CN105505863A - Culture method for heterocephalus glaber cardiac muscle cell - Google Patents
Culture method for heterocephalus glaber cardiac muscle cell Download PDFInfo
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- CN105505863A CN105505863A CN201610005208.1A CN201610005208A CN105505863A CN 105505863 A CN105505863 A CN 105505863A CN 201610005208 A CN201610005208 A CN 201610005208A CN 105505863 A CN105505863 A CN 105505863A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a culture method for a heterocephalus glaber cardiac muscle cell. In the culture method, the operation steps of separating and purifying the heterocephalus glaber cardiac muscle cell are simple, cell attachment is fast, the survival rate is high, and a cell culture system is high in stability. The culture method is an ideal culture method for the heterocephalus glaber primary cardiac muscle cell, can basically meet the requirements for heterocephalus glaber cardiac muscle cell related experiment research in all research fields such as medical science, biology, pharmacy and bioscience and is suitable for application and popularization in general laboratories.
Description
Technical field
The invention belongs to technical field of cell culture, specifically, is the cultural method of a kind of naked mole myocardial cell.
Background technology
Naked mole (Heterocephalusglaber, Nakedmole-rat) be a kind of Novel experimental animal having researching value, it has lower oxygen concentration resistance, many peculiar biological characteristicses such as anti-ageing and antitumor, in the medical science such as presbyatrics, oncology, immunology and life science field, play significant role, there is irreplaceable status.Especially in lower oxygen concentration resistance research, for the researchs such as the cardiovascular disorder that anoxic is correlated with provide desirable Study of Support and scientific tools.Myocardial cell is the basic composition unit of heart, is the Major cellular sources carrying out the aspect researchs such as physiology, pharmacology, pathology, toxicity and energy metabolism, is also one of main source of cardiac muscle tissue engineering seed cell simultaneously.At present, because naked mole animal-origin is precious, rare, and biological characteristics is unique, the domestic research that there is no for naked mole myocardial cells culture method, this greatly affects and limits the naked mole of Novel experimental animal life science applying especially in Cardiovascular Disease Study.Therefore, the relation technological researching carried out and cultivate naked mole myocardial cell is needed badly.
Chinese patent literature CN104087550A discloses a kind of cultural method of rat myocardial cell, it is characterized in that, it comprises the steps: (1) by rat heart muscle tissue be dipped in digestive ferment mixed solution carry out gradation digestion soft to cardiac muscular tissue after discard Digestive system, then DMEM substratum is added, blow and beat soft cardiac muscular tissue, get supernatant and carry out centrifugal cell harvesting precipitation, described digestive ferment mixed solution comprises 0.7 ~ 1.0g/L trypsinase and 0.5 ~ 0.8g/LII Collagenase Type; (2) with the cell precipitation of the resuspended step of DMEM substratum (1) gained, be transferred to after cell suspension being crossed 180 ~ 220 mesh sieves in culturing bottle and carry out differential velocity adherent 2 ~ 3 times, each 40 ~ 50 minutes; (3) cell suspension in culturing bottle after differential velocity adherent is got, by myocardial cell with 1.5 ~ 2.5 × 10
5the density of individual/mL is inoculated in culture vessel, after inoculation, culture vessel is placed in incubator and cultivates; Wherein, step (1) and the DMEM substratum described in (2) are the DMEM substratum containing 8 ~ 12%FBS, and described per-cent is volume percent.Preferred steps (1) digestion is carry out in the environment of 36.5 ~ 37.5 DEG C in temperature, the concentration of step (2) FBS is 10%, described cell suspension is transferred in culturing bottle after crossing 200 mesh sieves and carries out differential velocity adherent, the number of times of differential velocity adherent is 2 times, and the time of described differential velocity adherent is each 45 minutes.In preferred steps (3), the condition of cultivation is for be placed in 37 DEG C, CO by culture vessel
2concentration is cultivate in the incubator of 50mL/L.
