CN105505863B - A kind of cultural method of naked mole cardiac muscle cell - Google Patents
A kind of cultural method of naked mole cardiac muscle cell Download PDFInfo
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- CN105505863B CN105505863B CN201610005208.1A CN201610005208A CN105505863B CN 105505863 B CN105505863 B CN 105505863B CN 201610005208 A CN201610005208 A CN 201610005208A CN 105505863 B CN105505863 B CN 105505863B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention belongs to technical field of cell culture, the cultural method of specifically a kind of naked mole cardiac muscle cell.In cultural method of the present invention, cardiac muscle cell's separation of naked mole, purification procedures are simple, adherent fast, the survival rate height of cell, cell culture system stability is good, it is a kind of ideal naked mole primary myocardial cell culture method, substantially it can satisfy the requirement of medicine, biology, pharmacy and each research field of life science to naked mole cardiac muscle cell Related Experimental Study, be suitable for being promoted and applied in common laboratory.
Description
Technical field
The invention belongs to technical field of cell culture, specifically, being the cultural method of naked mole cardiac muscle cell a kind of.
Background technique
Naked mole (Heterocephalus glaber, Naked mole-rat) is a kind of the novel of great researching value
Experimental animal, it has lower oxygen concentration resistance, anti-aging and many peculiar biological characteristics such as antitumor, geratology, oncology,
Significant role is played in the medicine such as immunology and life science field, there is irreplaceable status.Especially resistance to
Hypoxemia research aspect, provides ideal Study of Support and scientific tools for researchs such as the relevant cardiovascular diseases of anoxic.Cardiac muscle
Cell is the basic composition unit of heart, is the main of progress physiology, pharmacology, pathology, toxicity and energetic supersession etc. research
Cell origin, while being also one of the main source of cardiac muscle tissue engineering seed cell.Currently, since naked mole animal origin is precious
Expensive, rare, and biological characteristics are unique, the country there is no the research for naked mole myocardial cells culture method, this greatly shadow
Ring and limit popularization and application of the naked mole of novel experimental animal in life science especially in Cardiovascular Disease Study.
Therefore, it needs to carry out the relation technological researching for cultivating naked mole cardiac muscle cell.
Chinese patent literature CN104087550A discloses a kind of cultural method of rat myocardial cell, which is characterized in that
Its include the following steps: (1) by rat heart muscle tissue be dipped in digestion enzyme mixation in carry out by several times digestion it is soft to cardiac muscular tissue
After discard digestive juice, DMEM culture medium is then added, blows and beats soft cardiac muscular tissue, takes supernatant be harvested by centrifugation cell heavy
It forms sediment, the digestion enzyme mixation includes 0.7~1.0g/L trypsase and 0.5~0.8g/L II Collagenase Type；(2) with
Step (1) resulting cell precipitation is resuspended in DMEM culture medium, is transferred in culture bottle after cell suspension is crossed 180~220 meshes
It carries out differential velocity adherent 2~3 times, every time 40~50 minutes；(3) take the cell suspension after differential velocity adherent in culture bottle, will cardiac muscle it is thin
Born of the same parents are with 1.5~2.5 × 105The density of a/mL is inoculated in culture vessel, and culture vessel is placed in incubator after inoculation and is trained
It supports；Wherein, DMEM culture medium described in step (1) and (2) is the DMEM culture medium containing 8~12%FBS, the percentage
For percent by volume.Preferred steps (1) digestion carries out in the environment that temperature is 36.5~37.5 DEG C, the concentration of step (2) FBS
It is 10%, the cell suspension, which is crossed after 200 meshes to be transferred in culture bottle, carries out differential velocity adherent, and the number of differential velocity adherent is 2 times,
The time of the differential velocity adherent is 45 minutes every time.In preferred steps (3), the condition of culture be culture vessel is placed in 37 DEG C,
CO2It is cultivated in the incubator that concentration is 50mL/L.
But the cultural method about a kind of naked mole cardiac muscle cell yet there are no report.
