CN108774630A - A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell - Google Patents
A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell Download PDFInfo
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- CN108774630A CN108774630A CN201810609047.6A CN201810609047A CN108774630A CN 108774630 A CN108774630 A CN 108774630A CN 201810609047 A CN201810609047 A CN 201810609047A CN 108774630 A CN108774630 A CN 108774630A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of primary culture methods of Microhyla ornata Skeletal Muscle Cell.Its steps is as follows：Adult Microhyla ornata is taken to carry out surface sterilization；Dissection is taken out leg flesh and is cleaned with HBSS balanced salt solutions；Successively through Collagenase I and trypsin digestion；Cell suspension is made through 100 μm of apertures and 40 μm of aperture cell filtration net filtrations successively after cutting sharp suction nozzle and not cutting sharp suction nozzle piping and druming extruding in tissue suspension；27 DEG C of constant temperature incubations, the cell culture complete medium renewed every 3-4 days.The present invention can quickly, repeatably establish the original cuiture of Microhyla ornata Skeletal Muscle Cell, and required cell cultivation equipment is simple, and operability is strong.The present invention is the important supplement to existing Skeletal Muscle Cell, and new cell material is provided for the critical biologicals such as terrestrial adaptability, development by metamorphosis, muscle development, muscle damage and medical problem.
Description
Technical field
The invention belongs to animal cell culture technology field more particularly to a kind of primary trainings of Microhyla ornata Skeletal Muscle Cell
The method of supporting.
Background technology
In 1907, Harrison (Harrison) uses drop culture by frog's embryo nerve tissues culture in lymph grumeleuse
It as long as several weeks, and observes and has grown aixs cylinder from the tissue block of culture, thus create cover plate covering recess glass hanging drop training
The method of supporting, has established the basis of animal tissue's in vitro culture.1913, Ka Leier (Carrel) will be stringent sterile in surgical operation
Operating technology is introduced into the in vitro culture of zooblast, makes cell in vitro being capable of long term growth.1916, Louth (Rous)
Trypsinization is passed on for tissue digestion and cell with Jones (Jones), indicates that animal truly is thin
The beginning of born of the same parents' culture.1948, ell (Earle) was from the separation of the subcutaneous tissue of mouse and continuous passage culture the first cell
System：L fibroblasts.2006, the l cell reprogramming for more (Yamanaka) inducing in vitro culture is stretched in mountain
For induced multi-potent stem cell (iPSC).With the development of genetic engineering and other cell engineerings, cell culture technology is
As the basis of transgenic technology, bio-pharmaceuticals and other many technologies, play an important role in modern biotechnology.
Amphibian, especially batrachian cell culture have had certain Research foundation, from the leopard frog (Rana
Pipiens), bufo gargarizans Cantor (Bufo bufo gargarizans), Rana plancyi (Pelophylax plancyi), heat
Embryo with Xenopus laevis (Xenopus tropicalis), particularly Africa xenopus (Xenopus laevis), in tadpole or adult
It has been separately cultured out the primary cell and cell line (Balls and Worley 1973 of Various Tissues；Freed and
Mezger-Freed 1970；Sinzelle et al.2012；Smith and Tata 1991；Wu Zhengan, 1978).However with
Upper amphibian cell culture studies are concentrated mainly on the early stage of animal cell culture history, since the last century 90's
Just not big progress later.The research recent decades of animal cell culture technology focus primarily upon insect and mammal, right
The concern of amphibian is less.The research of Skeletal Muscle Cell (skeletal muscle cells) original cuiture technology mainly collects
In in mouse and people, have research (the Charge and Rudnicki 2004 of a small amount of rabbit, dog, chicken and Africa xenopus；Danoviz
and Yablonka-Reuveni 2012；Parker MH et al.2012；Shefer and Yablonka-Reuveni
2008；Shibota et al.2000；Yablonka-Reuveni and Day2011).Since skeletal muscle fibre has lost
Growth passage capacity, therefore usually take the myogenic cells (myogenic cell) to be cultivated to obtain Skeletal Muscle Cell, flesh
Protogonocyte is mainly muscle satellite cell (skeletal muscle satellite cell), is on a small quantity sarcoblast
(myoblast).Early stage thinks that muscle satellite cell is cultivated in vitro inevitably into terminal differentiation state, therefore can not
It passes on (Yablonka-Reuveni and Day 2011), finds that P38 inhibitor (SB 203580) is added to be defended in people's flesh in recent years
In astrocyte culture medium (Charville et al.2015) or be added four kinds of cell factors (IL-1 α, IL-13, TNF-α and
IFN-γ) in mouse muscle satellite cell culture medium (Fu et al.2015), it can repeatedly pass on.Part is by sarcoblast
(myoblast) cell line built can be passed on repeatedly, such as rabbit source L6 and L8 cell lines, mouse source C2, C2C12 and MM14 cell line.
The Skeletal Muscle Cell of amphibian animal source culture can not pass on report.
Microhyla ornata (Microhyla fissipes) is under the jurisdiction of Amphibia (Amphibia) Anura (Anura) Microhylidae
(Microhylidae), it is distributed in East Asia and Southeast Asia, population quantity is a lot of, has the bodily form small, survival ability is strong, and species are steady
Fixed, individual difference is small, and male and female are easier to differentiate, and oviposition is more, and tadpole is transparent, several times a year lays eggs and breeding period is long, and sexal maturity is very fast,
Ovum is relatively large in diameter the advantages that (0.8-1.0 millimeters) (Fei et al., 2009) and diploid (Li, 2006), it is made to study
Embryonic development, Adaptive mechanism, human diseases and Environmental Health etc. have important value (Liu et al., 2016).
In addition, the traditional mode amphibian Xenopus laevis relative to aquatic life, Microhyla ornata is terrestrial species, mainly in land after metamorphosis
It lives on the ground, can be used as and study the aquatic good model to terrestrial adaptability, the above feature, which shows that Microhyla ornata is great, becomes mould
The potentiality of formula biology.The physiological and biochemical research of individual level generally requires that a large amount of genetic backgrounds are consistent, the stage of development identical frog,
There are strict requirements, heavy workload to raising place, and precisely can easily study its correlation if the cell for having cultured in vitro
Function and mechanism.The culture of Microhyla ornata Skeletal Muscle Cell is blank at present.Therefore, through the invention, Microhyla ornata bone is established
The primary culture method of bone myocyte, is the important supplement to existing Skeletal Muscle Cell, amphibian cell culture into one
Step development, can not only promote the critical biologicals such as terrestrial adaptability, development by metamorphosis, muscle development, muscle damage and medical problem
Research, moreover it is possible to increase the quantity and type of China's cell bank, offer reference for the foundation of other amphibian cell lines in the future
Experience.
Invention content
The purpose of the present invention is to provide a kind of primary culture methods of Microhyla ornata Skeletal Muscle Cell, are skeletal muscle physiology
Biochemical Research provides technical support and guarantee.
The present invention completes by the following technical programs：
A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell, includes the following steps：
(1) solution is prepared；
(2) choose 0.5-2.0 grams of weight at frog crymoanesthesia, with 75% alcohol surface sterilization；
(3) frog is placed in plate, is dissected；
(4) clip leg flesh enters new culture dish, is cleaned 3 times with HBSS balanced salt solutions；
(5) it shifts cleaned leg flesh and enters centrifuge tube, siphon away remaining HBSS balanced salt solutions, 1.0-1.5mL glue is added
I solution of protoenzyme, 27 DEG C digest 90 minutes；
(6) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 7-15 minutes, centrifugal condition is：Centrifugal force
500-800g, 4-6 DEG C of temperature；
(7) supernatant is carefully sucked out and abandons, 1.0-1.5mL trypsin solutions are added, 27 DEG C digest 15 minutes；
(8) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 7-15 minutes, centrifugal condition is：Centrifugal force
500-800g, 4-6 DEG C of temperature；
(9) supernatant is carefully sucked out and abandons, 1.0mL cell culture complete mediums are then added；
(10) pipettor (matching 1mL suction nozzles, cutting off suction nozzle point makes tip aperture be about 2mm) is used firmly slowly to blow and beat muscle groups
Fragment is knitted, until tissue block can be easy, by suction nozzle, to stand 5 minutes；
It (11) will 100 μm of aperture cell filtration net filtrations of about 3/4 volume supernatant；
(12) pipettor (matching 1mL suction nozzles) is used slowly to blow and beat remaining 1/4 volume musculature fragment repeatedly, until tissue block
It can be easy to pass through suction nozzle；
(13) 1/4 volume musculature suspension of residue is transferred completely into 100 μm of aperture cell filtering net (see 11) filterings；
(14) filtrate (see 11,13) is refiltered once with 40 μm of aperture cell filtering nets；
(15) 1.0-4.0mL cell culture complete mediums are added and enter 40 μm of aperture cell filtering net (see 14) filterings；
(16) cell suspension that (14) (15) step obtains is transferred in tissue culture plate；
(17) tissue culture plate is placed in incubator, 27 DEG C of constant temperature incubations；
(18) cell culture complete medium was changed every 3-4 days.
Whole operation process aseptically carries out, and all consumptive materials, reagent are required to sterile.
The 70% of a concentration of standard HBSS balanced salt solutions of the HBSS balanced salt solutions.
The Collagenase I solution contains Collagenase I a concentration of 0.25%.
The trypsin solution is 0.175% containing trypsinase concentration.
The cell culture complete medium is Leibovitz L-15 culture mediums, fetal calf serum, Primocin and Y27632
Mixture, pH value 7.0-7.4.
Each component content is in the cell culture complete medium：Standard Leibovitz L-15 culture mediums 60%, tire ox
Serum 10%, pure water 30% and 100 μ g/ml Primocin and 10 μM of Y27632.
The invention further relates to a kind of Microhyla ornata Skeletal Muscle Cells obtained by above-mentioned cultural method.
The method of the present invention has the advantages that：
1. with double enzymes successively digestion method comparison mixing enzyme digestion or independent trypsinization, required trypsase
Digestion time it is shorter, it is light to the extent of damage of cell membrane, be conducive to cell survival；And individually Collagenase I digestion method almost extracts
Less than effective cell.
Cell, required enzymic digestion are obtained 2. squeezing fragment of tissue release myogenic cells (see 10,12) and comparing enzymolysis completely
Time is shorter, light to the extent of damage of cell membrane, is conducive to cell survival, and muscle fibre fragment is less, is conducive to observation.
3. cultural method is consistent, reproducible, condition of culture is simple, does not need carbon dioxide incubator, culture
Cellular morphology is uniform, well-grown.
4. the cell obtained has typical Skeletal Muscle Cell shape through the identification of micro- sem observation, qPCR and immunocytochemistry
State and feature are great for further the terrestrial adaptability of further investigation skeletal muscle, development by metamorphosis, muscle development, muscle damage etc.
Biology and medical problem provide abundant experiment material source.
Description of the drawings
The 2nd day Microhyla ornata Skeletal Muscle Cell of original cuiture under the common inverted phase contrast microscopes of Fig. 1 (200 ×)；
The 4th day Microhyla ornata Skeletal Muscle Cell of original cuiture under the common inverted phase contrast microscopes of Fig. 2 (200 ×)；
The 6th day Microhyla ornata Skeletal Muscle Cell of original cuiture under the common inverted phase contrast microscopes of Fig. 3 (200 ×)；
The qPCR of Fig. 4 Microhyla ornata Skeletal Muscle Cells identifies that the relative expression quantity of myoG genes is remote in Skeletal Muscle Cell
More than pneumonocyte and nephrocyte；
Microhyla ornata Skeletal Muscle Cell under Fig. 5 fluorescence microscopes (400 ×), the DAPI positives for occurring blue in core endochylema are glimmering
Light reaction, Skeletal Muscle Cell starch the reaction of Alpha Skeletal Muscle Actin antigen green-emitting Positive fluorescences；
Microhyla ornata Skeletal Muscle Cell under Fig. 6 fluorescence microscopes (400 ×), the DAPI positives for occurring blue in core endochylema are glimmering
Light reaction, Skeletal Muscle Cell starch the reaction of Desmin antigen green-emitting Positive fluorescences；
The growth curve chart of Fig. 7 Microhyla ornata Skeletal Muscle Cells.
Specific implementation mode
With reference to example and attached drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to
This, the above according to the present invention, according to the ordinary technical knowledge and customary means of this field, not departing from, the present invention is above-mentioned
Under the premise of basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.
The preparation of 1 solution of embodiment
(1) HBSS balanced salt solutions (Hanks ' balanced salt solution)：350mL standards HBSS is taken to balance salt
150mL pure water, 4 DEG C of preservation half a year are added in solution (Hyclone Cat.No.SH30030.02).
(2) Collagenase I solution (I solution of collagenase)：Dissolve 100mg Collagenase Is (Invitrogen
Cat.No.17100-017) in 40mL HBSS balanced salt solutions (see 1), 0.1 μm of filter filtering, -20 DEG C are kept in dark place 1 year.
(3) trypsin solution (Trypsin Solution)：35mL stostes (Gibco Cat.No.25200056) are taken,
15mL pure water is added, -20 DEG C preserve 1 year.
(4) Y27632 solution：2mg Y27632 (MCE Cat.No.HY-10583) are dissolved in 625 μ L DMSO (Sigma
Cat.No.D2650), 5.625mL pure water, -80 DEG C of preservation half a year are then added.
(5) L-15 culture mediums liquid storage：Take 335mL standard Leibovitz L-15 culture mediums (Hyclone
Cat.No.SH30525.01), 165mL pure water, 4 DEG C of preservation half a year are added.
(6) cell culture complete medium：Take 45mL L-15 culture mediums liquid storages (see 5) that 5mL fetal calf serums are added before use
(FBS) (WISENT Cat.No.086550), 0.1mL Primocin (Invitrogen Cat.No.ant-pm-1) and 0.5mL
Y27632 solution (see 4).
The original cuiture of 2 Microhyla ornata Skeletal Muscle Cell of embodiment
By the following steps：
(1) solution is prepared；
(2) adult one crymoanesthesia of the female frog for choosing 2.0 grams of weight, after aseptic water washing, with 75% cotton ball soaked in alcohol
Scrape skin；
(3) frog is placed in 3.5 cm diameter plates, is dissected；
(4) scissors and tweezers clip thigh flesh are used, about 30-40mm is cut to3The muscle masses of size are put into new 3.5 centimetres
In diameter dishes, cleaned 3 times with HBSS balanced salt solutions；
(5) cleaned muscle masses are shifted with Pasteur pipe and enter 15mL centrifuge tubes, siphon away remaining HBSS balanced salt solutions,
1.5mL Collagenase I solution is added, tightens centrifuge tube lid, 27 DEG C digest 90 minutes, and centrifuge tube was shaken 1 time every 30 minutes；
(6) whole digestive juices (containing non-digestion of solid musculature) are transferred to a 2mL microcentrifugation with Pasteur pipe
It manages in (EP pipes), merging refrigerated centrifuge centrifuges 7 minutes, and centrifugal condition is：Centrifugal force 800g, 6 DEG C of temperature；