But yet there are no report about the cultural method of a kind of naked mole myocardial cell.
Summary of the invention
The object of this invention is to provide the cultural method of a kind of naked mole myocardial cell.In this cultural method, the myocardial cell of naked mole is separated, purification procedures is simple, cell attachment is fast, survival rate is high, cell culture system good stability, it is a kind of ideal naked mole primary myocardial cell culture method, substantially can meet the requirement of each research field of medical science, biology, pharmacy and life science to naked mole myocardial cell Related Experimental Study, be suitable for applying in common laboratory.
For achieving the above object, the technical scheme that the present invention takes is: the cultural method of a kind of naked mole myocardial cell, comprises the following steps:
(A) in naked mole cardiac muscular tissue, add trypsinase and carry out digestion 1-2 time, abandoning supernatant;
(B) in the cell precipitation of the middle gained of step (A), add mixture slaking liquid, after piping and druming cardiac muscular tissue suspension digests gently, leave standstill;
(C) collect the supernatant liquor in step (B), with the sieved filter of 200 order cell after, add equivalent stop buffer immediately and stop digesting, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min;
(D) cell precipitation of the middle gained of step (C), the B of repetitive operation step successively, step C more than twice (as far as possible by the sufficient tissue suspension of filter sieve screening digestion);
(E) all cells is collected in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, appropriate stop buffer re-suspended cell is added in cell precipitation, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge again, abandoning supernatant, add in cell precipitation appropriate stop buffer resuspended, mixing cell;
(F) by gained cell suspension inoculation in step (E) in culture vessel, be placed in incubator and cultivate, by differential attachment method isolating cardiac inoblast and myocardial cell;
(G) collect myocardial cell suspensions not adherent in step (F), 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after substratum suspendible, is placed in incubator and cultivates.
Wherein, the preferred naked mole suckling mouse (hereinafter referred to as suckling mouse) of the naked mole described in step (A), described suckling mouse selects the Neonatal Mouse of 1-3 age in days, and birth age in days is the smaller the better, and animal male and female are not limit.
Naked mole cardiac muscular tissue described in step (A) prepares by following steps:
(1) get newborn naked mole suckling mouse, after 75% alcohol disinfecting, move in Bechtop;
(2) suckling mouse dorsal position placed, with eye scissors in close suckling mouse xiphoid-process upper left slightly partially, cut off thoracic cavity area skin, cut off breastbone and rib, heart jumps out naturally;
(3) with after another eye scissors clip apex of the heart tissue, be placed in D-hank ' the s solution of 4 DEG C of precoolings, fully washing 3 times, cleans hematocele residual in tissue;
(4) apex of the heart tissue is moved in penicillin bottle, be cut into cardiac muscular tissue's block of about 1mm × 1mm × 1mm size with eye scissors.Described penicillin bottle, before using first, first uses strong acid oxygenant washing lotion (12% potassium bichromate (K
2cr
2o
7) the vitriol oil (dense H
2sO
4) mixed solution) soaked overnight, then tap water 15-20 time, often all over all the water of bottle inwall being thrown out as possible, after oven dry, then rinse 3 times with distilled water, last masking foil sealing, 180 DEG C of sterilizing 2h post-dryings are for subsequent use.
Wherein, the trypsinase concentration described in step (A) is 0.08% (w/v), adopts 0.25% (w/v) trypsinase and D-hank ' s solution to mix formulated according to volume ratio 1:2.In the present invention, tryptic used in amounts is determined according to the amount of cardiac muscular tissue to be digested, need complete submergence cardiac muscular tissue in principle and have unnecessary in right amount.Trypsinase concentration is too high, and easily cause digestion excessively to cause damaged tissue, during subsequent cell is cultivated, cell attachment quantity is few, and functional cell quantity also can reduce, and specifically can refer to comparative example 1.
