats gently Uniformly.

4) with blood counting chamber, cell is counted, be inoculated in pre-by the density of 20000-30000/ square centimeter First it is covered with on 6 orifice plates of ECM, is placed in 37 DEG C, the CO of volume fraction 12%₂Incubator is cultivated.

5) second day basis of microscopic observation cell density and adherent situation (Figure 1A).Have between cell contact and Cell density is suitable, removes Y27632, renews fresh mTeSR1 culture medium and cultivate.

6) the 3rd day cell starts to assemble agglomerating (Figure 1B), and cell state is stable, renews fresh mTeSR1 and cultivates Base is cultivated.

7) the 4th day to the 5th day, cell active growth, density incrementally increased (Fig. 1 C, Fig. 1 D), to 75-85% During density, cell can carry out passing on or frozen.

Embodiment 2hiPSCs single cell clone

1) from incubator, taking-up converges the cell about rate 75%-85%, adds Accutase digestive enzyme and is placed in 37 DEG C, the CO of volume fraction 8-15%₂Digesting in incubator, cell is the most digested when getting off, and adds mTeSR1 Terminate digestion.

2) Trypan Blue, utilizes blood counting chamber meter number of viable cells, uses mTeSR1+Y27632 suppression Dilution agent cell suspension, to 5-15 cells/ml, adds 100ul cell in coated 96 orifice plates of ECM and hangs Liquid/hole, puts into the CO of 8-15%₂Incubator is cultivated.

3) microscopy after 10 days, passes on the long hiPSCs to clone's shape, amplification, i.e. obtains single cell source hiPSCs。

Embodiment 3-hiPSCs is unicellular pass on after versatility identify

HiPSCs use embodiment 1 method unicellular passed on for 20 generations after, take part cell and carry out embryoid body formation (EB formation) experiment and teratoma form experiment (Teratomas formation).

1) embryoid body is formed

On recovery hiPSCs to ECM, it is transferred to cell on feeder cultivate when cell length to 75%.When After cloning cluster grows 5-6 days on feeder, transfer cloning cluster suspension culture (Fig. 2 A).After suspension culture 8 days Transfer EB ball is to adherent growth in 6 orifice plates be coated Gelatin.When, after EB ball adherent growth 8 days, collecting The RNA of attached cell, utilizes the expression (Fig. 2 B) of qPCR three germinal layer labels of detection.Result shows Show that breaking up, by EB mode, the cell obtained all has growth than hiPSCs on the label of three endoderm cell, And compared with hiPSCs, the difference of each germinal layer at least label is more than 10 times.

2) teratoma is formed

On recovery hiPSCs to ECM, when cell length to 75%, it is expanded to 10cm culture dish, uses Accutase Peptic cell is unicellular, the centrifugal supernatant that goes, and then the ECM re-suspended cell of use 30% is injected to SCID Mice oxter and muscle, obtain teratoma, after using paraffin embedding, section after 8-12 week, H/E dyes, knot Fruit display teratoma contains the tissue (Fig. 2 C) of 3 germinal layers.

The karyotyping of embodiment 4hiPSCs

On recovery hiPSCs to ECM, by converge rate reach about 85% cell inhale abandon culture medium, DMEM/F12 Wash twice, add 10%MEF culture medium (90%DMEM height sugar, 10%FBS) and Colchicine mixed culture medium, It is placed in 37 degrees Celsius, 5%CO₂Middle process 2h, washes one time with DPBS after having processed, 0.25% trypsinization Rear collection cell, 200g is centrifuged 5min, abandons supernatant and adds the 0.075mol/L KCL solution 8ml of 37 DEG C of pre-temperature, Rapidly, fiercely blow and beat cell precipitation with suction pipe, mixing is placed on 37 DEG C of thermostat water bath Hypotonic treatment 18min, Adding the freshly prepared fixative of 1ml (methanol: glacial acetic acid=3: 1), softly blow with suction pipe even, room temperature pre-fixes 5min, 1200rpm are centrifuged 5min, abandon supernatant, along wall be slowly added to freshly prepared fixative (methanol: Glacial acetic acid=3: 1), blow even gently, be placed in 37 DEG C of pre-40min of water, repeat and fix once, 1200rpm is centrifuged 5min, inhales with suction pipe and abandons major part fixative, stay the soft re-suspended cell of about 1.5ml, draw a small amount of cell suspension, Drip 2-3 and drip on the slide that frozen water soaked, be at once placed in baking oven drying, specimen is placed in trypsinization 8s, It is put in rapidly in normal saline termination digestion, again Giemsa dyeing 8min, rinses slide, room with tap water gently Temperature after drying in basis of microscopic observation split coil method number and deployment conditions.Randomly select 20 split coil method, use G Banded karyotype analytic process analyzes the caryogram of all split coil method.It is 46 that statistical result showed has 18 split coil method Bar chromosome, and form is normal, 2 split coil method are abnormal.It can be assumed that this cell strain is through unicellular 20 generations Pass on rear caryogram normal, randomly select a split coil method and arrange out caryogram (Fig. 3).

