Efficient and high yield isolation of myoblasts from. Keep the medium to surface area ratio at 1ml per 5 cm 2. Negatively tested for mousepox ectromelia virus, the l6 cell line can fuse in a cell culture, creating multinucleated myotubes as well as striated fibers. The l6 cells are also used as host cells for trypanosoma cruzi and are therefore interesting to assess selectivity for this parasite. L6 a rat skeletal myoblast cell line or ht29 a human bladder carcinoma cell line. Pdf an alternate protocol for establishment of primary. Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. Primary cultures of fetal cells were plated at an initial cell density of 27x 10 cells per loomm dish, and sorted by facs 2 to 5 days later. The original cell line, c2, was obtained by yaffe and saxel in 1977 by establishing primary cultures from the thigh muscle of 2 month old normal mice, 70 hours after crush injury2. Human myoblast viability depends greatly on the use of suitable media, reagents, and. Isolation of human myoblasts with the fluorescence.
A myoblast laden collagen bioink with fully aligned au nanowires for muscletissue regeneration. Simultaneous isolation of enriched myoblasts and fibroblasts. Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Two kinds of myoblasts were employed in these studies. Here, we describe an improved protocol for culturing muscle cells to promote the formation of complex achr clusters. Isolation of human myoblasts with the fluorescenceactivated. C2c12 cells go through distinct differentiation steps after serum withdrawal. Remove the medium from culture flasks by aspiration. Simultaneous isolation of enriched myoblasts and fibroblasts for.
Mesenchymal stem cells and myoblast differentiation under hgf. Modified eagles minimum essential medium mem earles balanced salt solution bss 2 mm lglutamine 0. This is an established myoblast cell line with comparatively relaxed growth requirements dmem supplemented with 10% fetal bovine serum fbs. Conditions that promote primary human skeletal myoblast. Characterization of the purification and primary culture of adult. Cell cultures two cell lines are used for cytotoxicity determination. Towards a molecular pathway for myoblast fusion in. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the. An improved method for culturing myotubes on laminins for the. A simple method we have used for growing avian muscle in culture is also described, and the use of some mouse cell lines is. Tissue and organ regeneration via transplantation of cell bodies insitu has become an.
Dose and time dependent effect of dex on c2c12 myoblast cell line was analyzed at myoblast and myotube stage, respectively. Preparation and culture of myogenic precursor cellsprimary. The protocol describes the different steps for obtaining a primary cell line from biopsies of human muscle after enzyme digestion. Loner localizes to subcellular sites of fusion and acts downstream of cell surface fusion receptors by recruiting the. The level of differentiation was determined by myh1, pax7, atrogin1, and myostatin mrna expression and fusion index. Decontaminate the bottle with 70% alcohol in a sterile hood. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or r bio protoc. Myoblast fusion is essential for the formation and regeneration of skeletal muscle. One method involves enzymatic digestion of muscle tissues to liberate single. Isolation, culturing, and differentiation of primary. Rainbow trout slow myoblast cell culture as a model to. All experimentation involving human tissue described. Research paper dexamethasone treatment at the myoblast stage.
Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. Do this and all subsequent steps in a sterile tissue culture hood. Mesenchymal stem cells and myoblast differentiation under. Generation and use of cultured human primary myotubes. Mouse myoblast c2c12 cells were obtained from american type culture collection atcc, crl1772 and maintained in dmem with 10% fbs in a 37c and 5% co 2 incubator. Pdf current methods of isolation of muscle satellite cells from different animal species are highly variable making interspecies comparisons. In brief, the tissue was processed to remove the extraconnective tissues, digested twice with 0. Establishment of primary myoblast cell cultures from. C2c12 immortalised skeletal muscle cell line myoblasts were extracted from the thigh muscle of c3h mice 70 h after a crush injury and cultured. Which is the best protocol to differentiate c2c12 cells. It permits the analysis of cell specific functions which can relate to changes in certain disease states a t the single cell level. Oct 15, 2018 this protocol enabled us to obtain highly pure myoblast cell cultures with 90% desminpositive cells fig. Stable knockdown of genes encoding extracellular matrix.