(7) supernatant is carefully sucked out and abandons, 1.5mL trypsin solutions are added, 27 DEG C digest 15 minutes；
(8) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 7 minutes, centrifugal condition is：Centrifugal force 800g,
6 DEG C of temperature；
(9) supernatant is carefully sucked out and abandons, 1.0mL cell culture complete mediums are then added；
(10) pipettor (matching 1mL suction nozzles, cutting off suction nozzle point makes tip aperture be about 2mm) is used firmly slowly to blow and beat muscle groups
Fragment is knitted about 25 times, until tissue block can be easy, by suction nozzle, to stand 5 minutes；
(11) it places in 100 μm of aperture cell filtering nets to 50mL centrifuge tubes, about 3/4 volume supernatant is transferred to
Filter screen pats tube wall until supernatant is essentially by strainer；
(12) pipettor (matching 1mL suction nozzles) is used slowly to blow and beat remaining 1/4 volume musculature fragment repeatedly about 15 times, until
Tissue block can be easy to pass through suction nozzle；
(13) 1/4 volume musculature suspension of residue is transferred completely into 100 μm of aperture cell filtering nets (see 11), patted
Tube wall is until suspension is essentially by strainer；
(14) it places in 40 μm of aperture cell filtering nets to 50mL centrifuge tubes, filtrate was transferred to (see 11,13)
Strainer pats tube wall until suspension is essentially by strainer；
(15) be recycling more many cells, it is additional be added 4.0mL cell culture complete mediums to 40 μm of aperture cell filtering nets (see
14) tube wall, is patted until cell culture complete medium passes through strainer；
(16) cell suspensions of 5.0mL in total that (14) (15) step obtains are dispensed into 24 porocyte culture plates (TC processing)
Kong Zhong, per hole 1.0mL；
(17) tissue culture plate is placed in mold incubator, 27 DEG C of constant temperature incubations, mold incubator is put into one in bottom
The copper-bath of basin 5% is to maintain humidity in case；
(18) subculture was gently blown and beaten every 3 days and discarding is sucked out, and then the isometric new cell of change is trained completely
Support base.
The original cuiture of 3 Microhyla ornata Skeletal Muscle Cell of embodiment
By the following steps：
(1) solution is prepared；
(2) adult one crymoanesthesia of the female frog for choosing 0.5 gram of weight, after aseptic water washing, with 75% cotton ball soaked in alcohol
Scrape skin；
(3) frog is placed in 3.5 cm diameter plates, is dissected；
(4) scissors and tweezers clip thigh flesh are used, about 30-40mm is cut to3The muscle masses of size are put into new 3.5 centimetres
In diameter dishes, cleaned 3 times with HBSS balanced salt solutions；
(5) cleaned muscle masses are shifted with Pasteur pipe and enter 15mL centrifuge tubes, siphon away remaining HBSS balanced salt solutions,
1.0mL Collagenase I solution is added, tightens centrifuge tube lid, 27 DEG C digest 90 minutes, and centrifuge tube was shaken 1 time every 30 minutes；
(6) whole digestive juices (containing non-digestion of solid musculature) are transferred to a 2mL microcentrifugation with Pasteur pipe
It manages in (EP pipes), merging refrigerated centrifuge centrifuges 15 minutes, and centrifugal condition is：Centrifugal force 500g, 4 DEG C of temperature；
(7) supernatant is carefully sucked out and abandons, 1.0mL trypsin solutions are added, 27 DEG C digest 15 minutes；
(8) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 15 minutes, centrifugal condition is：Centrifugal force
500g, 4 DEG C of temperature；
(9) supernatant is carefully sucked out and abandons, 1.0mL cell culture complete mediums are then added；
(10) pipettor (matching 1mL suction nozzles, cutting off suction nozzle point makes tip aperture be about 2mm) is used firmly slowly to blow and beat muscle groups
Fragment is knitted about 25 times, until tissue block can be easy, by suction nozzle, to stand 5 minutes；
