CN105505858A - Separation and purification method for very small embryonic-like stem cells (VSELs) of pigs - Google Patents
Separation and purification method for very small embryonic-like stem cells (VSELs) of pigs Download PDFInfo
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Abstract
The invention discloses a rapid and effective separation and purification method for very small embryonic-like stem cells (VSELs). The method comprises the following steps: (1) cracking erythrocytes of pig bone marrow, and then, subjecting the cracked erythrocytes to resuspension cell separation by an FBS containing RPMI 1640 culture medium so as to obtain total single karyocytes of the pig bone marrow; (2) adding FcR confining liquid, coupling immune magnetic beads of an antibody CD133 or Lin, carrying out column separation, so as to obtain a CD133 (+) or Lin (-) cell mass of the pig bone marrow; (3) adopting a CD133, CD45 and Lin antibody labeling flow cell sorting technique, so as to obtain CD133 (+) Lin (-) CD45 (-) very small embryonic-like stem cells of the pig bone marrow. The method has the advantages of high efficiency, high purity, high stability and the like, the VSELs of artiodactyl pigs are separated and purified for the first time, the existing stem cell bank is enriched, the further research on the VSELs of large-sized artiodactyls is facilitated, and thus the VSELs can be closer to clinical application.
Description
Technical field
The present invention relates to the fields such as Stem Cell Engineering, organizational project and biological pace-making, particularly relate to minimum embryonic-like stem cell isolation and purification field.
Background technology
Minimum embryonic-like stem cell (verysmallembryonic-likestemcells, VSELs) is that a class volume is very tiny, quantity few (accounting for the 0.01%-0.04% of BMNC), have the non-hematopoietic stem cell of versatility.VSELs was successfully separated in 2006 by people such as Kentucky, USA University of Louisville MariuszRatajczak and is named from bone marrow mononuclear cells, and cell phenotype is Sca-1(+) lin(-) CD45(-), diameter 2-4 μm.This team is studied in the tissues such as finder's marrow, Cord blood, peripheral blood, brain, cardiac muscle, kidney, pancreas further to be existed equally, and cell phenotype is SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin-/CD45-, and diameter is 4-6 μm.The biological characteristics of people and mouse VSELs mainly comprises: large compared with thrombocyte, red corpuscle is little, nucleus is large, containing euchromatin, express primitive multipotent stem cells mark Oct-4 and Nanog, there is many differentiation potentials characteristic, have similarity with embryonic stem cell (ESCs) or the embryonic-like stem cell (iPSs) of inducing, the cell of all three kinds of embryonal systems can be divided in vivo as hemocyte, osteocyte, myocyte and neurocyte etc.This cell is relevant with aging, and its content can further reduce with advancing age, and in vitro single culture when be difficult to amplification.Because VSELs can to three differentiation of germinal layers, and without immunity rejection, without ethics problem, be considered to have the potentiality of alternative embryonic stem cell, it is paid attention to deeply in scientific circles.Show that cyclical level increases greatly in the rat animal model of myocardial infarction and myocardial infarction and paralytic, also demonstrates VSELs and repairs injury of myocardium, bone, pancreas islet, nervous tissue and immune validity in vivo.Possessor infers that VSELs likely changes regenerative medicine to the greatest extent, but in view of the Adult multipotent stem cells that VSELs is very rare in body, be separated, culture technique is complicated, and as California Stanford University Weissman, the professors such as the Dulak of Ya Jielong university of Poland repeat experiment and extensive analysis according to the initial described method of Ratajczak team, but cannot from the marrow of mouse, minimum embryonic-like stem cell is separated in the Cord blood of people, or be less than in the mouse medullary cell of 7 microns at diameter, fail to find the molecular label relevant with versatility, its existence of numerous and confused query and value, therefore, how improving isolation and purification technology is urgent problem in VSELs research.
Because VSELs volume is little, quantity is few and adherent ability is weak, the cell yield that common marrow non-hematopoietic stem cell separation method and adherent partition method and density gradient centrifugation obtain is quite low, therefore is not suitable for the separation of VSELs.Though cells by red blood cell lysis method, fluidic cell separating method or immunological magnetic bead sorting method may be used for being separated of people and mouse VSELs, a kind of independent method all can not reach promising result, and adopt immunomagnetic beads method merely, cell is numerous and diverse, and purity is inadequate; Simple employing airflow classification especially processes the target cell that a large amount of cell sample need reach some amount, once must 24-36 hour, easy damaged and contamination of cells.In addition, up to now, the isolated or purified report of large-scale artiodactyl VSELs is still lacked.
