{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T10:50:20Z","timestamp":1791024620740,"version":"4.1.0"},"reference-count":29,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2003,3,5]],"date-time":"2003-03-05T00:00:00Z","timestamp":1046822400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2003,5,20]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Tissue engineering of 1\u2010 to 5\u2010mm\u2010thick, 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\u2013cell 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\u20131] \u00d7 10<jats:sup>8<\/jats:sup> cells\/cm<jats:sup>3<\/jats:sup>), into collagen sponges (13 mm \u00d7 3 mm discs), using Matrigel\u00ae as a vehicle for rapid cell delivery. Scaffolds inoculated with cell\u2013gel 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\u2010up (medium perfusion at 0.5 and 1.5 mL\/min; orbitally mixed dishes) were investigated using a randomized three\u2010factor 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 \u00b1 0.8% and 56.3 \u00b1 4%, respectively, for 12 million cells at 4.5 h post\u2010seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100\u2013200 \u03bcm) 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\u2010sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function. \u00a9 2003 Wiley Periodicals, Inc. 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