Summary of the invention
The object of the present invention is to provide the cultural methods of naked mole cardiac muscle cell a kind of.In the cultural method, naked mole
Cardiac muscle cell's separation, purification procedures are simple, and adherent fast, the survival rate height of cell, cell culture system stability is good, is a kind of
Ideal naked mole primary myocardial cell culture method, it is each to can satisfy medicine, biology, pharmacy and life science substantially
Requirement of the research field to naked mole cardiac muscle cell Related Experimental Study, is suitable for being promoted and applied in common laboratory.
To achieve the above object, the technical solution adopted by the present invention is that: a kind of cultural method of naked mole cardiac muscle cell, packet
Include following steps:
(A) trypsase is added into naked mole cardiac muscular tissue and carries out digestion 1-2 times, discard supernatant liquid；
(B) in the cell precipitation obtained in step (A), be added mixture slaking liquid, gently blow and beat cardiac muscular tissue's suspension into
After row digestion, stand；
(C) termination of equivalent terminate liquid is added immediately and disappears after the filtering of 200 mesh cell sieves for the supernatant in collection step (B)
Change, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min；
(D) cell precipitation obtained in step (C) is repeated in operating procedure B, step C (passed through as far as possible more than twice
Screen screening digests sufficient tissue suspension)；
(E) all cells are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and 4 in refrigerated centrifuge
DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, appropriate terminate liquid be added in cell precipitation, cell is resuspended, again in freezing
4 DEG C in centrifuge, 1000rpm, it is centrifuged 5min, discards supernatant liquid, appropriate terminate liquid is added in cell precipitation and is resuspended, mixes carefully
Born of the same parents；
(F) gained cell suspension inoculation in step (E) is placed in culture in incubator, passes through differential in culture vessel
Adherent method isolating cardiac fibroblast and cardiac muscle cell；
(G) myocardial cell suspensions not adherent in collection step (F), 4 DEG C, 1000rpm, centrifugation in refrigerated centrifuge
5min after gained cell precipitation is suspended with culture medium, is placed in culture in incubator.
Wherein, the preferably naked mole suckling mouse (hereinafter referred to as suckling mouse) of naked mole described in step (A), the suckling mouse select 1-
The Neonatal Mouse of 3 ages in days, birth age in days is the smaller the better, and animal male and female are unlimited.
Naked mole cardiac muscular tissue described in step (A) can be prepared by following steps:
(1) take newborn naked mole suckling mouse, after 75% alcohol disinfecting, move into superclean bench；
(2) suckling mouse dorsal position is placed, with eye scissors in close to the slightly inclined upper left of suckling mouse xiphoid-process, cuts off thoracic cavity position skin
Skin, cuts breastbone and rib cage, heart jump out naturally；
(3) with after another eye scissors clip apex of the heart tissue, being placed in D-hank ' the s solution of 4 DEG C of pre-coolings, 3 are sufficiently washed
It is secondary, clean remaining hematocele in tissue；
(4) apex of the heart tissue is moved into penicillin bottle, is cut into about 1mm × 1mm × 1mm size heart with eye scissors
Muscular tissue block.The penicillin bottle, for the first time before use, first using strong acid oxidant washing lotion (12% potassium bichromate (K2Cr2O7)
The concentrated sulfuric acid (dense H2SO4) mixed liquor) soaked overnight, then tap water rinses 15-20 times, every all over the water of bottle inner wall will being use up
Power is thrown out, after drying, then is rinsed 3 times with distilled water, the sealing of last masking foil, is dried for standby after 180 DEG C of sterilizing 2h.
Wherein, trypsinase concentration described in step (A) is 0.08% (w/v), using 0.25% (w/v) trypsase and
D-hank ' s solution is uniformly mixed according to volume ratio 1:2 and is formulated.The used in amounts of trypsase is according to wait digest in the present invention
The amount of cardiac muscular tissue be determined, need to be totally submerged in principle cardiac muscular tissue and have it is appropriate extra.Trypsinase concentration mistake
Height, easily causing digestion excessively leads to injury tissue, and in subsequent cell culture, cell number of adherent is few, functional cell number
Amount can also be reduced, and specifically can refer to comparative example 1.
