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Fabrication of Myogenic Engineered Tissue Constructs

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Journal of Visualized Experiments : JoVE

MyJove Corporation

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      Abstract

      Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols [2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel™, and NaHCO 3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size [1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures [6]. Once the tissue has solidified at 37°C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

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      Most cited references 6

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      Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

      The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.
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        Isolation and characterization of human muscle cells.

        We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations of muscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, alpha-actin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.
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          Tissue-engineered skeletal muscle organoids for reversible gene therapy.

          Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.
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            Author and article information

            Affiliations
            Department of Anesthesiology, Children’s Hospital Boston and Harvard Medical School
            Perioperative and Pain Medicine, Children’s Hospital Boston and Harvard Medical School
            Author notes

            Correspondence to: Douglas B. Cowan at douglas.cowan@ 123456childrens.harvard.edu

            Journal
            J Vis Exp
            JoVE
            Journal of Visualized Experiments : JoVE
            MyJove Corporation
            1940-087X
            2009
            1 May 2009
            : 27
            19412158
            2794293
            1137
            10.3791/1137
            Copyright © 2009, Journal of Visualized Experiments

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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            Cellular Biology

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