(11) it places in 100 μm of aperture cell filtering nets to 50mL centrifuge tubes, about 3/4 volume supernatant is transferred to
Filter screen pats tube wall until supernatant is essentially by strainer；
(12) pipettor (matching 1mL suction nozzles) is used slowly to blow and beat remaining 1/4 volume musculature fragment repeatedly about 15 times, until
Tissue block can be easy to pass through suction nozzle；
(13) 1/4 volume musculature suspension of residue is transferred completely into 100 μm of aperture cell filtering nets (see 11), patted
Tube wall is until suspension is essentially by strainer；
(14) it places in 40 μm of aperture cell filtering nets to 50mL centrifuge tubes, filtrate was transferred to (see 11,13)
Strainer pats tube wall until suspension is essentially by strainer；
(15) be recycling more many cells, it is additional be added 1.0mL cell culture complete mediums to 40 μm of aperture cell filtering nets (see
14) tube wall, is patted until cell culture complete medium passes through strainer；
(16) cell suspensions of 2.0mL in total that (14) (15) step obtains are dispensed into 24 porocyte culture plates (TC processing)
Kong Zhong, per hole 1.0mL；
(17) tissue culture plate is placed in mold incubator, 27 DEG C of constant temperature incubations, mold incubator is put into one in bottom
The copper-bath of basin 5% is to maintain humidity in case；
(18) subculture was gently blown and beaten every 4 days and discarding is sucked out, and then the isometric new cell of change is trained completely
Support base.
The Observation of biological characteristics and measurement of 4 Microhyla ornata Skeletal Muscle Cell of embodiment
(1) morphological feature：It is observed under inverted phase contrast microscope, which is adherent growth, and it is mostly ball to cultivate the 2nd day
Shape, it is slightly raised, it is about 15-35 μm, individual to extend, it is dispersed in distribution, (Fig. 1, arrow at)；Cultivating the 4th day has proliferation, is in long shuttle
Shape, triangle is about 50-200 μm, wide about 3-25 μm (Fig. 2)；The 6th day apparent growing multiplication of cell is cultivated, in spindle shape and is handed over
Connection, is about 100-200 μm, wide about 3-30 μm (Fig. 3).
(2) qPCR is identified：It is thin with the primer pair Microhyla ornata lung with skeletal development marker gene myoG design primers
Born of the same parents, nephrocyte and Skeletal Muscle Cell of the present invention carry out qPCR respectively.MyoG genes are carried out in the relative expression quantity of these three cells
Welch variance analyses, the results showed that the differential expression of myoG extremely significantly (p in Skeletal Muscle Cell of the present invention, pneumonocyte and nephrocyte
=0.020)；Compare two-by-two (Games-Howell test), the expression quantity of myoG is noticeably greater than pneumonocyte (p in Skeletal Muscle Cell
=0.038) expression quantity indifference (the p=0.130) (figure of myoG and nephrocyte (p=0.038), and in pneumonocyte and nephrocyte
4).Above analysis shows cell of the present invention derives from skeletal muscle tissue rather than the pollution of its hetero-organization really.
(3) cellular immunofluorescence is identified：Primary antibody is respectively mouse monoclonal antibody anti-Alpha Skeletal
Muscle Actin (Abcam Cat.No.ab28052) and anti-Desmin (Abcam Cat.No.ab8976), secondary antibody is mountain
Sheep polyclonal antibody anti-mouse IgG H&L (Alexa488)(Abcam Cat.No.ab 150113).After antibody dyeing,
All nucleus are dyed with DAPI again.In fluorescence microscopy microscopic observation, it is seen that anti-Alpha Skeletal
The cytoplasmic antigen fluoresced green of Muscle Actin (Fig. 5) or anti-Desmin (Fig. 6) marker muscle cell, DAPI marks
The nucleus hair blue-fluorescence (Fig. 5-6) of note, expression positive cell account for the ratio of total cell number, i.e. cell purity average out to 95%
More than.Above analysis shows cell of the present invention is muscle cell property.