In step (A) otherwise digestion number may digest no more than 2 times and excessively be easy to damage cardiac muscular tissue；Disappear every time
Change 5~10min of time, preferably 8min；35~37 DEG C of the temperature of digestion, preferably 35 DEG C.
Wherein, mixture slaking liquid is 0.125% (w/v) trypsase and 0.3% (w/v) II Collagenase Type in step (B)
It is uniformly mixed and is formulated according to volume ratio 2:1, trypsase final concentration of 0.08% (w/v) in mixture slaking liquid, II type glue
Protoenzyme final concentration of 0.1% (w/v).Each digestion time 5~10min, preferably 8min in step (B)；The temperature 35 of digestion~
37 DEG C, preferably 35 DEG C.Comparative test with mixture slaking liquid concentration in the prior art is referring to comparative example 2.
Wherein, terminate liquid described in step (C) and step (E) is containing 8~12% (v/v) fetal calf serum (Fetal
Bovine Serum, FBS) DMEM culture medium, preferably contain 8~12% (v/v) fetal calf serums DMEM low sugar culture medium.With
DMEM low sugar culture medium, cell state can be more preferable, and the speed of growth is more preferably, and the culture effect of DMEM high glucose medium then poor one
A bit.With the comparative test using DMEM high glucose medium referring to comparative example 3.Concentration of glucose in the DMEM low sugar culture medium
For 1000ml/L, concentration of glucose is 4500ml/L in DMEM high glucose medium.
Wherein, repetitive operation number described in step (D) needs the amount according to cardiac muscular tissue to be digested to be determined, by several times
Until cardiac muscular tissue is soft, completely invisible tissue is in granular form for digestion, repeats to digest number to be 6~10 times, preferably 8 times.
Wherein, the differential attachment method cell culture time described in step (F) is 60~90min, preferably 70min.Utilize difference
Fast adherent method carries out purifying cells, the basic principle is that being easier to be attached at culture dish table than cardiac muscle cell using fibroblast
Cell suspension pre-vaccination is incubated in culture bottle, in this way, most fibroblasts are adherent, remains in by the characteristic in face
In suspension mainly be exactly cardiac muscle cell (Liu Jiaqi, Sun Yunyun neonatal rat myocardial cell primary culture method [J] microelement and
Health research, 2012,29 (4): 7-9.).After cultivating 60~90min, it is easy adherent to be cardiac fibroblast, it is not adherent
Be mostly cardiac muscle cell, in cell suspension collect after, the culture for subsequent cell；And adherent cardiac fibroblast quilt
Discard processing.
The environment temperature of incubator is 35~37 DEG C in the step (F) and step (G), O2Volumetric concentration is 5%, CO2
Volumetric concentration is 5%.
Routine experimentation animal cell culture temperature is generally at 37 DEG C or so, and present invention process gropes to find, naked mole institute
There are cell dissociation and cultivation temperature to be set in 35-37 DEG C preferably, preferably 35 DEG C, too high or too low, cell is not easy to be proliferated, or prolongs
Slow cell growth cycle.Comparative test is referring to comparative example 4.
Incubator CO2Concentration and other animals are the same, the difference is that controlling O by three gas incubators2It is dense
Degree, O2Concentration setting is 5% in volumetric concentration, this has been low-oxygen environment for other animals, because of the carrier of oxygen of atmosphere
Product concentration is about 20%, and the culture environment oxygen concentration of general zooblast is also about 20%.Comparative test is referring to comparative example 5.
Wherein, culture medium described in step (G) is containing 8~12% (v/v) FBS, the 1% dual anti-antibacterial of (v/v) penicillin and streptomycin
The DMEM low sugar culture medium of liquid, 0.1mmol/L 5-Brdu (5-Bromo-2-deoxyUridine).With DMEM low sugar culture medium,
Cell state can be more preferable, and the speed of growth is more preferably, and the culture effect of DMEM high glucose medium is then weaker.With use DMEM high
The comparative test of sugar culture-medium is referring to comparative example 3.
