Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis

To isolate mitochondrial complexes, we have combined elements from the classic Laemmli protocol and blue native polyacrylamide gel electrophoresis (BN–PAGE) methods to develop a straightforward modified native electrophoresis protocol. This modified protocol presented good resolution for native electrophoresis of inner mitochondrial membrane proteins, where bands were easily visualized with no leftover stain or gel lanes overlap. Enzymatic tests revealed that complexes I and V remain active in the gel. This protocol, designed to overcome specific limitations of the standard protocols, provides a potential methodology to study membrane proteins in their functional form.     

A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods

Background  

Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.     

In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro

The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The basic steps involve creating a "scratch" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. Besides monitoring migration of homogenous cell populations, this method has also been adopted to measure migration of individual cells in the leading edge of the scratch. Not taking into account the time for transfection of cells, in vitro scratch assay per se usually takes from several hours to overnight.     

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.     

An introduction to the wound healing assay using live-cell microscopy

Abstract

The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.     

A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment

In vitro systems that mimic organ functionality have become increasingly important tools in drug development studies. Systems that measure the functional properties of skeletal muscle are beneficial to compound screening studies and also for integration into multiorgan devices. To date, no studies have investigated human skeletal muscle responses to drug treatments at the single myotube level in vitro. This report details a microscale cantilever chip-based assay system for culturing individual human myotubes. The cantilevers, along with a laser and photo-detector system, enable measurement of myotube contractions in response to broad-field electrical stimulation. This system was used to obtain baseline functional parameters for untreated human myotubes, including peak contractile force and time-to-fatigue data. The cultured myotubes were then treated with known myotoxic compounds and the resulting functional changes were compared to baseline measurements as well as known physiological responses in vivo. The collected data demonstrate the system's capacity for screening direct effects of compound action on individual human skeletal myotubes in a reliable, reproducible, and noninvasive manner. Furthermore, it has the potential to be utilized for high-content screening, disease modeling, and exercise studies of human skeletal muscle performance utilizing iPSCs derived from specific patient populations such as the muscular dystrophies.     

Optimization of human skeletal muscle precursor cell culture and myofiber formation in vitro

Muscle bioengineering is proposed as a treatment option for various conditions requiring restoration of muscle function. In order to allow for rapid clinical translation culture conditions have to be optimized for human application. The optimal isolation and culture technique should be able to support cell growth and differentiation using defined media only. Therefore, we have evaluated alternative culture conditions to determine the optimal growth condition for the engineering of human skeletal muscle. In this research, we present protocols for consistent isolation and growth of human muscle precursor cells (MPCs). MPCs were grown from human biopsies and expanded in culture using defined media and collagen coated dishes only. The best results were achieved using a one-step pre-plating and by supplementing the growth medium with insulin, dexamethasone, human basic fibroblast growth factor (hFGF) and human epithelial growth factor (hEGF). Detailed cell characterization using fluorescence-activated cell-sorting analysis and morphological analysis at different passages were performed. Further, the applicability of these cells for tissue engineering purposes was assessed by measuring expansion potential, formation of myofibers and fused myotubes. We have established a culture technique for human MPCs that allows for reliable cell growth and expansion using collagen coated dishes and defined media only. Cell characterization demonstrated a muscle phenotype and the ability to form myofibers in vitro.     

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay

The interaction between myosin and actin in striated muscle tissue is regulated by Ca²⁺ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, β-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, β-, and Γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.     

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay

Molecular Biology Reports - The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at...     

Heparin inhibits skeletal muscle growth in vitro

Heparin or heparan sulfate proteoglycan (HeSPG), but not chondroitin sulfate or hyaluronic acid, exerts a pronounced inhibitory effect on muscle growth in vitro, as determined by total protein, myosin accumulation or synthesis, and [3H]thymidine incorporation studies. Primary muscle fibroblast culture growth is also inhibited by heparin but to a substantially lesser degree compared to muscle (30% and over 90% inhibition of growth, respectively). Heparin-induced inhibition of skeletal muscle growth is a consequence of its interaction with a growth factor(s) present in the media used to support myogenesis; heparin-Sepharose column absorbed horse serum can support muscle growth only in the presence of added heparin-binding growth factors like fibroblast growth factor (FGF) or chicken muscle growth factor (CMGF). Furthermore, heparin prevents the binding of iodinated FGF to the myoblast surface. We also show that the extent of muscle growth is a function of the relative amounts of heparin and FGF in culture. Finally, we provide evidence indicating that FGF can combine with endogenously occurring heparin-like components: immobilized FGF binds sodium-[35S]sulfate labeled components secreted in muscle culture conditioned medium, an interaction inhibited by anti-HeSPG antibodies or heparin, but not by other sulfated glycosaminoglycans. Since heparin binding growth factors not only stimulate myoblast proliferation but also actively inhibit the onset of muscle differentiation (G. Spitzz, D. Roman, and A. Strauss (1986). J. Biol. Chem. 261, 9483-9488), their interaction with naturally occurring heparin-like components may be an important physiological mechanism for modulating muscle growth and differentiation in development and regeneration.     

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.     

Mathematical analysis of a basic model for epidermal wound healing

The stimuli for the increase in epidermal mitosis during wound healing are not fully known. We construct a mathematical model which suggests that biochemical regulation of mitosis is fundamental to the process, and that a single chemical with a simple regulatory effect can account for the healing of circular epidermal wounds. The numerical results of the model compare well with experimental data. We investigate the model analytically by making biologically relevant approximations. We then obtain travelling wave solutions which provide information about the accuracy of these approximations and clarify the roles of the various model parameters.     

