CN104152409B - Method for simultaneous isolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells - Google Patents

Abstract

The invention relates to a canine stem cell isolated culture technology, and especially relates to a method for in vitro simultaneous insolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells. The method is characterized in that adherent culture of primary mesenchymal stem cells is carried out based on the separation of bone marrow mononuclear cells, half medium change is adopted, and a mononuclear cell culture suspension is added, that is, hematopoiesis microenvironment provided by the adherent-growth mesenchymal stem cells is used to culture the suspending-growth multifunctional hematopoietic stem cells, so the achieve the simultaneous isolated culture of the canine bone marrow mesenchymal stem cells and the multifunctional hematopoietic stem cells is realized.

Description

Dog mesenchymal stem cells MSCs and multi-functional candidate stem cell are separately cultured simultaneously Method

Technical field

The present invention relates to dog stem cell is separately cultured technology, especially the external dog medulla mesenchyma that is separately cultured simultaneously does thin Born of the same parents and the technology of multi-functional candidate stem cell.

Background technology

Mesenchymal stem cells MSCs (Bone marrow mesenchymal stemcells, BMMSCs) is that one kind is deposited It is the non-hematopoietic stem cell of marrow, convenient sources, without immunological rejection, the differentiation of tool polyphyly and high proliferative potentials exist in recent years Field is repaired in basic research field, clinical tissue organ and disease treatment field presents extremely tempting prospect；It is multi-functional to make Hemocytoblast (Hematopoietic stem cells, HSCs) is a blastoid cell in the hematopoietic tissues such as marrow, is had Self directed differentiation (being divided into all kinds of mature blood cells) and go back to the nest that (i.e. Jing Peripheral Circulations enter after venoclysis Enter medullary sinus, then across blood vessel endothelium barrier and be positioned at the extravascular space gap of marrow) etc. biological function, for blood cell Regeneration and reconstruction play an important role.

Tradition separates the method for BMMSCs and HSCs mainly includes density-gradient centrifugation method, flow cytometric sorting method, magnetic Pearl separating method etc., but action required complex steps are confined to, long, big to cellular damage, human factor is more for required time, and The in vitro culture of HSCs needs to add various growth factors and inducible factor.Therefore need when carrying out mass cell and being separately cultured The method for wanting a kind of time saving and energy saving and efficient cell separation culture.

The content of the invention

It is an object of the invention to set up one kind be separately cultured suitable for carrying out mass cell in vitro, can the efficiently same time-division It is that the studies and clinical application of follow-up dog class stem cell provides material base and technology from the method for culture dog BMMSCs and HSCs Support.

The invention discloses a kind of method for being separately cultured dog mesenchymal stem cells MSCs and multi-functional candidate stem cell, its It is characterised by the basis of separating bone marrow single nuclear cell, while adhere-wall culture primary mescenchymal stem cell, measures using half Liquid is changed, the method for adding mononuclearcell culture suspension, i.e., the hematopoiesis for being provided using the mescenchymal stem cell of adherent growth The multi-functional candidate stem cell of microenvironment culture suspension growth, so as to reach and meanwhile be separately cultured dog mesenchymal stem cells MSCs and The purpose of multi-functional candidate stem cell, concrete operation step is as follows：

The in-vitro separation of step 1, BMMSCs and HSCs：General anaesthesia experimental dog, posterior superior iliac spine preserved skin, sterilization, drape, Divide 2～3 points of bone marrow extraction about 10ml altogether with the marrow puncture needle containing 1ml liquaemins, mixing is anti-condensation, and 50ml sterilizations are proceeded to rapidly Centrifuge tube, is mixed with the dilution of equivalent DMEM/F12；The centrifuge tube that 10 bottoms add 5ml lymphocyte separation mediums is taken, addition Lymphocyte separation medium concentration is 1.077 g/ml, and the adherent inflow equivalent marrow dilution of upper strata difference, sealing controls temperature 4 DEG C, centrifuge speed is 2000 r/min, and centrifugation 20min removes upper-layer fat cell, draws middle intersection tunica albuginea confluent monolayer cells, Then washing interlayer cell is mixed with 4 DEG C of 10 ml DMEM/F12,20min, rotating speed 2000r/min, in removal is centrifuged again Clear liquid, the above-mentioned washing interlayer cell step 1 time of repetition；With containing 15% FBS, 100 U/ml penicillin, 100 U/ml streptomysins The resuspended deposits of DMEM/F12 culture medium 5ml；