Cardiac muscle cell is a kind of functional cell, and the feature with spontaneous beat determines whether cardiac muscle cell with this, but
In the step for being, it would still be possible to which remaining cardiac fibroblast, only quantity greatly reduces, and inhibits so being added in culture solution
The 5-Brdu of cardiac fibroblast growth, provides best culture fluid environment to cardiac muscle cell to reach.
Agents useful for same and consumptive material of the present invention are commercially available.
The present invention has the advantages that
Zhong Luo mole of the present invention cardiac muscular tissue materials are easy to operate, do not require Animal Sex；In Process of in vitro
The separation of cardiac muscle cell, purification procedures are simple and easy, time-consuming shorter；Adherent fast, the survival rate height of cell, cell culture system
Stability is good；Naked mole cardiac muscle cell obtained by being separately cultured has automaticity and shrinkage, can satisfy subsequent experimental
Requirement.
Detailed description of the invention
Fig. 1 is the photo of cardiac muscle cell under 1 microscope of embodiment.
Fig. 2 is the photo of cardiac muscle cell under 2 microscope of embodiment.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
In following embodiments, the source of part Experiment material and reagent is as follows:
Naked mole suckling mouse is from The 2nd Army Medical College Experimental Animal Center；
Trypsase (0.25%Typsin-EDTA) is purchased from U.S. Gibco company；
II Collagenase Type (II-collagenase) is purchased from Sigma Co., USA；
5- bromodeoxyuridine (5-Brdu) is purchased from Sigma Co., USA；
Fetal calf serum (fetal bovine serum, FBS) is purchased from U.S. Gibco company；
D-hank ' s solution is purchased from U.S. Gibco company；
Other experimental materials or reagent are such as not specifically noted, and are that conventional commercial can obtain.
Embodiment 1
(1) trypsase of 6ml 0.08% is added into the naked mole cardiac muscular tissue block of 10 new life 1-2 ages in days, is placed in
35 DEG C, 5%CO2After digesting 6min in cell incubator, liquid is discarded supernatant, aforesaid operations is repeated and digests again once；
(2) in the cell precipitation obtained by aforesaid operations, 6ml mixture slaking liquid (pancreatin final concentration of 0.08%, II type is added
Clostridiopetidase A is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To digest 6min in 5% cell incubator, stand；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 8 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration is that 60min is cultivated in 5% cell incubator, passes through differential attachment method point
From cardiac fibroblast and cardiac muscle cell；
(6) myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugation in refrigerated centrifuge are collected
5min, gained cell precipitation is with containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, the DMEM low sugar of 0.1mmol/L5-Brdu
After culture medium is suspended, it is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration is to continue to cultivate in 5% cell incubator；36h
Afterwards, that mycardial contractility can be observed under microscope is strong.See Fig. 1.
Embodiment 2
(1) trypsase of 6.5ml 0.08% is added into the naked mole cardiac muscular tissue block of 12 new life 1-3 ages in days, sets
In 35 DEG C, O2Volumetric concentration is 5%, CO2After digesting 7min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, weight
Multiple aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, and addition 6.5ml mixture slaking liquid (pancreatin final concentration of 0.08%, II
Collagenase Type is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volume is dense
Degree is to digest 7min in 5% cell incubator, is stood；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 9 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 5%, CO270min is cultivated in the cell incubator that volumetric concentration is 5%, passes through differential attachment method
Isolating cardiac fibroblast and cardiac muscle cell；
(6) myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C, 1000rpm, centrifugation in refrigerated centrifuge are collected
5min, gained cell precipitation is with containing 12%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, the DMEM low sugar of 0.1mmol/L5-Brdu
After culture medium is suspended, it is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Continue to cultivate in the cell incubator that volumetric concentration is 5%；
After 36h, cardiac muscle cell can be observed under microscope Rythmic contractions characteristic, beating.See Fig. 2.
