An In Vitro System to Evaluate the Effects of Ischemia on Survival of Cells Used for Cell Therapy
Department of Biomedical Engineering, Duke University, Durham, NC, USA.Annals of Biomedical Engineering (Impact Factor: 3.2). 09/2007; 35(8):1414-24. DOI: 10.1007/s10439-007-9301-2
Maintaining cell viability is a major challenge associated with transplanting cells into ischemic myocardium to restore function. A likely contributor to significant cell death during cardiac cell therapy is hypoxia/anoxia. We developed a system that enabled quantification and association of cell survival with oxygen and nutrient values within in vitro constructs. Myoblasts were suspended in 2% collagen gels in 1 cm diameter x 1 cm deep constructs. At 48 +/- 3 h post-seeding, oxygen levels were measured using microelectrodes and gels were snap-frozen. Bioluminescence metabolite imaging and TUNEL staining were performed on cryosections. Oxygen and glucose consumption and lactate production rates were calculated by fitting data to Fick's second law of diffusion with Michaelis-Menten kinetics. Oxygen levels dropped to 0 mmHg and glucose levels dropped from 4.28 to 3.18 mM within the first 2000 mum of construct depth. Cell viability dropped to approximately 40% over that same distance and continued to drop further into the construct. We believe this system provides a reproducible and controllable test bed to compare survival, proliferation, and phenotype of various cell inputs (e.g., myoblasts, mesenchymal stem cells, and cardiac stem cells) and the impact of different treatment regimens on the likelihood of survival of transplanted cells.
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ABSTRACT: Skeletal muscle stem cells known as satellite cells are responsible for muscle regeneration. Upon muscle injury, previously quiescent satellite cells become activated as proliferating myogenic precursors, differentiate into myoblasts, and ultimately fuse into new, multinucleated myofibers. Unfortunately, this paradigm breaks down with aging and instead of becoming activated upon injury, satellite cells remain quiescent. Recent work, however, has shed light on the mechanisms behind this impaired regeneration and these findings suggest several therapeutic avenues. Skeletal muscle tissue engineering aims to create functional muscle in vitro followed by engraftment in vivo for the replacement or repair of missing or pathological tissue in various dystrophies or myopathies. Biomaterials have rapidly become central to these regeneration efforts and numerous repair strategies already exist. However, optimization of these biomaterial platforms in order to more fully mimic the in vivo adult skeletal muscle niche is still necessary. More importantly perhaps, the effects of aged or pathological environments on skeletal muscle engraftment has yet to be fully characterized. Furthermore, debate still remains over whether or not all satellite cells (considered to be heterogeneous in genetic markers and functional properties) are in fact stem cells, and what implications this could have on in vitro regeneration strategies. Novel uses and advances in biomaterials show promise in tackling these problems. Therefore, after a discussion of muscle regeneration in both the ‘young’ and ‘aged’ niches, this chapter will examine the most up-to-date strategies for in vitro skeletal muscle regeneration and will discuss how current efforts in biomaterial technologies might be used to accurately determine the significance of satellite cells as muscle stem cells, and control robust production of myofibers in vitro by mimicking the in vivo niche.01/1970: pages 275-308;
- IEEE Engineering in Medicine and Biology Magazine 01/2008; 27(5):109-13. · 26.30 Impact Factor
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ABSTRACT: Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate the repair and reconstruction of a variety of tissues in clinical and pre-clinical studies. The clinical utility of such scaffolds can be limited by the geometric and mechanical properties of the tissue or organ from which the ECM is harvested. An injectable gel form of ECM could potentially conform to any three-dimensional shape and could be delivered to sites of interest by minimally invasive techniques. The objectives of the present study were to prepare a gel form of ECM harvested from the urinary bladder (urinary bladder matrix or UBM), to characterize the rheological properties of the gel, and finally to evaluate the ability of the gel to support in vitro growth of smooth muscle cells. Following enzymatic solubilization with pepsin, UBM was induced to self-assemble into a gel when brought to physiological conditions. The UBM gel supported the adhesion and growth of rat aortic smooth muscle cells when cultured under static in vitro conditions. The present study showed that an intact form of UBM can be successfully solubilized without purification steps and induced to repolymerize into a gel form of the UBM biologic scaffold material.Biomaterials 05/2008; 29(11):1630-7. DOI:10.1016/j.biomaterials.2007.12.014 · 8.56 Impact Factor
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