Step 2, determination of trypan blue staining cell viability：0.4g trypan blues are taken, the 0.1 M PBS solutions of 100 ml are added In, it is configured to 0.4% trypan blue solution, 0.1M PBS solutions formula：NaCl 8g, KCl 0.2g, Na₂HPO₄.12H₂O 3.488g, KH₂PO₄0.2g, plus three and boils off ionized water to 1000 ml, and adjustment pH value is that 7.4,0.22 m membrane filtrations are degerming. Take the ml of 0.4% trypan blue solution, 0.5 ml, DMEM/F12 culture medium 0.3 and the ml of cell suspension 0.2 is fully mixed, use blood cell Tally living cell counting (non-pigmented cells) and dead cell (pigmented cells), altogether 500 cells of number, calculate cell viability, Cell viability (%)=(TCS-pigmented cells number)/TCS × 100%.As Trypan Blue cell viability >= 95%, then can carry out next step operation, otherwise repeat step 1（The in-vitro separation of repeat step 1, BMMSCs and HSCs）；

The adherent BMMSCs of step 3, culture：With containing 15% FBS, 100 U/ml penicillin, 100 U/ml when cultivating first The DMEM/F12 nutrient solutions adjustment cell density of streptomysin (dual anti-), with 1 × 10⁶The nutrient solution inoculation of the ml of/ml cell densities 5 In 25 cm²Disposable Tissue Culture Flask in, be placed in 37 DEG C of temperature, 5% CO₂, saturated humidity cell culture incubator in cultivate, training Change liquid after supporting 3 days first, remove red blood cell, the marrow hemopoietic stem cells of suspension growth and other not adherent stem cells, Changed liquid 1 time every 3 days later, that is, renew fresh containing 15% FBS, 100 U/ml penicillin, 100 U/ml streptomysins DMEM/F12 nutrient solutions, when cell growth is to 70%～80% fusion, with 0.25% 2～3min of Trypsin Induced, with containing 10% FBS and dual anti-DMEM/F12 terminate digestion, bottle wall cell is blown and beaten repeatedly, make single cell suspension by 1:3 pass on, and are inverted aobvious Micro mirror is observed day by day, until attached cell is fusion together when being paved with bottom of bottle, repeats aforesaid operations, amplification is passed on repeatedly, collection；

The HSCs of step 4, culture suspension growth：Due to HSCs suspension growths in vitro, and need what stroma cell was supported Growing environment, therefore during step 3 culture BMMSCs, when liquid is changed for the second time, discard the whole supernatants in cell bottle Liquid, adds by the cell suspension obtained by step 1,2, close with cell is adjusted containing 10% FBS and dual anti-DMEM/F12 nutrient solutions Spend for 1 × 10⁶/ ml, draws 5 ml cell suspensions and is directly inoculated in above adherent layer, hereafter changes liquid per 6 days half amounts, i.e., 2.5 ml suspension cultures are taken out from blake bottle, 20min, rotating speed 2000r/min is centrifuged, collected cell precipitation and be HSCs, then add 2.5 ml containing 10% FBS and dual anti-DMEM/F12 nutrient solutions, continue culture by this method and go down, Microscope observation of cell growing state is put, when adherent BMMSCs grows to 80% fusion, suspension growth can be simultaneously collected The HSCs and BMMSCs of adherent growth；