Comparative example 1
Research culture is compared using different trypsinase concentrations, condition is with embodiment 1, and difference is mainly in pancreas egg
The digestion concentration of white enzyme is different.
(1) trypsase of 6ml 0.12% is added into the naked mole cardiac muscular tissue block of 10 new life 1-2 ages in days, is placed in
35℃、O2Volumetric concentration is 5%, CO2After digesting 6min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, is repeated
Aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, 6ml mixture slaking liquid (pancreatin final concentration of 0.08%, II type is added
Clostridiopetidase A is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To digest 6min in 5% cell incubator, stand；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 8 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 5%, CO260min is cultivated in the cell incubator that volumetric concentration is 5%；
(6) adherent cardiac fibroblast quantity can be observed after 60min under microscope to disappear than 0.08% trypsase
The cell quantity for changing processing significantly reduces, and collects myocardial cell suspensions not adherent in 6 orifice plates, 4 DEG C in refrigerated centrifuge,
1000rpm, centrifugation 5min, gained cell precipitation, which is used, contains 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L 5-
After the DMEM low sugar culture medium of Brdu is suspended, it is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2The cell culture that volumetric concentration is 5%
Continue to cultivate in case；After 48h, just observe that cardiac muscle cell starts to shrink in microscope.
Comparative example 2
Research culture is compared using various concentration mixture slaking liquid, condition is with embodiment 2, and difference is mainly in pancreas
The digestion concentration of protease is different.
(1) trypsase of 6.5ml 0.08% is added into the naked mole cardiac muscular tissue block of 12 new life 1-3 ages in days, sets
In 35 DEG C, O2Volumetric concentration is 5%, CO2After digesting 7min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, weight
Multiple aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, and addition 6.5ml mixture slaking liquid (pancreatin final concentration of 0.12%, II
Collagenase Type is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volume is dense
Degree stands to digest 7min in 5% cell incubator；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 9 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration is that 70min is cultivated in 5% cell incubator；
(6) adherent cardiac fibroblast quantity can be observed after 70min under microscope than final concentration of 0.08% pancreas
The cell quantity of the mixture slaking liquid processing of protease significantly reduces, and collects myocardial cell suspensions not adherent in 6 orifice plates, Yu Leng
Freeze in centrifuge 4 DEG C, 1000rpm, centrifugation 5min, gained cell precipitation, which is used, contains 12%FBS, the dual anti-antibacterial of 1% penicillin and streptomycin
Liquid, 0.1mmol/L 5-Brdu DMEM low sugar culture medium be suspended after, be placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To continue to cultivate in 5% cell incubator；After 48h, microscopically observation is to there is cardiac muscle cell to occur beating and shrink.
Comparative example 3
Research culture is compared using the DMEM culture medium containing different glucose, condition with embodiment 1, difference
Place is mainly different in the concentration of glucose of DMEM culture medium.
(1) trypsase of 6ml 0.08% is added into the naked mole cardiac muscular tissue block of 10 new life 1-2 ages in days, is placed in
35℃、O2Volumetric concentration is 5%, CO2After digesting 6min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, is repeated
Aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, 6ml mixture slaking liquid (pancreatin final concentration of 0.08%, II type is added
Clostridiopetidase A is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To digest 6min in 5% cell incubator, stand；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM high glucose medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 8 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 5%, CO260min is cultivated in the cell incubator that volumetric concentration is 5%；
(6) adherent cardiac fibroblast can be observed after 60min under microscope to be not in good state, shape is not full enough,
Myocardial cell suspensions not adherent in 6 orifice plates are collected, 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained cell is heavy
It forms sediment after being suspended with the DMEM high glucose medium containing 10%FBS, the dual anti-antimicrobial fluid of 1% penicillin and streptomycin, 0.1mmol/L 5-Brdu,
It is placed in 35 DEG C, O2Volumetric concentration is 5%, CO2Continue to cultivate in the cell incubator that volumetric concentration is 5%；After 48h, just micro-
Sem observation is started to shrink to there is cardiac muscle cell.