In step (A), digestion number of times is no more than 2 times, otherwise may digest excessively easily injury of myocardium tissue; Each digestion time 5 ~ 10min, preferred 8min; The temperature 35 ~ 37 DEG C of digestion, preferably 35 DEG C.
Wherein, in step (B) mixture slaking liquid be 0.125% (w/v) trypsinase and 0.3% (w/v) II Collagenase Type mix formulated according to volume ratio 2:1, in mixture slaking liquid, trypsinase final concentration is 0.08% (w/v), and II Collagenase Type final concentration is 0.1% (w/v).Each digestion time 5 ~ 10min, preferred 8min in step (B); The temperature 35 ~ 37 DEG C of digestion, preferably 35 DEG C.With the simultaneous test of mixture slaking liquid concentration in prior art see comparative example 2.
Wherein, step (C) and the stop buffer described in step (E) are for containing 8 ~ 12% (v/v) foetal calf serum (FetalBovineSerum, FBS) DMEM substratum, preferably containing the low sugar culture-medium of DMEM of 8 ~ 12% (v/v) foetal calf serum.With the low sugar culture-medium of DMEM, cell state can be better, and the speed of growth is better, and the culture effect of DMEM high glucose medium is then weaker.With adopt the simultaneous test of DMEM high glucose medium see comparative example 3.In the low sugar culture-medium of described DMEM, glucose concn is that in 1000ml/L, DMEM high glucose medium, glucose concn is 4500ml/L.
Wherein, the repetitive operation number of times described in step (D) needs to determine according to the amount of cardiac muscular tissue to be digested, and gradation digestion is until cardiac muscular tissue is soft, and cannot see tissue is completely particulate state, and repeating digestion number of times is 6 ~ 10 times, preferably 8 times.
Wherein, the differential attachment method cell cultures time described in step (F) is 60 ~ 90min, preferred 70min.Differential attachment method is utilized to carry out purifying cells, its ultimate principle utilizes the inoblast characteristic that be attached at culture dish surface easier than myocardial cell, cell suspension pre-vaccination is hatched in culturing bottle, like this, most inoblast is all adherent, remain in suspension be exactly mainly myocardial cell (Liu Jiaqi, Sun Yunyun. neonatal rat myocardial cell primary culture method [J]. trace elements and health is studied, 2012,29 (4): 7-9.).Cultivate after 60 ~ 90min, easily adherent is cardiac fibroblast, does not have adherent mostly to be myocardial cell, after collecting in cell suspension, for the cultivation of subsequent cell; And adherent cardiac fibroblast is dropped process.
The envrionment temperature of described step (F) and the middle incubator of step (G) is 35 ~ 37 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is 5%.
Routine experimentation animal cell culture temperature is generally at about 37 DEG C, and the present invention is through groping to find, the digestion of naked mole all cells and culture temperature be set in 35-37 DEG C better, preferably 35 DEG C, too high or too low, cell is not easily bred, or delays cell growth cycle.Simultaneous test is see comparative example 4.
Incubator CO
2concentration is the same with other animals, and difference is to control O by three gas incubators
2concentration, O
2it is 5% that concentration is arranged on volumetric concentration, and this is to other animals, has been low-oxygen environment, because the carrier of oxygen volume concentrations of air is about 20%, the culture environment oxygen concentration of general zooblast is also about 20%.Simultaneous test is see comparative example 5.
Wherein, the substratum described in step (G) is the low sugar culture-medium of DMEM containing 8 ~ 12% (v/v) FBS, the dual anti-antimicrobial fluid of 1% (v/v) penicillin and streptomycin, 0.1mmol/L5-Brdu (5-Bromo-2-deoxyUridine).With the low sugar culture-medium of DMEM, cell state can be better, and the speed of growth is better, and the culture effect of DMEM high glucose medium is then weaker.With adopt the simultaneous test of DMEM high glucose medium see comparative example 3.
Myocardial cell is a kind of functional cell, there is the feature of spontaneous beat, myocardial cell is determine whether with this, but in this step, the still remaining cardiac fibroblast of possibility, just quantity greatly reduces, so add the 5-Brdu suppressing cardiac fibroblast growth in nutrient solution, to offer the best cultivation fluid environment to myocardial cell to reach.
Agents useful for same of the present invention and consumptive material are all commercially available.
The invention has the advantages that:
Zhong Luo mole cardiac muscular tissue of the present invention draws materials easy and simple to handle, to Animal Sex not requirement; Separation, the purification procedures of Process of in vitro cardiac myocyte are simple, consuming time shorter; Cell attachment is fast, survival rate is high, cell culture system good stability; Separation and Culture and the naked mole myocardial cell obtained has automaticity and shrinkability, can meet the service requirements of subsequent experimental.
Accompanying drawing explanation
Fig. 1 is the photo of myocardial cell under embodiment 1 microscope.
Fig. 2 is the photo of myocardial cell under embodiment 2 microscope.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
In following embodiment, the source of part Experiment material and reagent is as follows:
Naked mole suckling mouse comes from The 2nd Army Medical College Experimental Animal Center;
Trypsin 0.25%Typsin-EDTA) purchased from American Gibco company;
II Collagenase Type (II-collagenase) purchased from American Sigma company;
5-bromodeoxyuridine (5-Brdu) purchased from American Sigma company;
Foetal calf serum (fetalbovineserum, FBS) purchased from American Gibco company;
D-hank ' s solution purchased from American Gibco company;
Other experiment materials or reagent are as being not specifically noted, and being conventional commercial can obtain.
Embodiment 1
(1) in the naked mole cardiac muscular tissue block of 10 newborn 1-2 ages in days, add the trypsinase of 6ml0.08%, be placed in 35 DEG C, 5%CO
2after digesting 6min in cell culture incubator, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 6min in 5% cell culture incubator, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 8 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is cultivate 60min in 5% cell culture incubator, by differential attachment method isolating cardiac inoblast and myocardial cell;
(6) myocardial cell suspensions not adherent in 6 orifice plates is collected, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is continue in 5% cell culture incubator to cultivate; After 36h, can be observed mycardial contractility under microscope strong.See Fig. 1.
Embodiment 2
(1) in the naked mole cardiac muscular tissue block of 12 newborn 1-3 ages in days, add the trypsinase of 6.5ml0.08%, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is after digesting 7min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6.5ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 7min in 5% cell culture incubator, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 9 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture incubator interior cultivation 70min of 5%, by differential attachment method isolating cardiac inoblast and myocardial cell;
(6) myocardial cell suspensions not adherent in 6 orifice plates is collected, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 12%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the interior continuation cultivation of cell culture incubator of 5%; After 36h, can be observed myocardial cell under microscope has Rythmic contractions characteristic, beats.See Fig. 2.
Comparative example 1
Adopt different trypsinase concentration to carry out comparative study cultivation, condition is with embodiment 1, and difference is mainly different in tryptic digestion concentration.
(1) in the naked mole cardiac muscular tissue block of 10 newborn 1-2 ages in days, add the trypsinase of 6ml0.12%, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is after digesting 6min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 6min in the cell culture incubator of 5%, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 8 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture incubator interior cultivation 60min of 5%;
(6) cell quantity that can be observed adherent cardiac fibroblast number ratio 0.08% tryptic digestion process after 60min under microscope obviously reduces, collect myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the interior continuation cultivation of cell culture incubator of 5%; After 48h, just start to shrink to there being myocardial cell in microscopic examination.
Comparative example 2
Adopt different concns mixture slaking liquid to carry out comparative study cultivation, condition is with embodiment 2, and difference is mainly different in tryptic digestion concentration.