Step 5, cellular identification：(1) Morphological Identification：The BMMSCs that adherent growth is observed under inverted microscope is in long shuttle Shape or polygonal fibrocyte shape, the HSCs of suspension growth is rounded, and cell light, refractivity is good；(2) flow cytometry Identification：The BMMSCs and HSCs of culture are taken respectively, and the single cell suspension that 300 l contain 2.5 × 106 is made with PBS；Dispense to In the Eppendof pipes of 6 1.5ml, 50 l sheep blood serums closing 30min is separately added into；Be separately added into again the anti-CD14 in mouse source one, CD34, CD44, CD11a, HLA2DR and each 5 l of control PBS, mix, and react 30min；PBS centrifuge washings 2 times, remove unmarked On antibody；Each effective PBS is configured to 99 l cell suspensions；Often pipe adds 1 l FITC marks goat anti-mouse IgG (H+L) Two resist, and mix, lucifuge reaction 15min at 37 DEG C；PBS centrifuge washings 2 times；Often pipe adds 1ml PBS and is suspended again cell, uses The expression of flow cytomery cell surface antigen CD14, CD34, CD44, CD11a and HLA2DR；BMMSCs strongly expressed CD44 (mescenchymal stem cell specific molecular antigen), HSCs strongly expressed CD34 (candidate stem cell specific molecular antigen);

The frozen and recovery of step 6, BMMSCs and HSCs：(1) it is frozen：The every BMMSCs and HSCs in generation, except for continuing Cultivate and carry out related experiment all outer, it is all frozen.Cells frozen storing liquid：10% DMSO, 40% FBS, 50% DMEM/F12 are tieed up It is 1 × 10 to hold nutrient solution adjustment cell concentration⁶/ ml, in adding aseptic cell cryopreservation tube, the every ml of pipe 1.5, application program drop Warm instrument is frozen in liquid nitrogen；(2) recover：It is rapid from liquid nitrogen to take out cell cryopreservation tube, 37 DEG C of water baths are put immediately into, gently Shake makes it melt as early as possible, takes out cryopreservation tube, is opened with after 75% ethanol disinfection, suctions out cell suspension, injects centrifuge tube, rotating speed 2000r/min, is centrifuged 20min, removes supernatant, uses resuspended thin containing 10% FBS and dual anti-DMEM/F12 nutrient solution nutrient solutions Born of the same parents are precipitated, and put 37 DEG C of temperature, 5% CO₂, saturated humidity cell culture incubator in cultivate.

This method is innovated and improved in the method that tradition is separately cultured, and is overcome loaded down with trivial details when dog stem cell separates Screening step, the multi-functional candidate stem cell of dog is suspended in nutrient solution, is difficult to grow, needs to add various growth factors and induction The defect of the factor, can simultaneously be separately cultured dog mesenchymal stem cells MSCs and multi-functional Hematopoietic Stem is thin using the method for the present invention Born of the same parents, resulting cellular morphology is homogeneous, shortens the time required to cultured cells, and two kinds of isolated stem cell purity are higher, increases Grow very fast.The inventive method provides abundant material source for the research and application of dog class stem cell, with extensive dog class Clinical medicine application prospect.

Specific embodiment

The in-vitro separation of embodiment 1, step 1, BMMSCs and HSCs：General anaesthesia experimental dog, posterior superior iliac spine preserved skin, disappears Malicious, drape, with 2～3 points of common bone marrow extraction about 10ml of the marrow puncture needle containing 1ml liquaemins point, mixing is anti-condensation, proceeds to rapidly 50ml sterilization centrifuge tubes, are mixed with the dilution of equivalent DMEM/F12；Take 10 bottoms and add 5ml lymphocyte separation mediums (1.077 G/ml centrifuge tube), upper strata is carefully added into respectively (adherent inflow) equivalent marrow dilution, sealing.4 DEG C, 2000 r/min, Centrifugation 20min removes upper-layer fat cell, careful to draw middle intersection tunica albuginea confluent monolayer cells.It is mixed with 4 DEG C of 10ml DMEM/F12 Even washing interlayer cell, 2000r/min is centrifuged 20min, removes supernatant, repeated washing 1 time；With containing 15% FBS, 100 U/ The resuspended deposits of DMEM/F12 culture medium 5ml of ml penicillin, 100 U/ml streptomysins；