Summary of the invention
Prepare for current minimum embryonic-like stem cell that difficulty is large, the cycle is long, efficiency is low, purity is not high, and above-mentioned any one method all can not reach satisfied effect, the invention provides a boar minimum embryonic-like stem cell isolation and purification method, comprise the following steps:
(1) the total mononuclearcell of Medulla Sus domestica is isolated through the RMPI1640 substratum re-suspended cell containing FBS after Medulla Sus domestica liquid erythrocyte splitting;
(2) according to 1 × 10
8/ 100ul cell concn adds FcR confining liquid, coupling CD133 antibody or the immunomagnetic beads of Lin antibody, crosses post sorting, obtains Medulla Sus domestica CD133(+) or Lin(-) cell mass;
(3) Flow cytometry of CD133, CD45, Lin antibody labeling is adopted to obtain Medulla Sus domestica CD133 (+) Lin (-) CD45 (-) minimum embryonic-like stem cell.
The present invention is by cells by red blood cell lysis method, immunological magnetic bead sorting method (MACS) and Flow cytometry (FACS) three road program scientific combination, 50ml Medulla Sus domestica liquid disengaging time foreshortens to 4-6 hour, and purity reaches more than 95%, cell yield is high, cell quantity reaches 1 × 10
5above.3-4 is improved doubly with employing cells by red blood cell lysis method and classical fluidic cell sorting (publication number 103396988A) phase specific efficiency, simple employing airflow classification is as reached the target cell of some amount level, each palpus 24-36 hour, length consuming time, cell damages in a large number and a small amount of VSELs has likely been divided into comparatively mature cell; Purity significantly increases compared with a certain monoclonal antibody magnetic bead sorting method (publication number 102229910A) of employing.The separation of the efficient VSELs that invention proposes, method have huge application prospect, and the clinical study for VSELs provides important technical guarantee.
Preferred as present method, cells by red blood cell lysis method is adopted to be separated the total mononuclearcell of Medulla Sus domestica, first of the right age pig anesthesia is fixed in cleaning stations, in pig posterior superior iliac spine or the capable bone marrow puncture of anterior superior spine under aseptic technique, obtain q.s and the marrow of abundant heparinization, centrifugal degrease, adds erythrocyte cracked liquid, absorb supernatant liquor, add 0.02%EDTA at 4 DEG C and wash to obtain cell suspension.With the RPMI1640 substratum re-suspended cell containing 2%FBS, blow and beat gently, centrifugal 10 minutes of 500rpm at 4 DEG C.With 2ml containing the RPMI1640 substratum re-suspended cell of 2%FBS, be separated after 40um strainer filtering, so centrifugal, washing, filter, remove fragment after obtain total monocyte.
Preferred as present method, selected is the small-sized adult pig at 2-4 monthly age for the of the right age pig of anaesthetizing, body weight (35 ± 5kg), male and female are not limit, thered is provided by Yangzhou agriculture university Experimental Animal Center, all laboratory animal are all subject to humanity and treat, and meet " management of laboratory animal and instruction manual " that NIH promulgates.Experimental program obtains laboratory animal Ethics Committee of Yangzhou University and the approval of Ethics Committee of northern Suzhou, Jiangsu Province the People's Hospital.
Preferred as present method, described acquisition Medulla Sus domestica CD133(+) cell mass, step comprises:
(1) FcR non-specific adsorption agent closing cell is added
By centrifugal 10 minutes of 500rpm at total mononuclearcell suspension 4 DEG C of above-mentioned separation, according to 1 × 10
8/ 300ul cell concn adds damping fluid re-suspended cell, according to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) CD133 antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds the immunomagnetic beads labeled cell of coupling CD133 antibody.Abundant mixing, hatches 30 minutes for 2 DEG C ~ 8 DEG C.According to 1 × 10
8/ 2ml cell concn adds buffer solution for cleaning cell, centrifugal 10 minutes of 4 DEG C of 500rpm.According to 1 × 10 after careful absorption supernatant liquor
8/ 500ul cell concn adds damping fluid re-suspended cell;
(3) cell is crossed post immunological magnetic bead sorting and is obtained CD133(+) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL injection sorting post to infiltrate, sorting post fully infiltrates rear injection 1ml cell suspension, after cell suspension flows to end, add PBS damping fluid 1ml again to rinse from separator column upper end, access the cell suspension of outflow by 15ml sterile tube, the cell that in sorting post, magnetic bead combines is CD133(+) cell.