(1) in the naked mole cardiac muscular tissue block of 12 newborn 1-3 ages in days, add the trypsinase of 6.5ml0.08%, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is after digesting 7min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6.5ml mixture slaking liquid (pancreatin final concentration is 0.12%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 7min in the cell culture incubator of 5%, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 9 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is cultivate 70min in 5% cell culture incubator;
(6) can be observed adherent cardiac fibroblast number ratio final concentration after 70min under microscope is that the cell quantity of 0.08% tryptic mixture slaking liquid process obviously reduces, collect myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 12%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is continue in 5% cell culture incubator to cultivate; After 48h, basis of microscopic observation occurs beating and shrinking to there being myocardial cell.
Comparative example 3
Adopt the DMEM substratum containing different glucose to carry out comparative study cultivation, condition is with embodiment 1, and difference is mainly different at the glucose concn of DMEM substratum.
(1) in the naked mole cardiac muscular tissue block of 10 newborn 1-2 ages in days, add the trypsinase of 6ml0.08%, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is after digesting 6min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 6min in the cell culture incubator of 5%, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the DMEM high glucose medium containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 8 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture incubator interior cultivation 60min of 5%;
(6) can be observed adherent cardiac fibroblast after 60min under microscope to be not in good state, profile is full not, collect myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the DMEM high glucose medium suspendible containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the interior continuation cultivation of cell culture incubator of 5%; After 48h, just start to shrink to there being myocardial cell in microscopic examination.
Comparative example 4
Adopt different culture temperature to carry out comparative study cultivation, condition is with embodiment 1, and difference is mainly that the envrionment temperature residing for cell cultures is different.
(1) in the naked mole cardiac muscular tissue block of 10 newborn 1-2 ages in days, add the trypsinase of 6ml0.08%, be placed in 39 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is after digesting 6min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 39 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is digest 6min in the cell culture incubator of 5%, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 8 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 39 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture incubator interior cultivation 60min of 5%;
(6) can be observed adherent cardiac fibroblast quantity after 60min under microscope and be obviously less than 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture environment of 5%.Collect myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 39 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the interior continuation cultivation of cell culture incubator of 5%; After 48h, start to shrink to there being myocardial cell in microscopic examination.
Comparative example 5
Adopt different culture environment to carry out comparative study cultivation, condition is with embodiment 1, and difference is mainly that the carrier of oxygen volume concentrations residing for cell cultures is different.
(1) in the naked mole cardiac muscular tissue block of 10 newborn 1-2 ages in days, add the trypsinase of 6ml0.08%, be placed in 35 DEG C, O
2volumetric concentration is 20%, CO
2volumetric concentration is after digesting 6min in the cell culture incubator of 5%, abandoning supernatant, repeats aforesaid operations and digests once again;
(2) in aforesaid operations gained cell precipitation, add 6ml mixture slaking liquid (pancreatin final concentration is 0.08%, II Collagenase Type final concentration is 0.1%), blow and beat cardiac muscular tissue's suspension gently, be placed in 35 DEG C, O
2volumetric concentration is 20%, CO
2volumetric concentration is digest 6min in the cell culture incubator of 5%, leaves standstill;
(3) supernatant liquor in step (2) is collected, after the sieved filter of 200 order cell, add equivalent stop buffer (the low sugar culture-medium of the DMEM containing 10%FBS) immediately and stop digestion, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, gained cell precipitation, adds mixture slaking liquid and repeats digestion 8 times;
(4) collect all cells be deposited in same centrifuge tube, with appropriate stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, repeats once;