Step 2, determination of trypan blue staining cell viability：Take 0.4 % trypan blues (PBS preparations) 0.5 ml, DMEM/F12 trainings The ml of foster base 0.3 and the ml of cell suspension 0.2 are fully mixed, with blood counting chamber living cell counting (non-pigmented cells) and extremely Cell (pigmented cells), altogether 500 cells of number, calculate cell viability, and cell viability (%)=(TCS-coloring is thin Born of the same parents' number)/TCS × 100%.Such as Trypan Blue cell viability >=95%, then next step operation can be carried out, otherwise repeatedly be walked Rapid 1；

The adherent BMMSCs of step 3, culture：With the DMEM/ containing 15% FBS and dual anti-(mycillin) when cultivating first F12 culture mediums adjust cell density, with the culture that the nutrient solution of the ml of 1 × 106/ml cell densities 5 is inoculated in a diameter of 25 cm2 In bottle, 37 DEG C are put, cultivated in 5% CO2, the cell culture incubator of saturated humidity.Culture changes first liquid after 3 days, remove red blood cell, The marrow hemopoietic stem cells of suspension growth and other not adherent stem cells.Changed liquid 1 time, cell growth every 3 days later When 70%～80% fusion, 2～3min is digested with the ml of 0.25% trypsase+0.03%EDTA 3, with containing 10% FBS and double Anti- DMEM/F12 terminates digestion, and bottle wall cell is blown and beaten repeatedly, makes single cell suspension by 1:3 pass on, and inverted microscope is day by day Observation, until attached cell is fusion together when being paved with bottom of bottle, repeats aforesaid operations, amplification is passed on repeatedly, collection.

The HSCs of step 4, culture suspension growth：Due to HSCs suspension growths in vitro, and need what stroma cell was supported Growing environment, therefore during step 3 culture BMMSCs, when liquid is changed for the second time, discard the whole supernatants in cell bottle Liquid, adds by the cell suspension obtained by step 1,2.With the DMEM/F12 nutrient solutions containing 10% FBS and dual anti-(mycillin) Adjustment cell density is 1 × 10⁶/ ml, draws 5 ml cell suspensions and is directly inoculated in above adherent layer.Hereafter per 6 days Half amount changes liquid, i.e., 2.5 ml suspension cultures are taken out from blake bottle, and 2000r/min is centrifuged 20min, collects cell precipitation As HSCs.Then 2.5 ml fresh mediums are added, is continued culture by this method and is gone down, the growth of inverted microscope observation of cell Situation, when adherent BMMSCs grows to 80% fusion, can simultaneously collect the HSCs of suspension growth and adherent growth BMMSCs。

Step 5, cellular identification：(1) Morphological Identification：The BMMSCs that adherent growth is observed under inverted microscope is in spindle shape Or polygonal fibrocyte shape, the HSCs of suspension growth is rounded, and cell light, refractivity is good.(2) flow cytometry mirror It is fixed：The BMMSCs and HSCs of culture are taken respectively, 300 l are made with PBS and contains 2.5 × 10⁶Individual single cell suspension；Dispense to 6 In the Eppendof pipes of 1.5ml, 50 l sheep blood serums closing 30min is separately added into；Be separately added into again the anti-CD14 in mouse source one, CD34, CD44, CD11a, HLA2DR and each 5 l of control PBS, mix, and react 30min；PBS centrifuge washings 2 times, remove unmarked On antibody.Each effective PBS is configured to 99 l cell suspensions；Often pipe adds 1 l FITC marks goat anti-mouse IgG (H+L) Two resist, and mix, lucifuge reaction 15min at 37 DEG C；PBS centrifuge washings 2 times；Often pipe adds 1ml PBS and is suspended again cell, uses The expression of flow cytomery cell surface antigen CD14, CD34, CD44, CD11a and HLA2DR.BMMSCs strongly expressed CD44 (mescenchymal stem cell specific molecular antigen), HSCs strongly expressed CD34 (candidate stem cell specific molecular antigen).