(4) it draws a conclusion in conjunction with (2) (3)：Cell of the present invention is Microhyla ornata Skeletal Muscle Cell.
(5) growth of cell：Slowly growth in 2nd day to the 4th day, the 4th day to the 6th day molecular marker for increased proliferation are in logarithmic growth
Phase, the 7th day drastically dead, is in the decline phase (Fig. 7).
Claims (6)
1. a kind of primary culture method of Microhyla ornata Skeletal Muscle Cell, which is characterized in that this approach includes the following steps：
(1) solution is prepared；
(2) choose 0.5-2.0 grams of weight at frog crymoanesthesia, with 75% alcohol surface sterilization；
(3) frog is placed in plate, is dissected；
(4) clip leg flesh enters new culture dish, is cleaned 3 times with HBSS balanced salt solutions；
(5) it shifts cleaned leg flesh and enters centrifuge tube, siphon away remaining HBSS balanced salt solutions, 1.0-1.5mL Collagenase Is are added
Solution, 27 DEG C digest 90 minutes；
(6) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 7-15 minutes, centrifugal condition is：Centrifugal force 500-
800g, 4-6 DEG C of temperature；
(7) supernatant is carefully sucked out and abandons, 1.0-1.5mL trypsin solutions are added, 27 DEG C digest 15 minutes；
(8) by whole digestive juices (containing non-digestion of solid musculature) centrifugation 7-15 minutes, centrifugal condition is：Centrifugal force 500-
800g, 4-6 DEG C of temperature；
(9) supernatant is carefully sucked out and abandons, 1.0mL cell culture complete mediums are then added；
(10) using pipettor (matching 1mL suction nozzles, cutting off suction nozzle point makes tip aperture be about 2mm), firmly slowly piping and druming musculature is broken
Piece, until tissue block can be easy, by suction nozzle, to stand 5 minutes；
It (11) will 100 μm of aperture cell filtration net filtrations of about 3/4 volume supernatant；
(12) pipettor (matching 1mL suction nozzles) is used slowly to blow and beat remaining 1/4 volume musculature fragment repeatedly, until tissue block can hold
Easily pass through suction nozzle；
(13) 1/4 volume musculature suspension of residue is transferred completely into 100 μm of aperture cell filtering net (see 11) filterings；
(14) filtrate (see 11,13) is refiltered once with 40 μm of aperture cell filtering nets；
(15) 1.0-4.0mL cell culture complete mediums are added and enter 40 μm of aperture cell filtering net (see 14) filterings；
(16) cell suspension that (14) (15) step obtains is transferred in tissue culture plate；
(17) tissue culture plate is placed in incubator, 27 DEG C of constant temperature incubations；
(18) cell culture complete medium was changed every 3-4 days.
Whole operation process aseptically carries out, and all consumptive materials, reagent are required to sterile.
2. according to the method described in claim 1, it is characterized in that, a concentration of standard HBSS of the HBSS balanced salt solutions is flat
The 70% of weighing apparatus salting liquid.
3. according to the method described in claim 1, it is characterized in that, the Collagenase I solution is a concentration of containing Collagenase I
0.25%.
4. according to the method described in claim 1, it is characterized in that, the trypsin solution is containing trypsinase concentration
0.175%.
5. according to the method described in claim 1, it is characterized in that, the cell culture complete medium is Leibovitz L-15
The mixture of culture medium, fetal calf serum, Primocin and Y27632, pH value 7.0-7.4.
6. according to the method described in claim 5, it is characterized in that, each component content is in the cell culture complete medium：
Standard Leibovitz L-15 culture mediums 60%, fetal calf serum 10%, pure water 30% and 100 μ g/ml Primocin and 10 μM
Y27632。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108774629A (en) * | 2018-04-16 | 2018-11-09 | 中国科学院成都生物研究所 | A kind of primary culture method of Microhyla ornata alveolar epithelial cells |
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