(5) add in cell precipitation appropriate stop buffer resuspended, mixing cell, by cell suspension inoculation in 6 orifice plates, be placed in 35 DEG C, O
2volumetric concentration is 20%, CO
2volumetric concentration is the cell culture incubator interior cultivation 60min of 5%;
(6) can be observed adherent cardiac fibroblast quantity after 60min under microscope and be obviously less than 35 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is the cell culture environment of 5%.Collect myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after the low sugar culture-medium suspendible of DMEM containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L5-Brdu, is placed in 35 DEG C, O
2volumetric concentration is 20%, CO
2volumetric concentration is the interior continuation cultivation of cell culture incubator of 5%; After 48h, start to shrink to there being myocardial cell in microscopic examination.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (8)
1. a naked mole myocardial cell's cultural method, comprises the following steps:
(A) in naked mole cardiac muscular tissue, add trypsinase and carry out digestion 1-2 time, abandoning supernatant;
(B) in the cell precipitation of the middle gained of step (A), add mixture slaking liquid, after piping and druming cardiac muscular tissue suspension digests gently, leave standstill;
(C) collect the supernatant liquor in step (B), with the sieved filter of 200 order cell after, add equivalent stop buffer immediately and stop digesting, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min;
(D) cell precipitation of the middle gained of step (C), the B of repetitive operation step successively, step C more than twice;
(E) all cells is collected in same centrifuge tube, use stop buffer re-suspended cell, and in refrigerated centrifuge 4 DEG C, 1000rpm, centrifugal 5min, abandoning supernatant, stop buffer re-suspended cell is added in cell precipitation, 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge again, abandoning supernatant, add in cell precipitation stop buffer resuspended, mixing cell;
(F) by gained cell suspension inoculation in step (E) in culture vessel, be placed in incubator and cultivate, by differential attachment method isolating cardiac inoblast and myocardial cell;
(G) collect myocardial cell suspensions not adherent in step (F), 4 DEG C, 1000rpm, centrifugal 5min in refrigerated centrifuge, gained cell precipitation, with after substratum suspendible, is placed in incubator and cultivates;
The envrionment temperature of described step (F) and the middle incubator of step (G) is 35 ~ 37 DEG C, O
2volumetric concentration is 5%, CO
2volumetric concentration is 5%.
2. the cultural method of naked mole myocardial cell according to claim 1, it is characterized in that, in described step (A), trypsinase concentration is 0.08% (w/v), adopts 0.25% (w/v) trypsinase and D-hank ' s solution to mix formulated according to volume ratio 1:2.
3. the cultural method of naked mole myocardial cell according to claim 1, it is characterized in that, in described step (B) mixture slaking liquid be 0.125% (w/v) trypsinase and 0.3% (w/v) II Collagenase Type mix formulated according to volume ratio 2:1, in mixture slaking liquid, trypsinase final concentration is 0.08% (w/v), and II Collagenase Type final concentration is 0.1% (w/v).
4. the cultural method of naked mole myocardial cell according to claim 1, is characterized in that, in described step (A) and step (B), each digestion time is 5 ~ 10min, and the temperature of digestion is 35 ~ 37 DEG C.
5. the cultural method of naked mole myocardial cell according to claim 1, is characterized in that, described step (C) and the middle stop buffer of step (E) are the DMEM substratum containing 8 ~ 12% (v/v) foetal calf serum.
6. the cultural method of naked mole myocardial cell according to claim 1, is characterized in that, repeating digestion number of times in described step (D) is 6 ~ 10 times.
7. the cultural method of naked mole myocardial cell according to claim 1, is characterized in that, in described step (F), the differential attachment method cell cultures time is 60 ~ 90min.
8. the cultural method of naked mole myocardial cell according to claim 1, it is characterized in that, the substratum in described step (G) is the low sugar culture-medium of DMEM containing 8 ~ 12% (v/v) FBS, the dual anti-antimicrobial fluid of 1% (v/v) penicillin and streptomycin, 0.1mmol/L5-Brdu.
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| CN113502260A (en) * | 2021-07-16 | 2021-10-15 | 新疆医科大学第一附属医院 | Method for separating and culturing primary myocardial cells of mice suckling mice and application of method |
| CN113652395A (en) * | 2021-08-18 | 2021-11-16 | 深圳职业技术学院 | Preparation method of MOFs (metal-organic frameworks) -coated cardiac muscle cell core-shell structure |
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