Preferred as present method, described acquisition Medulla Sus domestica Lin(-) cell mass, step comprises:
(1) FcR non-specific adsorption agent closing cell is added
By total mononuclearcell suspension of above-mentioned separation, centrifugal 10 minutes of 500rpm at 4 DEG C, according to 1 × 10
8/ 400ul cell concn adds damping fluid re-suspended cell.According to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) Lin antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds the immunomagnetic beads of coupling Lin antibody, labeled cell.Abundant mixing, hatches 30 minutes for 2 DEG C ~ 8 DEG C.According to 1 × 10
8/ 5ml cell concn adds buffer solution for cleaning cell, centrifugal 10 minutes of 4 DEG C of 500rpm.According to 1 × 10 after careful absorption supernatant liquor
8/ 500ul cell concn adds damping fluid re-suspended cell;
(3) cell is crossed post immunological magnetic bead sorting and is obtained Lin(-) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL and infiltrate sorting post, inject 1ml cell suspension, cell suspension crosses post post-flush sorting post three times, add 1ml damping fluid, access the cell suspension of outflow by 15ml sterile tube, in sterile tube, cell suspension is Lin(-at every turn) cell mass.
Preferred as present method, adopts flow cytometry sorting to obtain the minimum embryonic-like stem cell of Medulla Sus domestica, with 10
7/ 100uL cell concn adds CD133 mark, CD45 mark, Lin mark and CD133/CD45/Lin mark respectively, the microballon (1,2,4,6,10 and 15 μm) of size will be determined, run in order on flow cytometer, namely can sub-elect the minimum embryonic-like stem cell that CD133 (+) Lin (-) CD45 (-) is qualified.The VSELs obtained after airflow classification is added in the culture plate without feeder layer cells, is placed in 37 DEG C, 5%CO
2incubator in, add dual anti-(green grass or young crops-Streptomycin sulphate storage liquid), the RPMI1640 conditioned medium of the cytokines such as LIF, bFGF, SCF is cultivated simultaneously.
As present method further preferably, described erythrocyte cracked liquid is mixed according to volume ratio 1:10 ratio by FACS hemolysin and ultrapure water, and use 0.22um filtering with microporous membrane is degerming, and 4 DEG C of preservations, are preheated to room temperature during use.
As present method further preferably, Na2HPO412H2O1.4425g got by described PBS damping fluid (not containing Ca, Mg), KH2PO40.1g, NaCl4g, KCl0.1g, be dissolved in the freshly prepd ultrapure water of 500mL, adjust PH to 7.2 after constant volume, after autoclaving, preserve in 4 DEG C of refrigerators.
Further preferential as present method, described 0.02%EDTA is dissolved in by 0.02gEDTA in 100mLPBS damping fluid, uses 0.22um filtering with microporous membrane degerming, be placed in after packing in-20 DEG C of refrigerators for subsequent use after PH being adjusted to 7.2.
As present method further preferably, the immunomagnetic beads of CD133 antibody is CD133MicroBeadKit-HematopoieticTissue.
As present method further preferably, the immunomagnetic beads of Lin antibody is LineageCellDepletionkit.
As present method further preferably, fluorescein-labeled antibody stoste is PerCP-Cy5.5-anti-CD45 monoclonal antibody, anti-Lin-FITC monoclonal antibody and anti-CD133/2PE monoclonal antibody.
As present method further preferably, described dual anti-(green grass or young crops-Streptomycin sulphate storage liquid): get in each 1,000,000 molten 100mLPBS damping fluids of unit of penicillin and streptomycin.
For implementing present method, the major experimental instrument related to, comprises Bechtop, CO
2constant incubator, inverted fluorescence microscope, thermostat water bath, micropipette rifle, vortex oscillation instrument, refrigerator, electronic balance ice-making machine, ultrapure water apparatus, immunological magnetic bead sorting instrument, flow cytometer, 5810R type low-temperature and high-speed whizzer.
For implementing present method, the major experimental apparatus related to, comprises (1) instruments: bone marrow aspiration bag, 1mL, 5mL, 20mL syringe; (2) cell cultures related experiment equipment: T25 Tissue Culture Flask, 6 holes, 24 holes and 96 porocyte culture plates, 15mL and 50mL centrifuge tube, 0.22um filter membrane and plastics filter, 40um filter screen, beaker, graduated cylinder, cell counting count board etc.
Superiority of the present invention is:
(1) by increasing cell purity, improve isolated cell efficiency, for people provide a kind of VSELs separation purification method efficiently, simultaneously, also be the VSELs isolating artiodactyl pig first, its biological characteristics and people and muroid seemingly, are conducive to studying further large-scale artiodactyl VSELs, make it closer to clinical application;
(2) preparation cycle is short, and preparation efficiency is high, and by cells by red blood cell lysis method, MACS and FACS tri-road program scientific combination, 50ml Medulla Sus domestica liquid disengaging time foreshortens to 4-6 hour;
(3) method is practical, easy, repeatable strong;
(4) reliability obviously increases, and cell yield is high, cell quantity reaches 1 × 10
5above;
(5) separation purity significantly increases, and purity reaches more than 95%, and adopts a kind of immunomagnetic beads method of monoclonal antibody bag quilt, and cell is numerous and diverse, and purity is inadequate;
(6) for the preparation of other tissue of artiodactyls as VSELs such as peripheral blood, brain, cardiac muscle, kidney, pancreas provides new method, also provide method and access for the follow-up study of artiodactyls VSELs cell simultaneously;
(7) this technology is also applicable to the preparation of other species as VSELs such as mouse, people, birds, can be used for the research and development of precious species.
