Micro-perfusion for cardiac tissue engineering: development of a bench-top system for the culture of primary cardiac cells.
Tissue-engineered constructs have high metabolic requirements during in vitro culture necessitating the development of micro-perfusion systems to maintain high functional performance. In this study, we describe the design, fabrication, and testing of a novel micro-perfusion system to support the culture of primary cardiac cells. Our system consists of a micro-incubator with independent stages for 35-mm tissue culture plates with inflow/outflow manifolds for fluid delivery and aspiration. A peristaltic pump is utilized for fluid delivery and vacuum for fluid aspiration. Oxygen saturation, pH, and temperature are regulated for the media while temperature is regulated within the micro-incubator, fluid reservoir, and oxygenation chamber. Validation of the perfusion system was carried out using primary cardiac myocytes, isolated from 2- to 3-day-old neonatal rat hearts, plated on collagen-coated tissue culture plates. Two million cells/plate were used and the perfusion system was run for 1 h (without the need for a cell culture incubator) while controls were maintained in a standard cell culture incubator. We evaluated the cell viability, cell adhesion, total protein, total RNA, and changes in the expression of SERCA2 and phospholamban using RT-PCR, with N = 6 for each group. We found that there was no significant change in any variable during the 1-h run in the perfusion system. These studies served to demonstrate the compatibility of the perfusion system to support short-term culture of primary cardiac cells.
Khait
Luda
L
Section of Cardiac Surgery, University of Michigan, MSRB I, A510E, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA.
Hecker
Louise
L
Radnoti
Desmond
D
Birla
Ravi K
RK
D000818:Animals; D001705:Bioprosthesis; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D006354:Heart, Artificial; D007260:Infusion Pumps; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D010477:Perfusion / Q000295:instrumentation* / Q000379:methods; D051381:Rats; D011916:Rats, Inbred F344; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods