of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 280: C288-C295, 2001.-The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C 2 C 12 myoblast and 10T 1 ⁄2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1-1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After ϳ30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from C 2 C 12 -10T 1 ⁄2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C 2 C 12 -10T 1 ⁄2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures. tissue engineering; myooid; myogenesis; isometric force; rodent tissue culture IN VITRO SKELETAL MUSCLE TISSUE engineering involves culturing isolated myoblasts and fibroblasts in an environment conducive to the formation of a three-dimensional tissue construct. Cell sources employed to engineer such constructs include the C 2 C 12 mouse muscle sarcoma cell line (15,26,29), embryonic avian muscles (1, 21-23, 25, 28), and skeletal muscles of neonatal (21) and adult (5, 17, 27) mammals. To promote formation of a three-dimensional structure, cells have been cultured in collagen gels (1,15,21,26,27,30), on an elastic membrane (1, 21, 28), or between two stationary anchor points to which the cell monolayer remains attached (5,21,23). Collagen gels provide a scaffold for the cells and define the size and shape of the construct but may interfere with the force and power production of the myotubes. In the absence of an artificial scaffold, embryonic avian cells were developed into cylindrical organoids when subjected to uniaxial mechanical strain (28) or when cultured on prestrained elastic substrates (21). In one report, the isometric force of organoids was measured by lateral displacement of the organoids with a calibrated microneedle after elevation of the extracellular K ϩ concentration (28). Cylindrical musclelike structures, termed myooids, were engineered from cocultures of primary mammalian myoblasts and fibroblasts (5), without a preexisting scaffold, the application of uniaxial mechanical strain, or prestressed substrates. Myooids were supported under tension between artificial tendons of laminin-coated silk suture; organoids were attached to stainless steel screens. In the case of myooids, the laminin-coated silk suture segments served as flexurally compl...

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Cellular meshworks and topography of gerbil fibroma cells can be preserved by gentle extraction procedures using Triton X-100. We determined the stability and specificity of these cytoskeletal frameworks by measuring extraction rate and its sensitivity to exogenous protein. Two buffers were used, which mimicked the intracellular and extracellular ionic environments. With both buffers, extraction was nearly complete at 5 min. This pattern of extraction was seen both in 5- and 9-day-old cultures. The same pattern of extraction was seen when three different dilutions of cells were examined the second day after plating. Thus, extraction rate was largely independent of minor variations in ionic composition, age in culture, or cell density. Specificity of the cytoskeletal frameworks so produced was determined by competition with two different exogenous proteins (bovine serum albumin or ovalbumin), which did not remove any additional material from the cytoskeletal frameworks, even with over 10% exogenous protein in the extraction buffer. This pattern of extraction is not unique to gerbil fibroma cells. A similar pattern of extraction was seen for a series of cells: mouse 3T3 cells, 3T6 cells and SVPY 3T3 cells. These experiments indicate that the cytoskeletal framework produced by Triton extraction under appropriate conditions is stable after extraction for a period of 10 min or longer, and that the structures are specific, in that they are not disrupted by the presence of exogenous proteins.

This project and my time at the University of Redlands would never have been possible without the help and support of many important people in my life. First, thank you to my wonderful husband, Don R. Cecil. Without his support and belief in me, I would never have submitted my admissions application in the first place. Don, you were always there with words of encouragement and support, to make edits to writing assignments and presentations, and to pull me back from the edge when things got a little out of control. You are a wise and successful man, and I strive to be more like you every day. This was a challenging year for us personally, but you were always able to take control of the chaos in our lives and keep me focused on my studies. I also owe a debt of gratitude to my family (Rose, Sid, Scott, and Heather), who rallied around me in my time of need and insisted that I not give up. Your support was essential to my success. Thank you! I want to give a big thankyou to my advisor and committee chair, Ruijin Ma, Ph.D. Your technical guidance during the project implementation was critical to my success. I greatly appreciate that you challenged me on my deadlines, and I am convinced this was the only reason I was able to finish on time. Also, I'd like to thank to my second reader and MS GIS faculty member, Mark Kumler, Ph.D. You introduced me to peanut butter pizza, and-on more than one occasion-the free Wi-Fi at Gourmet Pizza Shoppe prevented late assignments when the university network was down. I'm grateful to my client Levi Vincent, executive director of the Greater Palm Springs Film Alliance. Working with you could not have been easier. You are a true professional, and you were always so understanding and accommodating as we worked through the pitfalls and learning experiences this project provided. Your interesting and engaging project idea reignited my love for classic Hollywood movies and introduced me to the world of filmmaking history. Another thankyou goes to University of Redlands alumnus and Cohort 14 member David Robinson, from the Coachella Valley Economic Partnership (CVEP), for bringing this project and for providing me with little doses of reality as someone who has been in this position before. Last but certainly not least, thank you to Andrea Barrios, MS GIS project coordinator. You managed the chaos with a smile every day and made it look easy to keep multiple cohorts moving forward at the same time. From your gentle reminders about turning something in to your always having candy at your desk, your support was appreciated every day. It was such a quick year but I am looking forward to running into you on the Big Thunder Mountain Railroad.