Accompanying drawing explanation
Fig. 1 shows the abstraction and purification schematic diagram of Medulla Sus domestica VSELs.
Fig. 2 adopts the positive separation results of CD133.
Fig. 3 adopts the negative separation results of Lin.
Fig. 4 is through immunological magnetic bead sorting Lin (-) cell.
Fig. 5 airflow classification result.
After Fig. 6 inverted fluorescence microscope observes airflow classification, RPMI1640 conditioned medium cultivates Medulla Sus domestica VSELs after 2 days.Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further details.
Figure 1 shows that the operating process schematic diagram of pig minimum embryonic-like stem cell isolation and purification method, wherein show cells by red blood cell lysis method, immunological magnetic bead sorting method and Flow cytometry three road program successively, the combination of this three roads program can significantly improve separation, the purification efficiency of pig VSELs.Embodiment is as follows.
One. total mononuclearcell is separated
(1) anesthesia of pig
Choose 3-4 monthly age, the small-sized adult pig of 35 ± 5kg, after gluteus maximus muscle place 10cm scope preserved skin, adopt 1% iodophor solution to sterilize 3 times, give Su Mian Xin II and the appropriate intramuscular injection of diazepam, after 20 minutes when pig is anaesthetized, be fixed on the clean operator's console in animal surgery room;
(2) bone marrow aspiration
With after ilium or anterior superior spine for point of puncture, with containing the asepsis injector bone marrow extraction 50mL of heparin water in the sterile test tube of anti-freezing in advance, and repeatedly shake test tube with Anti-solidification;
(3) fat and foam is removed
Bone marrow fluid average mark is loaded in four 50mL centrifuge tubes, centrifugal 10 minutes of 1000rpm under 4 DEG C of conditions;
(4) erythrocyte splitting
Mixed according to volume ratio 1:10 ratio by FACS hemolysin and ultrapure water, use 0.22um filtering with microporous membrane degerming, make erythrocyte cracked liquid, 4 DEG C of preservations, are preheated to room temperature during use, add erythrocyte cracked liquid according to the ratio of erythrocyte cracked liquid and bone marrow fluid 4:1, abundant mixing, incubated at room temperature 10 minutes, at 4 DEG C, centrifugal 10 minutes of 500rpm, slowly absorbs supernatant liquor.Again add lysate re-suspended cell according to lysate and bone marrow fluid 2:1 ratio again, incubated at room 10 minutes, at 4 DEG C, centrifugal 10 minutes of 500rpm, carefully absorbs supernatant liquor, adds 0.02%EDTA and wash to obtain cell suspension at 4 DEG C;
0.02gEDTA is dissolved in 100mLPBS damping fluid by the 0.02%EDTA added, and uses 0.22um filtering with microporous membrane degerming, be placed in after packing in-20 DEG C of environment, use front taking-up after PH being adjusted to 7.2;
(5) separation of total mononuclearcell
With the RPMI1640 substratum re-suspended cell of 30mL containing 2%FBS, blow and beat gently, centrifugal 10 minutes of 500rpm at 4 DEG C; With the RPMI1640 substratum re-suspended cell of 2ml containing 2%FBS, after 40um strainer filtering, count for subsequent use.
Two, immunological magnetic bead sorting CD133(+) or Lin(-) cell mass
(1) immunomagnetic beads positive sorting CD133 cell mass
(1) FcR non-specific adsorption agent closing cell is added
By centrifugal 10 minutes of 500rpm at total mononuclearcell suspension 4 DEG C of above-mentioned separation, according to 1 × 10
8/ 300ul cell concn adds damping fluid re-suspended cell.According to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) CD133 antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds the immunomagnetic beads labeled cell of coupling CD133 antibody; Hatch 30 minutes, according to 1 × 10 for 2 DEG C ~ 8 DEG C
8/ 2ml cell concn adds PBS buffer solution for cleaning cell, centrifugal 10 minutes of 4 DEG C of 500rpm.According to 1 × 10
8/ 500ul cell concn adds damping fluid re-suspended cell; The immunomagnetic beads of described CD133 antibody is CD133MicroBeadKit-HematopoieticTissue;
(3) cell is crossed post immunological magnetic bead sorting and is obtained CD133(+) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL injection sorting post to infiltrate, sorting post fully infiltrates rear injection 1ml cell suspension, after cell suspension flows to end, add PBS damping fluid 1ml again to rinse from separator column upper end, access the cell suspension of outflow by 15ml sterile tube, sorting post inner cell is CD133(+) cell;
Described PBS damping fluid (not containing Ca, Mg), be get Na2HPO412H2O1.4425g, KH2PO40.1g, NaCl4g, KCl0.1g, be dissolved in the freshly prepd ultrapure water of 500mL, adjust PH to 7.2 after constant volume, after autoclaving, 4 DEG C of preservations.
(2) immunomagnetic beads negative sorting Lin cell mass
(1) FcR non-specific adsorption agent closing cell is added
By total mononuclearcell suspension of above-mentioned separation, centrifugal 10 minutes of 500rpm at 4 DEG C, according to 1 × 10
8/ 400ul cell concn adds damping fluid re-suspended cell.According to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) Lin antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds the immunomagnetic beads of coupling Lin antibody, labeled cell.Abundant mixing, hatches 30 minutes for 2 DEG C ~ 8 DEG C.According to 1 × 10
8/ 5ml cell concn adds 2%PBS and cleans cell, centrifugal 10 minutes of 4 DEG C of 500rpm.According to 1 × 10 after careful absorption supernatant liquor
8/ 500ul cell concn adds 2%PBS re-suspended cell; The immunomagnetic beads of described Lin antibody is LineageCellDepletionkit;
Figure 2 shows that cell quantity contrast before and after the positive sorting of CD133, Figure 3 shows that quantitative comparison before and after the negative sorting of Lin, Fig. 2,3 illustrates total mononuclearcell of equivalent, through the positive sorting of CD133 comparatively Lin negative sorting gained cell quantity significantly reduce, also namely the positive efficiency of separation of CD133 is higher;
(3) cell is crossed post immunological magnetic bead sorting and is obtained Lin(-) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL and infiltrate sorting post, inject 1ml cell suspension, cell suspension crosses post post-flush sorting post three times, add 1mlPBS, access the cell suspension of outflow by 15ml sterile tube, in sterile tube, cell suspension is Lin(-at every turn) cell mass.
Lin(-shown by Fig. 4) sorting acquisition cell quantity is more, but purity is inadequate.
Three, flow cytometry sorting obtains Medulla Sus domestica VSELs
(1) mark of cell
Determine the cell quantity in cell suspension, with 300rpm centrifugal 10 minutes, abandoning supernatant.Cell suspension is divided five groups: blank group, CD133 mark group, CD45 mark group, Lin mark group, CD133/CD45/Lin mark group.Every 10
7individual cell adds 100ul damping fluid and the fluorescein-labeled antibody stoste of 10ul, and piping and druming fully mixing gently, lucifuge is placed in hatches 10 minutes on ice.The RPMI1640 substratum cleaning adding 3mL2%FBS often organizes sample, with 300rpm centrifugal 10 minutes.With substratum re-suspended cell, filtration, be placed in ice preserve, for subsequent use;
Described fluorescein-labeled antibody stoste is PerCP-Cy5.5-anti-CD45 monoclonal antibody, anti-Lin-FITC monoclonal antibody and anti-CD133/2PE monoclonal antibody;
(2) determination of bead size
Before flow sorted cells sample, run the microballon (the size calibration spheroid with 1,2,4,6,10 and 15 μm of normal diameter) pre-determining size, the each 200ul of microballon drawing 5 kinds of diameters is placed in the aseptic EP pipe of 5 1.5ml respectively, run in order on flow cytometer, all objects that adjustment threshold value enables machine find between 2-10 μm;
(3) airflow classification
The high purity of selection standard divides lectotype, five groups of cells are run on flow cytometer in order, record CD133 (+), Lin (-), CD45 (-) cell subsets proportion in total cell respectively, screen and collect CD133 (+) Lin (-) CD45 (-) i.e. VSELs cell, during sorting, not with too fast separation velocity, to ensure the restorability that the cell of institute's sorting is higher and purity;
The VSELs obtained after airflow classification is added in the culture plate without feeder layer cells, is placed in 37 DEG C, 5%CO
2incubator in, add dual anti-(green grass or young crops-Streptomycin sulphate storage liquid), the cytokines such as LIF, bFGF, SCF are cultivated in RPMI1640 conditioned medium simultaneously;
Described dual anti-(green grass or young crops-Streptomycin sulphate storage liquid) refers to that getting each 1,000,000 units of penicillin and streptomycin is dissolved in 100mLPBS damping fluid and is made.
Shown in Fig. 5: A figure is that blank group P1 region shows 2-10 μm of cell mass, B figure is that experimental group P1 region shows 2-10 μm of cell mass, C figure is that blank group P2 region does not show positive cell, D figure is that experimental group P2 region shows CD133 (+) CD45 (-) cell, positive rate is 2.3%, E figure is that blank group P3 region does not show positive cell, F is that experimental group P3 region shows CD133 (+) Lin (-) CD45 (-) cell, and positive rate is 1.2%; Shown in Fig. 6: A figure cultivates the 2nd day result (× 100) after Medulla Sus domestica VSELs sorting, B figure cultivates the 2nd day result (× 400) after Medulla Sus domestica VSELs sorting.
