A novel perfusion bioreactor providing a homogenous milieu for tissue regeneration.

We developed a novel perfusion bioreactor that is capable of cultivating multiple 3-dimensional (3D) cellular constructs in one flow chamber with a total cross-section area of 20 cm(2). Two unique features integrated into the bioreactor provided a homogenous fluid flow along the bioreactor cross-section and maximal exposure of the cellular constructs to the perfusing medium. Mathematical modeling of the fluid flow regime in the perfusion bioreactor showed that integrating a flow-distributing mesh 1.5 cm upstream from the construct compartment imposed an equal medium flow and shear stress of 0.6 dynes /cm(2) along the entire cell construct cross-section area. The design of 95.8%open-pore-area fixing nets enabled the exposure of 99.88% of the cell construct volume to the perfusing medium. Cardiac cell constructs seeded with physiologically relevant cell density (0.7 x 10(8) cells/cm(3)) in alginate scaffolds developed into homogenous compacted cardiac tissue, as judged using cell staining with fluorescein diacetate and hematoxylin-eosin histology. The cell constructs maintained 80% viability for nearly 2 weeks, whereas in static-cultivated cell constructs, only 50% of the initial cells remained, as determined according to total DNA content and MTT viability assay. Medium perfusion resulted in better cell viability, presumably due to the convective-diffusive transport of oxygen, compared with oxygen diffusion within the static-cultivated cell constructs, as well as due to efficient removal of harmful cell secretions. It is envisioned that this bioreactor would be useful for 3D cultivation of different mammalian cells for purposes of tissue engineering or production of valuable biologicals.

[1]  Milica Radisic,et al.  Medium perfusion enables engineering of compact and contractile cardiac tissue. , 2004, American journal of physiology. Heart and circulatory physiology.

[2]  W. Zimmermann,et al.  Tissue Engineering of a Differentiated Cardiac Muscle Construct , 2002, Circulation research.

[3]  F J Schoen,et al.  Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. , 1999, Biotechnology and bioengineering.

[4]  Gordana Vunjak-Novakovic,et al.  Effects of oxygen on engineered cardiac muscle. , 2002, Biotechnology and bioengineering.

[5]  Robert E Guldberg,et al.  Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. , 2003, Tissue engineering.

[6]  M. Radisic,et al.  Cell seeding of polymer scaffolds. , 2004, Methods in molecular biology.

[7]  Milica Radisic,et al.  Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Milica Radisic,et al.  High-density seeding of myocyte cells for cardiac tissue engineering. , 2003, Biotechnology and bioengineering.

[9]  P. Greenfield,et al.  Shear sensitivity of three hybridoma cell lines in suspension culture , 1987 .

[10]  L. Griffith,et al.  Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. , 2002, Tissue engineering.

[11]  Smadar Cohen,et al.  Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. , 2002, Biotechnology and bioengineering.

[12]  C. Murry,et al.  Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. , 1999, Circulation.

[13]  L. Shapiro,et al.  Novel alginate sponges for cell culture and transplantation. , 1997, Biomaterials.

[14]  N. Stathopoulos,et al.  Shear stress effects on human embryonic kidney cells in Vitro , 1985, Biotechnology and bioengineering.

[15]  R. Fournier.  Basic Transport Phenomena In Biomedical Engineering , 1998 .

[16]  Feng Zhao,et al.  Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development. , 2005, Biotechnology and bioengineering.

[17]  Antonios G. Mikos,et al.  Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[18]  L. Griffith,et al.  A microfabricated array bioreactor for perfused 3D liver culture. , 2002, Biotechnology and bioengineering.

[19]  Smadar Cohen,et al.  Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication. , 2002, Biomaterials.

[20]  Gordana Vunjak-Novakovic,et al.  Perfusion improves tissue architecture of engineered cardiac muscle. , 2002, Tissue engineering.

[21]  Antonios G Mikos,et al.  Design of a flow perfusion bioreactor system for bone tissue-engineering applications. , 2003, Tissue engineering.

[22]  S. Silver,et al.  Heart Failure , 1937, The New England journal of medicine.