The frozen and recovery of step 6, BMMSCs and HSCs：(1) it is frozen：The every BMMSCs and HSCs in generation, except for continuing Cultivate and carry out related experiment all outer, it is all frozen.Cells frozen storing liquid：10% DMSO, 40% FBS, 50% DMEM/F12 are tieed up It is 1 × 10 to hold nutrient solution adjustment cell concentration⁶/ ml, in adding aseptic cell cryopreservation tube, the every ml of pipe 1.5, application program drop Warm instrument is frozen in liquid nitrogen；(2) recover：It is rapid from liquid nitrogen to take out cell cryopreservation tube, 37 DEG C of water baths are put immediately into, gently Shake makes it melt as early as possible, takes out cryopreservation tube, is opened with after 75% ethanol disinfection, suctions out cell suspension, injects centrifuge tube, rotating speed 2000r/min, is centrifuged 20min, removes supernatant, uses resuspended thin containing 10% FBS and dual anti-DMEM/F12 nutrient solution nutrient solutions Born of the same parents are precipitated, and put 37 DEG C of temperature, 5% CO₂, saturated humidity cell culture incubator in cultivate.

Claims (1)

1. a kind of method for being separately cultured dog mesenchymal stem cells MSCs and multi-functional candidate stem cell, it is characterised in that separating On the basis of BMNC, while adhere-wall culture primary mescenchymal stem cell, liquid is changed using half amount, added single The method of nucleus culture suspension, i.e., the hematopoieticmicroenviron-ment culture for being provided using the mescenchymal stem cell of adherent growth is suspended and is given birth to Long multi-functional candidate stem cell, so as to reach while being separately cultured dog mesenchymal stem cells MSCs and multi-functional candidate stem cell Purpose, concrete operation step is as follows：

The in-vitro separation of step 1, BMMSCs and HSCs：General anaesthesia experimental dog, posterior superior iliac spine preserved skin, sterilization, drape, with containing The common bone marrow extraction about 10ml of 2～3 points of the marrow puncture needle of 1ml liquaemins point, mixing is anti-condensation, and 50ml sterilization centrifugations are proceeded to rapidly Pipe, is mixed with the dilution of equivalent DMEM/F12；Take the centrifuge tube that 10 bottoms add 5ml lymphocyte separation mediums, the lymph of addition Cell separation liquid concentration is 1.077 g/ml, and the adherent inflow equivalent marrow dilution of upper strata difference, sealing controls 4 DEG C of temperature, from Scheming rotating speed is 2000 r/min, and centrifugation 20min removes upper-layer fat cell, draws middle intersection tunica albuginea confluent monolayer cells, then Washing interlayer cell is mixed with 4 DEG C of 10 ml DMEM/F12,20min, rotating speed 2000r/min are centrifuged again, remove supernatant Liquid, the above-mentioned washing interlayer cell step 1 time of repetition；With containing 15% FBS, 100 U/ml penicillin, 100 U/ml streptomysins The resuspended deposits of DMEM/F12 culture medium 5ml；

Step 2, determination of trypan blue staining cell viability：0.4g trypan blues are taken, in adding the 0.1 M PBS solutions of 100 ml, is matched somebody with somebody Make 0.4% trypan blue solution, 0.1M PBS solutions formula：NaCl 8g, KCl 0.2g, Na₂HPO₄.12H₂O 3.488g, KH₂PO₄0.2g, plus three and boils off ionized water to 1000 ml, and adjustment pH value is that 7.4,0.22 m membrane filtrations are degerming；

Take the ml of 0.4% trypan blue solution, 0.5 ml, DMEM/F12 culture medium 0.3 and the ml of cell suspension 0.2 is fully mixed, not Pigmented cells are living cells, and pigmented cells are dead cell, with blood counting chamber living cell counting and dead cell, number 500 altogether Cell, calculates cell viability, cell viability (%)=(TCS-pigmented cells number)/TCS × 100%；Such as platform Expect blue staining cell vigor >=95%, then can carry out next step operation, otherwise repeat step 1；