Earles balanced salt solution bss 2 mm lglutamine 0. Cell culture methods for the isolation of myoblast and fibroblast clones, and for the longterm propagation of highdensity cultures, have been described 2, 51. Pipette 5 ml of trypsinedta solution into the t75 flask. Bovine myoblast cell production in a microcarriersbased system. Myoblast cell culture satellite cells were isolated from hind limb muscles of male lewis rats charles river, wilmington, massachusetts, usa as described previously 18. A triple coculture method to investigate the effect of. The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Wash the monolayer of cells with hbss and remove the solution by aspiration. The cells were cultured with the dmem dulbeccos modified. This approach enables the isolation of an enriched population of primary myoblasts for functional studies. Collagen type i to coat cell culture plate see recipe 2.
Always wear gloves and work behind a protective screen when handling primary human cells. In goat, only a few protocols for establishing a myogenic cell culture are available and. Human skeletal muscle cells hskmc culture protocol sigma. Dexamethasone treatment at the myoblast stage enhanced c2c12. Silicon substrate as a novel cell culture device for myoblast cells mohammod k bhuyan1, jorge i rodriguezdevora2, kym fraser3,4 and tzuliang bill tseng5 abstract background. If there are 20% cells left in the flask, repeat steps 29.
Owing to the compartmentalization of fish muscle fibres, we have developed a slow myoblast cell culture for rainbow trout oncorhynchus mykiss. Caprine adult myoblast and fibroblast cell culture. Apr 03, 2018 this procedure generated low passage, pure myoblast and fibroblast populations. The c2c12 cell line differentiates rapidly, forming contractile myotubes and producing characteristic muscle proteins. May 17, 2011 c2c12 cell culture, differentiation treatment, and crosslinking protocol. For cell culture, ham s f10 medium gibco, carlsbad, california, usa containing 25% fcs biochrom gmbh, berlin, germany, 1. Many steps of myogenesis can be recapitulated through in vitro. Atmospheric oxygen tension slows myoblast proliferation via. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle. Somatic celllike features of cloned mouse embryos prepared.
Large scale cell production is not only necessary to achieve large. Low passage cells are normally smaller in size therefore use the higher end of the range to achieve desired confluency, whereas higher passage cells are more elongated and larger in size so the lower. Molecular medicine vol107 human cell culture protocols, 2nd ed. All media, supplements, and tissue cultureware used in this protocol should be sterile. We stably knocked down adamtsl2 in the murine c2c12 myoblast cell line. Preparation and culture of myogenic precursor cells. Isolation, culture, and differentiation of primary. Human primary cultured skeletal muscle cells can directly reflect a. Rock the flask gently to ensure the solution covers all the cells. C 5% co2 incubator and change the medium every 2 days, using f10based primary myoblast growth medium. Myoblast fusion, by which mononucleated myoblasts fuse to form multinucleated muscle fibers, is an essential early step during skeletal muscle differentiation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient. C2c12 myoblasts were obtained from the american type culture collection atcc, manassas, va.
Pdf basic cell culture protocols mohamed ali academia. Aspirate transport media and wash specimen with 10 ml pbs. An alternate protocol for establishment of primary caprine fetal myoblast cell culture. For best longterm maintenance, cells should be grown on collagen coated dishes. Most studies of myoblast fusion during the past three decades have been carried out in mammalian cell culture systems in which myoblast fusion can be synchronized. Efficient and high yield isolation of myoblasts from skeletal muscle. Muscle is generated from human pluripotent stem cells in 3 steps, via satellite like. Research open access silicon substrate as a novel cell. This and other cell lines below were authenticated by each provider. An alternate protocol for establishment of primary caprine. Aspirate supernatant and resuspend the cell pellet carefully in freezing medium. C2c12 cell culture, differentiation treatment, and crosslinking protocol. Incubating the cells in 100% methanol chilled at 20c at room temperature for 5 min 2. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation.
Move the muscle tissue by gently agitating the tube every 5 10 min. Using primary myoblasts andor a c2c12 myoblast cell line, several. Myoblast myogenic differentiation but not fusion process is. Cell culture guidelines the following is a general guideline for culturing of cell lines. Culture of myoblast on micro coil spring with electric pulses. A number of cell culture models have been established.