Embodiment 5 transfects mRNA and plasmid to the hiPSCs of unicellular

1) peptic cell: use Accutase peptic cell, collects cell, centrifugal, abandons supernatant, mTeSR1/Y27632 Resuspended；

2) cell counting: count with trypan blue, calculates viable count；

3) appropriate living cells is taken to 1.5ml centrifuge tube, 115g, 3min；

4) use consideration convey transfection reagent resuspended；

5) in two rotaring redyeing systems, it is separately added into appropriate pMAX-GFP plasmid and GFP-mRNA, blows gently Play mixing；

6) consideration convey dye: transfer to cell and DNA mixed liquor, in electricity revolving cup (avoiding bubble), use Amaxa Nucleofactor II, selects corresponding program electric shock；

7) recover: 500ul culture medium (37 degree of preheatings in advance) is added electricity revolving cup, then cell is turned from electricity Sucking-off gently in Bei；

8) bed board: need that cell is taped against corresponding ECM according to experiment and be coated on flat board, generally 12 orifice plates 3 holes.

9) after 18 hours, test under microscope GFP expression (Fig. 4).

It is last it should be noted that, above example is only in order to illustrate technical scheme rather than to the present invention The restriction of protection domain, although the present invention being explained in detail with reference to preferred embodiment, the common skill of this area Art personnel should be appreciated that and can modify technical scheme or equivalent, without deviating from this The spirit and scope of inventive technique scheme.

Claims (11)

1. the propagating method of a people induced multi-potent stem cell, it is characterised in that the method comprises the steps:

A. before passage, 1-2h spreads extracellular matrix to porous plate；

B. take out from incubator and converge the cell that rate 75%-85% needs pass on, wash once by DMEM/F12 culture medium, add Accutase digestive enzyme is placed in 37 DEG C, digests in the CO2 incubator of volume fraction 8-15%；Cell is the most digested to get off Time, add mTeSR1 culture medium and terminate digestion；

C. it is centrifuged, abandons supernatant, use the mTeSR1 culture medium re-suspended cell adding ROCK inhibitor, gently piping and druming uniformly note Number；

Cell is spread to the most extracellular matrix coated culture plate by the density D. pressing 20000-30000 cells/square cm, puts In 37 DEG C, volume fraction be 8-15% CO2 incubator cultivate；

E. second day observation of cell density, adherent situation, i.e. removes ROCK inhibitor if all going well, and renews fresh mTeSR1 training Foster base is cultivated, and later every day changes liquid, again reaches 75%-85% can pass on or frozen to converging rate.

Propagating method the most according to claim 1, it is characterised in that the extracellular matrix in described step A be ECM, MatrigelLDEV-Free hESC Qualified, Vitronectin or CELLstart^TMCTS^TM。

Propagating method the most according to claim 1, it is characterised in that need the cell passed on to make a living strong point in described step B Cell when logarithmic (log) phase.

Propagating method the most according to claim 1, it is characterised in that described ROCK inhibitor is Y27632, HA-100, Blebbistatin or Thiazovivin.

5. the propagating method of a people induced multi-potent stem cell, it is characterised in that the method uses institute any one of claim 1-3 The method stated, except described Accutase digestive enzyme replaces with pancreatin.

6. the propagating method of a people induced multi-potent stem cell, it is characterised in that the method uses institute any one of claim 1-3 The method stated, except described mTeSR1 culture medium replaces with TeSR2, Essential8^TMMedium, or NutriStem.

7. the propagating method as according to any one of claim 1-6 is applied to hiPSCs cell and expands on a large scale.

8. the propagating method as according to any one of claim 1-6 is applied to high-efficiency transfection hiPSCs and exogenous gene imports.

9. the propagating method as according to any one of claim 1-6 is applied to obtain the clone in individual cells source, with purifying cells Or screening has the cell colony of specific markers.

10. the propagating method as according to any one of claim 1-6 is applied to obtain the homogenic type cell of mutant allele Strain.

11. propagating methods as according to any one of claim 1-6 are applied to obtain the single cell suspension for genetic modification.