Determinants of cardiomyocyte development in long-term primary culture

Piper, Hans Michael; Jacobson, Stuart L.; Schwartz, Peter J.

doi:10.1016/s0022-2828(88)80007-5

Cited by 78 publications

(32 citation statements)

References 16 publications

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“…In concordance with the results ofothers using serum-free conditions and laminin as substrate (36), cell number at 72 h in control cultures was roughly one-third of the initial inoculum of 105 cells (35,458±4,458). No deterioration of cell number occurred in virus-infected cells (36,141±4,317; Fig.…”

Section: Methodssupporting

confidence: 86%

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Kirshenbaum¹,

MacLellan²,

Mazur³

et al. 1993

J. Clin. Invest.

157

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Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replicationdeficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of 6-galactosidase 48 h after infection. The frequency of lacZ + rodshaped myocytes was half-maximal at 4 x i05 plaque-forming units (PFU) and approached 90% at 1 X 108 PFU. Uninfected cells and cells infected with lacZ -virus remained colorless. fi-galactosidase activity concurred with the proportion of lacZ + cells and was contingent on the exogenous lacZ gene. At 108 PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells. (J. Clin. Invest. 1993. 92:381-387.)

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Section: Methodssupporting

confidence: 86%

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Kirshenbaum¹,

MacLellan²,

Mazur³

et al. 1993

J. Clin. Invest.

157

View full text Add to dashboard Cite

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“…These findings support earlier reports that isolated CMs cultured under 2D conditions undergo dedifferentiation due to the loss of their native biochemical and mechanical environment (3,26,35). The presence of non-CM cell types, such as fibroblasts, in the primary heart isolates has also been demonstrated to negatively affect CM contractility in 2D cul- ture (8,9,20).…”

Section: Discussionsupporting

confidence: 89%

“…Long-term primary cultures of CMs are rarely reported in the literature; generally experimental time frames are limited to 2-3 wk. Previous studies have shown that neonatal CMs lose their contractile phenotype within weeks in culture and that this is associated with CM dedifferentiation and cytoskeleton remodeling (3,26). One potential reason for this could be the cellular heterogeneity of the heart, containing both contractile CMs and noncontractile cells such as fibroblasts, endothelial cells, and smooth muscle cells.…”

mentioning

confidence: 99%

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

Nguyen

Hsiao

Sivakumaran

et al. 2012

American Journal of Physiology-Cell Physiology

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Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro. Am J Physiol Cell Physiol 303: C1220 -C1228, 2012. First published August 29, 2012; doi:10.1152/ajpcell.00449.2011.-Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.cardiomyocyte; tetramethylrhodamine methyl ester perchlorate; enrichment; contractility CARDIOVASCULAR DISEASE is a leading cause of morbidity and mortality in the world (21). The ongoing pandemic of cardiovascular disease has fueled much basic science research to achieve a better understanding of the pathophysiological function of cardiomyocytes (CMs) and for effective strategizing of potential therapies. Cell cultures of cardiomyocytes are commonly used in vitro model for investigation at a cellular level. Primary cardiomyocytes have been successfully isolated from hearts of a range of species [e.g., mouse (16), rat (4), guinea pig (15), rabbit (12), cat (31), and human (27)] using a simple enzymatic method and maintained as primary cultures for a short duration (13).Neonatal rat CM cultures have been widely employed as a cost-effective experimental model to study the contractility, electrophysiology, and morphology of CMs. CMs from postnatal hearts do not normally proliferate in culture (30), thus limiting the number of CMs available to the starting material. Long-term primary cultures of CMs are rarely reported in the literature; generally experimental time frames are limited to 2-3 wk. Previous studies have shown that neonatal CMs lose their contractile phenotype within weeks in culture and that this is associated with CM dedifferentiation and cytoskeleton remodeling (3, 26). One potential reason for this could be the cellular heterogeneity of the heart, containing both contractile CMs and noncontractile cells such as fibroblasts, endothelial cells, and smooth muscle cells. The noncontractile cells, especially fibroblasts, can proliferate rapidly in culture and typically overgrow CMs to a point where it is belie...

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“…Alternating flow was utilized to minimize cell washout during attachment and to maintain unrestricted flow through the scaffold pores. Matrigel® was selected because it contains laminin and collagen IV, known to promote surface attachment of cardiac myocytes (Borg et al, 1984;Lundgren et al, 1985;Piper et al, 1988). Feasibility studies were carried out using neonatal rat cardiac myocytes because these are the most frequently used cells in cardiac tissue engineering.…”

Section: Discussionmentioning

confidence: 99%

“…The seeding time was set to 1.5 h (low) and 4.5 h (high), based on the attachment times of 1-4 h reported for cardiomyocytes seeded on surfaces coated with collagen IV, laminin, and fetal bovine serum (Borg et al, 1984;Lundgren et al, 1985;Piper et al, 1988) and the attachment times of 1-5 h reported for C2C12 cells seeded on untreated glass surfaces (Acarturk et al, 1998) or laminin-and collagen-covered glass (unpublished data from our laboratory). Since Matrigel® contains both laminin and collagen type IV and the Ultrafoam contains collagens type I and II, it was expected that most cells would attach within 1.5 h.…”

Section: Experimental Designmentioning

confidence: 99%

High‐density seeding of myocyte cells for cardiac tissue engineering

Radišić

Euloth

Yang

et al. 2003

Biotech & Bioengineering

266

233

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Tissue engineering of 1- to 5-mm-thick, functional constructs based on cells that cannot tolerate hypoxia for prolonged time periods (e.g., cardiac myocytes) critically depends on our ability to seed the cells at a high and spatially uniform initial density and to maintain their viability and function. We hypothesized that rapid gel-cell inoculation in conjunction with direct medium perfusion through the seeded scaffold would increase the rate, yield, viability, and uniformity of cell seeding. Two cell types were studied: neonatal rat cardiomyocytes for feasibility studies of seeding and cultivation with direct medium perfusion, and C2C12 cells (a murine myoblast cell line) for detailed seeding studies. Cells were seeded at densities corresponding to those normally present in the adult rat heart ([0.5-1] x 10(8) cells/cm(3)), into collagen sponges (13 mm x 3 mm discs), using Matrigel as a vehicle for rapid cell delivery. Scaffolds inoculated with cell-gel suspension were seeded either in perfused cartridges with alternating medium flow or in orbitally mixed Petri dishes. The effects of seeding time (1.5 or 4.5 h), initial cell number (6 or 12 million cells per scaffold), and seeding set-up (medium perfusion at 0.5 and 1.5 mL/min; orbitally mixed dishes) were investigated using a randomized three-factor factorial experimental design with two or three levels and three replicates. The seeding cell yield was consistently high (over 80%), and it appeared to be determined by the rapid gel inoculation. The decrease in cell viability was markedly lower for perfused cartridges than for orbitally mixed dishes (e.g., 8.8 +/- 0.8% and 56.3 +/- 4%, respectively, for 12 million cells at 4.5 h post-seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100-200 microm) surface layer in dish seeded constructs. Over 7 days of cultivation, medium perfusion maintained the viability and differentiated function of cardiac myocytes, and the constructs contracted synchronously in response to electrical stimulation. Direct perfusion can thus enable seeding of hypoxia-sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function.

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Determinants of cardiomyocyte development in long-term primary culture

Cited by 78 publications

References 16 publications

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

High‐density seeding of myocyte cells for cardiac tissue engineering

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