In order to implement present method, the major experimental instrument that need possess comprises: Bechtop, CO
2constant incubator, inverted fluorescence microscope, thermostat water bath, micropipette rifle, vortex oscillation instrument, refrigerator, electronic balance ice-making machine, ultrapure water apparatus, immunological magnetic bead sorting instrument, flow cytometer, 5810R type low-temperature and high-speed whizzer.
In order to implement present method, the major experimental apparatus that must possess comprises, (1) instruments: bone marrow aspiration bag, 1mL, 5mL, 20mL syringe; (2) cell cultures related experiment equipment: T25 Tissue Culture Flask, 6 holes, 24 holes and 96 porocyte culture plates, 15mL and 50mL centrifuge tube, 0.22um filter membrane and plastics filter, 40um filter screen, beaker, graduated cylinder, cell counting count board.
The foregoing is only main embodiment of the present invention, all impartial improvements and modifications done according to the present patent application the scope of the claims also should be considered as the covering scope of patent of the present invention.
Claims (14)
1. the isolation and purification method of the minimum embryonic-like stem cell of a boar, is characterized in that, comprise the steps:
(1) the total mononuclearcell of Medulla Sus domestica is isolated through the RPMI1640 substratum re-suspended cell containing FBS after Medulla Sus domestica liquid erythrocyte splitting;
(2) according to 1 × 10
8/ 100ul cell concn adds FcR confining liquid, coupling CD133 antibody or the immunomagnetic beads of Lin antibody, crosses post sorting, obtains Medulla Sus domestica CD133(+) or Lin(-) cell mass;
(3) Flow cytometry of CD133, CD45, Lin antibody labeling is adopted to obtain Medulla Sus domestica CD133 (+) Lin (-) CD45 (-) minimum embryonic-like stem cell.
2. the isolation and purification method of the minimum embryonic-like stem cell of pig according to claim 1, is characterized in that, described isolates the total mononuclearcell of Medulla Sus domestica, and step comprises:
(1) anesthesia of pig
Choose 2-4 monthly age, the small-sized adult pig of body weight (35 ± 5kg), to give after Su Mian Xin II and the appropriate intramuscular injection of diazepam 20 minutes, when pig is in appropriateness anesthesia, be fixed on the clean operator's console in animal surgery room;
(2) bone marrow aspiration
With after ilium or anterior superior spine for point of puncture, 10cm scope preserved skin around this point, then 1% iodophor disinfection three times are used, bone marrow aspiration is carried out after wearing sterile gown, band sterile gloves, with the asepsis injector bone marrow extraction 50mL containing heparin water in the sterile test tube of anti-freezing in advance, and repeatedly shake test tube in case bone marrow fluid solidifies;
(3) fat and foam is removed
Be loaded on by bone marrow fluid average mark in four 50mL centrifuge tubes, under 4 DEG C of conditions, 1000rpm removes fat and foam in centrifugal 10 minutes;
(4) erythrocyte splitting
Add erythrocyte cracked liquid according to the ratio of erythrocyte cracked liquid and bone marrow fluid 4:1, rotating centrifugal pipe is fully to mix gently, incubated at room temperature 10 minutes, and at 4 DEG C, centrifugal 10 minutes of 500rpm, slowly absorbs supernatant liquor; Again add lysate re-suspended cell according to lysate and bone marrow fluid 2:1 ratio, incubated at room 10 minutes, centrifugal 10 minutes of 500rpm at 4 DEG C, absorb supernatant liquor;
Add 0.02%EDTA at 4 DEG C and wash to obtain cell suspension;
(5) separation of total mononuclearcell
With the RPMI1640 substratum re-suspended cell of 30mL containing 2%FBS, blow and beat gently, centrifugal 10 minutes of 500rpm at 4 DEG C, with the RPMI1640 substratum re-suspended cell of 2ml containing 2%FBS, after 40um strainer filtering, for subsequent use with cell counting count board counting.