Isolation, culture, and transplantation of muscle satellite cells jove. The cell line c2c12 is an immortal line of mouse skeletal myoblasts originally derived from satellite cells from the thigh muscle of a two month old female c3h mouse donor 70h after a crush injury yaffe and saxel, 1977. Yaffe using rat rattus norvegicus thigh muscle and resembles skeletal muscle tissue. L6 transfection reagent rat myoblast cells, crl1458. Rainbow trout slow myoblast cell culture as a model to study. Feb 15, 2014 culture conditions were chosen on the basis of myoblast response to growth factorreduced matrigel gfrmg coating, media serum level, application of cyclic stretch, and transient inhibition of mir3a compared with widely used c2c12 cells as reference. Cell culture is a widely used technique in biomedical research.
Corning costar, or greiner bioone cellstar tissue culture treated plates and flasks. Cell growth protocol and differentiation treatment for the. Cultures populations typically comprise of 7090% muscle myotybes and. Take the skeletal muscle cell growth medium from the refrigerator. The cell line c2c12 is an immortal line of mouse skeletal myoblasts. Human myoblast culture as muscle stem cells in medical and. Initially we showed that the inhibition of proliferation by hyperoxia occurs during culture of cells from both adult 1620 weeks old and aged 3240 weeks old mice.
Also, l6 cells have a propensity for high cellular. In a genetic screen for regulators of muscle development in drosophila, we discovered a gene encoding a guanine nucleotide exchange factor, called loner, which is required for myoblast fusion. Myoblast enrichment at each preplate level pp1pp5 was determined using the myoblast markers pax7 and desmin. The presence of pure myoblasts in the cell cultures was further confirmed by their differentiation capacity to form multinucleated myotubes in low serum conditions fig. Research paper dexamethasone treatment at the myoblast. Pipette 15 ml of skeletal muscle cell growth medium to a t75 flask. This protocol describes techniques for dissociation of cells from human skeletal.
Primary culture vs cell line primary culture freshly isolated from tissue source cell line finite cell line. This protocol enabled us to obtain highly pure myoblast cell cultures with 90% desminpositive cells fig. This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. February 16th, 2017 this protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. Silicon substrate as a novel cell culture device for. Cell culture basics handbook thermo fisher scientific. Gel preparation video gel filling video coating the media channels media channel hydration video cell seeding m edia replacement video cell staining.
Add appropriate aliquots of the cell suspension to new culture flasks a. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Tissue and organ regeneration via transplantation of cell bodies insitu has become an interesting strategy in regenerative medicine. We recently started to use cell and tissue culture for food application where the required scale is even orders of magnitudes higher. In vitro drug testing based on contractile activity of c2c12 cells in an. For cell culture, ham s f10 medium gibco, carlsbad, california, usa containing 25%. These cell lines have also been used to establish myotube cultures to. An in vitro model using spheroidsladen nanofibrous. C2c12 myoblasts and their differentiation into myotubes is a welldescribed and widely used cell culture model for skeletal muscle differentiation and skeletal muscle bioengineering 8, 9. Do not leave cells in the same culture dish for more than 5 days, regardless of cell density. Isolation of primary human skeletal muscle cells request pdf. Dmem high glucose invitrogen, ca, usa with 10% fetal bovine serum fbs hyclone, ut, usa. The tenth passage of c2c12 mouse myoblast cell line originated with crossstriated muscle of c3h mouse was seeded in the area inside of the pdms ring on the culture dish at the concentration of 0 cells per cm2.
Furthermore, we established a wound healing protocol that uses these two cell types in a co culture system to mimic the in vivo environment more closely. Primary cultures derived from skeletal muscle consist mainly of myoblasts and fibroblasts and. The muscles used here include foot and leg muscles. Human skeletal muscle cells hskmc culture protocol. Pdf a simplified but robust method for the isolation of avian and. Spin the cells at 600g for 5 mins and aspirate by gently pipetting 6. Myoblast introduction large scale culture of mammalian, anchor dependent cells is a necessary condition for cell therapy and tissue engineering.
Do not agitate the cells by hitting or shaking the flask while waiting for cells to detach. Control of myoblast fusion by a guanine nucleotide. The most established cell line enabling the study of muscle. Perform the following steps using a shaker for the incubation steps. Compared to other protocols, this method yields a higher number of myoblasts 1020 million by enabling the outgrowth of these cells from tissue fragments. Mar 29, 2018 following co culture with 20 x 10 3 cells, myoblast wound closure had increased significantly to 19. Isolation, culturing, and differentiation of primary myoblasts from. Immunocytochemistry and immunofluorescence protocol. The protocol describes the different steps for obtaining a primary cell line from biopsies of human muscle, cultured as explants.
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