Tissue dispersion and flow cytometry for the cellular analysis of wound healing

Injury induces a flux in the cellular composition of tissues as part of the wound healing response. There is no reliable and rapid method to quantify and characterize the cellular composition of the matrix-rich wound. Our aim was to develop a rapid method to quantify the cellular composition in wounds by tissue dispersion and flow cytometry. Age- and weight-matched mice were wounded on the dorsum using a 1.5 x 1.5 cm2 template, and the wounds were excised at predetermined time points. Tissues were dispersed into single-cell suspensions and labeled with antibodies to cell surfaces and intracellular antigens. Flow cytometry was performed to quantify the percentage of each cell population and cell death. We found that our tissue dispersion protocol resulted in low cell death (4%-6%) and very high yield (80-220 x 10(6) cells/g). Furthermore, cell surfaces and intracellular antigens were preserved to provide accurate identification of the different cell populations. With the appropriate modifications, this protocol is likely to be applicable for the viable retrieval and identification of cells from skin and other collagen-dense tissues.     

Androgen metabolism in rat skeletal muscle in vitro

Androgen metabolism by the cytosol fraction of rat skeletal muscle was investigated. Testosterone metabolism was low, the main metabolite being 4-androstene-3α, 17β-diol. In addition, small amounts of 5α-androstane-3a,17β-diol were formed, but no 17β-hydroxy-5α-androstane-3-one could be detected. 4-Androstene-3α,17β-diol was metabolized only to testosterone in this system of incubation. When 17β-hydroxy-5α-androstane-3-one was incubated with muscle cytosol, considerable metabolism to 5α-androstane-3α,17β-diol and to 5α-androstane-3β,17β-diol could be detected. Low 5α-reduction of testosterone and rapid conversion of formed 17α-hydroxy-5α-androstane-3-one to 5α-androstane-3α, 17β-diol and 5α-androstane-3β,17β-diol gave limited ability of the muscle preparation employed to accumulate 17β-hydroxy-5α-androstane-3-one.     

Myostatin induces autophagy in skeletal muscle in vitro

Myostatin is an important regulator of muscle mass that contributes to the loss of muscle mass in a number of chronic diseases. Myostatin is known to activate the expression of components of the ubiquitin-proteosomal pathway but its effect on the autophagic pathway is not known. We therefore analysed the effect of myostatin and TGF-β on autophagy in C2C12 cells by determining the effect of these proteins on LC3 processing, autophagosome formation and autophagy gene expression. Both myostatin and TGF-β increased LC3II expression and turnover as well as autophagosome formation (marked by the formation of puncta in LC3-GFP transfected cells). Myostatin also significantly increased the expression of ATG-4B and ULK-2 mRNA while TGF-β caused a trend towards an increase in these genes. We conclude that myostatin and TGF-β increase autophagy in skeletal muscle cells.     

A high throughput, interactive imaging, bright-field wound healing assay

The wound healing assay is a commonly used technique to measure cell motility and migration. Traditional methods of performing the wound healing assay suffer from low throughput and a lack of quantitative data analysis. We have developed a new method to perform a high-throughput wound healing assay that produces quantitative data using the LEAP? instrument. The LEAP? instrument is used to create reproducible wounds in each well of a 96-well plate by laser ablation. The LEAP? then records bright field images of each well at several time points. A custom texture segmentation algorithm is used to determine the wound area of each well at each time point. This texture segmentation analysis can provide faster and more accurate image analysis than traditional methods. Experimental results show that reproducible wounds are created by laser ablation with a wound area that varies by less than 10%. This method was tested by confirming that neuregulin-2β increases the rate of wound healing by MCF7 cells in a dose dependent manner. This automated wound healing assay has greatly improved the speed and accuracy, making it a suitable high-throughput method for drug screening.     

In vitro studies of skeletal muscle membranes

Summary Sarcolemmal membranes were prepared from slow-twitch (red) and fasttwitch (white) skeletal muscle of the rat. A sensitive adenylate cyclase assay was used and basal, fluoride- and catecholamine-stimulated activities measured. The greaterin vivo sensitivity of red muscle to the effects of catecholamines correlates, in the present study, with approximately a twofold stimulation of its sarcolemmal adenylate cyclase with isoproterenol (10 m). The white muscle enzyme, on the other hand, is only minimally stimulated (20%) at the same concentration of -adrenergic agonist. Fast-twitch muscle is known to be physiologically insensitive to catecholaminein vivo.A course of sciatic nerve denervation was followed to further distinguish these two metabolic types of skeletal muscle and their respective adenylate cyclases. The slow-twitch muscle enzyme activities were completely and permanently lost on denervation. The white muscle enzyme, however, recovered almost completely after an initial reduction in specific activity the first week. Interestingly, the NaF-stimulated activity lagged behind both the basal and hormone-stimulated activities of the white muscle enzyme, in returning to control levels. The activities of cyclic nucleotide phosphodiesterase were evaluated in homogenates of the two muscle types in innervated rats and following denervation, in order to further define the neural influence on skeletal muscle cyclic nucleotide metabolism.The results suggest that the motor nerve may regulate some of the metabolic properties of slow-twitch muscle (which may involve cyclic AMP) by controlling the responsiveness of its sarcolemmal-bound adenylate cyclase system.Presented in part at the 60th Annual Meeting, Federation of American Societies for Experimental Biology, April, 1976, Anaheim, California.     

Biogenesis of transverse tubules in skeletal muscle in vitro

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.