3. the isolation and purification method of the minimum embryonic-like stem cell of pig according to claim 1, is characterized in that, described acquisition Medulla Sus domestica CD133(+) cell mass, step comprises:
(1) FcR non-specific adsorption agent closing cell is added
By centrifugal 10 minutes of 500rpm at total mononuclearcell suspension 4 DEG C of above-mentioned separation, according to 1 × 10
8/ 300ul cell concn adds damping fluid re-suspended cell, according to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) CD133 antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds the immunomagnetic beads labeled cell of coupling CD133 antibody, fully mixes, hatches 30 minutes, according to 1 × 10 for 2 DEG C ~ 8 DEG C
8/ 2ml cell concn adds buffer solution for cleaning cell, centrifugal 10 minutes of 4 DEG C of 500rpm, carefully absorbs after supernatant liquor according to 1 × 10
8/ 500ul cell concn adds damping fluid re-suspended cell;
(3) cell is crossed post immunological magnetic bead sorting and is obtained CD133(+) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL injection sorting post to infiltrate, sorting post fully infiltrates rear injection 1ml cell suspension, after cell suspension flows to end, add PBS damping fluid 1ml again to rinse from separator column upper end, access the cell suspension of outflow by 15ml sterile tube, the cell that in sorting post, magnetic bead combines is CD133(+) cell.
4. the isolation and purification method of the minimum embryonic-like stem cell of pig according to claim 1, is characterized in that, described acquisition Medulla Sus domestica Lin(-) cell mass, step comprises:
(1) FcR non-specific adsorption agent closing cell is added
By total mononuclearcell suspension of above-mentioned separation, centrifugal 10 minutes of 500rpm at 4 DEG C, according to 1 × 10
8/ 400ul cell concn adds damping fluid re-suspended cell, according to 1 × 10
8/ 100ul cell concn adds FcR confining liquid;
(2) Lin antibody immune magnetic beads labeled cell is added
According to 1 × 10
8/ 100ul cell concn adds coupling Lin antibody immune magnetic beads labeled cell, fully mixes, hatches 30 minutes, according to 1 × 10 for 2 DEG C ~ 8 DEG C
8/ 5ml cell concn adds buffer solution for cleaning cell, centrifugal 10 minutes of 4 DEG C of 500rpm, absorbs after supernatant liquor according to 1 × 10
8/ 500ul cell concn adds damping fluid re-suspended cell;
(3) cell is crossed post immunological magnetic bead sorting and is obtained Lin(-) cell
Cell suspension is placed on immunological magnetic bead sorting instrument and crosses post, every 1ml cell suspension uses the sorting post of a MS model, draw PBS damping fluid 500uL and infiltrate sorting post, inject 1ml cell suspension, cell suspension crosses post post-flush sorting post three times, add 1ml damping fluid, access the cell suspension of outflow by 15ml sterile tube, in sterile tube, cell suspension is Lin(-at every turn) cell mass.
5. the isolation and purification method of the minimum embryonic-like stem cell of pig according to claim 1, is characterized in that, the described minimum embryonic-like stem cell of acquisition Medulla Sus domestica CD133 (+) Lin (-) CD45 (-), and step comprises:
(1) mark of cell
Determine the cell quantity in cell suspension, with 1000rpm centrifugal 10 minutes, abandoning supernatant, divides five groups: blank group by cell suspension, CD133 mark group, CD45 mark group, Lin mark group, CD133/CD45/Lin mark group;
Every 10
7individual cell adds 100ul damping fluid and the fluorescein-labeled antibody stoste of 10ul, piping and druming fully mixing gently, and lucifuge is placed in hatches 10 minutes on ice; Add the RPMI1640 substratum cleaning that 3mL contains the FBS of 2% and often organizes sample, with 300rpm centrifugal 10 minutes, use substratum re-suspended cell, filter, be placed in ice and preserve, for subsequent use;
(2) determination of bead size
Before flow sorted cells sample, run the microballon (the size calibration spheroid with 1,2,4,6,10 and 15 μm of normal diameter) pre-determining size, the each 200ul of microballon drawing 5 kinds of diameters is placed in the sterile tube of 5 1.5ml respectively, run in order on flow cytometer by five groups of cells, adjustment threshold value can find all objects between 2-10 μm;
(3) airflow classification
Record CD133 (+), Lin (-), CD45 (-), CD133 (+) Lin (-) CD45 (-) cell subsets proportion in total cell respectively, the high purity of selection standard divides lectotype, to ensure the restorability that the cell of institute's sorting is higher and purity, collect CD133 (+) Lin (-) CD45 (-) i.e. VSELs;
The VSELs obtained after airflow classification is added in the culture plate without feeder layer cells, is placed in 37 DEG C, 5%CO
2incubator in, add dual anti-(green grass or young crops-Streptomycin sulphate storage liquid), R/ minute I1640 conditioned medium of the cytokines such as LIF, bFGF, SCF is cultivated simultaneously.
6. the isolation and purification method of the minimum embryonic-like stem cell of pig as claimed in claim 2, it is characterized in that, described erythrocyte cracked liquid: mixed according to volume ratio 1:10 ratio by FACS hemolysin and ultrapure water, use 0.22um filtering with microporous membrane degerming, 4 DEG C of preservations, are preheated to room temperature during use.