The adherent BMMSCs of step 3, culture：With containing 15% FBS, 100 U/ml penicillin, 100 U/ml chains when cultivating first The DMEM/F12 nutrient solutions adjustment cell density of mycin, with 1 × 10⁶The nutrient solution of the ml of/ml cell densities 5 is inoculated in 25 cm²'s In disposable Tissue Culture Flask, 37 DEG C of temperature, 5% CO are placed in₂, saturated humidity cell culture incubator in cultivate, it is first after culture 3 days It is secondary to change liquid, red blood cell, the marrow hemopoietic stem cells of suspension growth and other not adherent stem cells are removed, later every 3 It changes liquid 1 time, that is, renew the fresh cultures of the DMEM/F12 containing 15% FBS, 100 U/ml penicillin, 100 U/ml streptomysins Liquid, when cell growth is to 70%～80% fusion, with 0.25% 2～3min of Trypsin Induced, with containing 10% FBS and dual anti- DMEM/F12 terminates digestion, and bottle wall cell is blown and beaten repeatedly, makes single cell suspension by 1:3 pass on, and inverted microscope is observed day by day, When being paved with bottom of bottle up to attached cell is fusion together, repeat aforesaid operations, amplification is passed on repeatedly, collect；

The HSCs of step 4, culture suspension growth：Due to HSCs suspension growths in vitro, and need the growth that stroma cell supports Environment, therefore during step 3 culture BMMSCs, when liquid is changed for the second time, the whole supernatants in cell bottle are discarded, plus Enter by the cell suspension obtained by step 1,2, be 1 with cell density is adjusted containing 10% FBS and dual anti-DMEM/F12 nutrient solutions ×10⁶/ ml, draws 5 ml cell suspensions and is directly inoculated in above adherent layer, hereafter changes liquid per 6 days half amounts, i.e., from training 2.5 ml suspension cultures are taken out in foster bottle, 20min, rotating speed 2000r/min is centrifuged, collected cell precipitation and be HSCs, so Add afterwards 2.5 ml containing 10% FBS and dual anti-DMEM/F12 nutrient solutions, continue culture by this method and go down, inverted microscope Observation of cell growing state, when adherent BMMSCs grows to 80% fusion, can simultaneously collect the HSCs and note of suspension growth The BMMSCs of wall growth；

Step 5, cellular identification：(1) Morphological Identification：The BMMSCs of adherent growth is observed under inverted microscope in spindle shape or Polygonal fibrocyte shape, the HSCs of suspension growth is rounded, and cell light, refractivity is good；(2) flow cytometry identification： The BMMSCs and HSCs of culture are taken respectively, and the single cell suspension that 300 l contain 2.5 × 106 is made with PBS；Dispense to 6 In the Eppendof pipes of 1.5ml, 50 l sheep blood serums closing 30min is separately added into；Be separately added into again the anti-CD14 in mouse source one, CD34, CD44, CD11a, HLA2DR and each 5 l of control PBS, mix, and react 30min；PBS centrifuge washings 2 times, remove on unmarked Antibody；Each effective PBS is configured to 99 l cell suspensions；Often pipe adds 1 l FITC marks goat anti-mouse IgG (H+L) two It is anti-, mix, lucifuge reaction 15min at 37 DEG C；PBS centrifuge washings 2 times；Often pipe adds 1ml PBS and is suspended again cell, with stream Formula cell instrument detects the expression of cell surface antigen CD14, CD34, CD44, CD11a and HLA2DR；BMMSCs strongly expressed CD44, HSCs strongly expressed CD34；

The frozen and recovery of step 6, BMMSCs and HSCs：(1) it is frozen：The every BMMSCs and HSCs in generation, except for Continuous education It is all outer with related experiment is carried out, it is all frozen；

Cells frozen storing liquid：It is 1 × 10 that 10% DMSO, 40% FBS, 50% DMEM/F12 maintain nutrient solution adjustment cell concentration⁶/ ml, In adding aseptic cell cryopreservation tube, the every ml of pipe 1.5, application program cooling instrument is frozen in liquid nitrogen；(2) recover：From liquid nitrogen In it is rapid take out cell cryopreservation tube, be put immediately into 37 DEG C of water baths, be shaken gently for making it melt as early as possible, take out cryopreservation tube, use 75% Open after ethanol disinfection, suction out cell suspension, inject centrifuge tube, rotating speed 2000r/min is centrifuged 20min, removes supernatant, uses Containing 10% FBS and dual anti-DMEM/F12 nutrient solution nutrient solutions re-suspended cell precipitation, 37 DEG C of temperature, 5% CO are put₂, saturated humidity Cell culture incubator in cultivate.