Comparative example 4
Research culture is compared using different cultivation temperatures, for condition with embodiment 1, difference essentially consists in cell training
It is different to support locating environment temperature.
(1) trypsase of 6ml 0.08% is added into the naked mole cardiac muscular tissue block of 10 new life 1-2 ages in days, is placed in
39℃、O2Volumetric concentration is 5%, CO2After digesting 6min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, is repeated
Aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, 6ml mixture slaking liquid (pancreatin final concentration of 0.08%, II type is added
Clostridiopetidase A is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 39 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To digest 6min in 5% cell incubator, stand；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 8 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 39 DEG C, O2Volumetric concentration is 5%, CO260min is cultivated in the cell incubator that volumetric concentration is 5%；
(6) adherent cardiac fibroblast quantity can be observed after 60min under microscope and is considerably less than 35 DEG C, O2Volume
Concentration is 5%, CO2The cell culture environment that volumetric concentration is 5%.Collect myocardial cell suspensions not adherent in 6 orifice plates, Yu Leng
Freeze in centrifuge 4 DEG C, 1000rpm, centrifugation 5min, gained cell precipitation, which is used, contains 10%FBS, the dual anti-antibacterial of 1% penicillin and streptomycin
Liquid, 0.1mmol/L 5-Brdu DMEM low sugar culture medium be suspended after, be placed in 39 DEG C, O2Volumetric concentration is 5%, CO2Volumetric concentration
To continue to cultivate in 5% cell incubator；After 48h, observe that cardiac muscle cell starts to shrink in microscope.
Comparative example 5
Research culture is compared using different culture environments, for condition with embodiment 1, difference essentially consists in cell training
It is different to support locating oxygen volumetric concentration.
(1) trypsase of 6ml 0.08% is added into the naked mole cardiac muscular tissue block of 10 new life 1-2 ages in days, is placed in
35℃、O2Volumetric concentration is 20%, CO2After digesting 6min in the cell incubator that volumetric concentration is 5%, liquid is discarded supernatant, is repeated
Aforesaid operations digest once again；
(2) in the cell precipitation obtained by aforesaid operations, 6ml mixture slaking liquid (pancreatin final concentration of 0.08%, II type is added
Clostridiopetidase A is final concentration of 0.1%), gently blows and beats cardiac muscular tissue's suspension, is placed in 35 DEG C, O2Volumetric concentration is 20%, CO2Volume is dense
Degree stands to digest 6min in 5% cell incubator；
(3) equivalent terminate liquid is added immediately and (contains after the filtering of 200 mesh cell sieves for the supernatant in collection step (2)
The DMEM low sugar culture medium of 10%FBS) digestion is terminated, and 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min, gained is thin
Born of the same parents' precipitating is added mixture slaking liquid and repeats digestion 8 times；
(4) all cell precipitations are collected, cell is resuspended with appropriate terminate liquid in same centrifuge tube, and in refrigerated centrifuge
In 4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, be repeated once；
(5) appropriate terminate liquid resuspension is added in cell precipitation, mixing cell is set by cell suspension inoculation in 6 orifice plates
In 35 DEG C, O2Volumetric concentration is 20%, CO260min is cultivated in the cell incubator that volumetric concentration is 5%；
(6) adherent cardiac fibroblast quantity can be observed after 60min under microscope and is considerably less than 35 DEG C, O2Volume
Concentration is 5%, CO2The cell culture environment that volumetric concentration is 5%.Collect myocardial cell suspensions not adherent in 6 orifice plates, Yu Leng
Freeze in centrifuge 4 DEG C, 1000rpm, centrifugation 5min, gained cell precipitation, which is used, contains 10%FBS, the dual anti-antibacterial of 1% penicillin and streptomycin