7. the isolation and purification method of the minimum embryonic-like stem cell of pig as claimed in claim 2, it is characterized in that, described PBS damping fluid (not containing Ca, Mg): get Na2HPO412H2O1.4425g, KH2PO40.1g, NaCl4g, KCl0.1g, be dissolved in the freshly prepd ultrapure water of 500mL, PH to 7.2 is adjusted after constant volume, after autoclaving, 4 DEG C of preservations.
8. the isolation and purification method of the minimum embryonic-like stem cell of pig as claimed in claim 2, it is characterized in that, described 0.02%EDTA: 0.02gEDTA is dissolved in 100mLPBS damping fluid, use 0.22um filtering with microporous membrane degerming after PH being adjusted to 7.2, be placed in after packing in-20 DEG C of environment for subsequent use.
9. as right wants the isolation and purification method of the minimum embryonic-like stem cell of the pig as described in 3, it is characterized in that, the immunomagnetic beads of described CD133 antibody is CD133MicroBeadKit-HematopoieticTissue.
10. the isolation and purification method of the minimum embryonic-like stem cell of pig as claimed in claim 4, it is characterized in that, the immunomagnetic beads of described Lin antibody is LineageCellDepletionkit.
The isolation and purification method of the minimum embryonic-like stem cell of 11. pig as claimed in claim 5, it is characterized in that, described fluorescein-labeled antibody stoste is PerCP-Cy5.5-anti-CD45 monoclonal antibody, anti-Lin-FITC monoclonal antibody and anti-CD133/2PE monoclonal antibody.
The isolation and purification method of the minimum embryonic-like stem cell of 12. pig as claimed in claim 5, is characterized in that, described dual anti-(green grass or young crops-Streptomycin sulphate storage liquid): get each 1,000,000 units of penicillin and streptomycin and be dissolved in 100mLPBS damping fluid.
13. the method for claim 1, is characterized in that, implement the major experimental instrument that present method relates to, comprise Bechtop, CO
2constant incubator, inverted fluorescence microscope, thermostat water bath, micropipette rifle, vortex oscillation instrument, refrigerator, electronic balance ice-making machine, ultrapure water apparatus, immunological magnetic bead sorting instrument, flow cytometer, 5810R type low-temperature and high-speed whizzer.
The isolation and purification method of the minimum embryonic-like stem cell of 14. pig as claimed in claim 1, is characterized in that, implements the major experimental apparatus that present method relates to, comprises (1) instruments: bone marrow aspiration bag, 1mL, 5mL, 20mL syringe; (2) cell cultures related experiment equipment: T25 Tissue Culture Flask, 6 holes, 24 holes and 96 porocyte culture plates, 15mL and 50mL centrifuge tube, 0.22um filter membrane and plastics filter, 40um filter screen, beaker, graduated cylinder, cell counting count board.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047794A (en) * | 2016-06-23 | 2016-10-26 | 中国人民解放军第三军医大学第三附属医院 | Method for sorting senile renal tubular cells |
| CN108588009A (en) * | 2018-05-10 | 2018-09-28 | 广州四叶草健康科技有限公司 | A method of it detaches and activates the minimum embryonic-like stem cell of human peripheral |
| CN108753685A (en) * | 2018-06-20 | 2018-11-06 | 首都医科大学 | A kind of separation, screening, culture and the Function Identification method of the human aorta vascular wall stem cell of expression c-Kit |
| CN114574439A (en) * | 2022-01-29 | 2022-06-03 | 国药集团动物保健股份有限公司 | Preparation method of pig bone marrow cells and pig cancellous bone crushing device |
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2015
- 2015-12-10 CN CN201510909419.3A patent/CN105505858A/en active Pending
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| 孙加斌: "猪骨髓极小胚胎样干细胞的分离、鉴定及生物学特性探究", 《万方数据》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047794A (en) * | 2016-06-23 | 2016-10-26 | 中国人民解放军第三军医大学第三附属医院 | Method for sorting senile renal tubular cells |
| CN108588009A (en) * | 2018-05-10 | 2018-09-28 | 广州四叶草健康科技有限公司 | A method of it detaches and activates the minimum embryonic-like stem cell of human peripheral |
| CN108753685A (en) * | 2018-06-20 | 2018-11-06 | 首都医科大学 | A kind of separation, screening, culture and the Function Identification method of the human aorta vascular wall stem cell of expression c-Kit |
| CN108753685B (en) * | 2018-06-20 | 2022-06-28 | 首都医科大学 | Separation, screening, culture and function identification method of human aortic vessel wall stem cells expressing c-Kit |
| CN114574439A (en) * | 2022-01-29 | 2022-06-03 | 国药集团动物保健股份有限公司 | Preparation method of pig bone marrow cells and pig cancellous bone crushing device |
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