Liquid, 0.1mmol/L 5-Brdu DMEM low sugar culture medium be suspended after, be placed in 35 DEG C, O2Volumetric concentration is 20%, CO2Volume is dense
Continue to cultivate in the cell incubator that degree is 5%；After 48h, observe that cardiac muscle cell starts to shrink in microscope.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (5)
1. a kind of cultural method of naked mole cardiac muscle cell, comprising the following steps:
(A) trypsase is added into naked mole cardiac muscular tissue and carries out digestion 1-2 times, discard supernatant liquid；The trypsase
Concentration is 0.08% (w/v), is uniformly mixed using 0.25% (w/v) trypsase and D-hank ' s solution according to volume ratio 1:2
It is formulated；
(B) in the cell precipitation obtained in step (A), mixture slaking liquid is added, gently blows and beats cardiac muscular tissue's suspension and disappears
After change, stand；The mixture slaking liquid is 0.125% (w/v) trypsase and 0.3% (w/v) II Collagenase Type according to body
Product is uniformly mixed than 2:1 and is formulated, trypsase final concentration of 0.08% (w/v) in mixture slaking liquid, and II Collagenase Type is whole
Concentration is 0.1% (w/v)；
(C) equivalent terminate liquid is added immediately and terminates digestion after the filtering of 200 mesh cell sieves for the supernatant in collection step (B),
And 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min；
(D) cell precipitation obtained in step (C) is repeated in operating procedure B, step C, repeats to digest number to be 6~10 times；
(E) all cells are collected in same centrifuge tube, cell is resuspended with terminate liquid, and 4 DEG C in refrigerated centrifuge,
1000rpm, centrifugation 5min, discard supernatant liquid, terminate liquid are added in cell precipitation, cell is resuspended, again in refrigerated centrifuge
4 DEG C, 1000rpm, centrifugation 5min, discard supernatant liquid, and terminate liquid is added in cell precipitation and is resuspended, mixes cell；
(F) gained cell suspension inoculation in step (E) is placed in culture in incubator, passes through differential velocity adherent in culture vessel
Method isolating cardiac fibroblast and cardiac muscle cell；
(G) myocardial cell suspensions not adherent in collection step (F), 4 DEG C in refrigerated centrifuge, 1000rpm, centrifugation 5min,
After gained cell precipitation is suspended with culture medium, it is placed in culture in incubator；
The environment temperature of incubator is 35 DEG C in the step (F) and step (G), O2Volumetric concentration is 5%, CO2Volumetric concentration
It is 5%.
2. the cultural method of naked mole cardiac muscle cell according to claim 1, which is characterized in that the step (A) and
Each digestion time is 5~10min in step (B), and the temperature of digestion is 35~37 DEG C.
3. the cultural method of naked mole cardiac muscle cell according to claim 1, which is characterized in that the step (C) and
Terminate liquid is the DMEM culture medium containing 8~12% (v/v) fetal calf serums in step (E).
4. the cultural method of naked mole cardiac muscle cell according to claim 1, which is characterized in that in the step (F)
The differential attachment method cell culture time is 60~90min.
5. the cultural method of naked mole cardiac muscle cell according to claim 1, which is characterized in that in the step (G)
Culture medium be containing 8~12% (v/v) FBS, 1% (v/v) penicillin and streptomycin dual anti-antimicrobial fluid, 0.1mmol/L 5-Brdu
DMEM low sugar culture medium.
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| CN106676061A (en) * | 2016-11-19 | 2017-05-17 | 河南医学高等专科学校 | Myocardial cell separation method |
| CN107142242B (en) * | 2017-05-08 | 2021-05-07 | 中国人民解放军第二军医大学 | Isolated culture method of naked mole rat skeletal muscle myoblasts |
| CN107022520B (en) * | 2017-05-08 | 2021-05-07 | 中国人民解放军第二军医大学 | Isolated culture method of naked mole rat alveolar II type epithelial cells |
| CN108949743A (en) * | 2018-06-15 | 2018-12-07 | 翁炳焕 | A kind of full-length genome hybridizing clones method |
| CN108949673A (en) * | 2018-08-13 | 2018-12-07 | 武汉华联科生物技术有限公司 | A kind of primary separation method of Fetal Rat rat cardiomyocyte |
| 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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