ABSTRACT
Blood vessel engineering requires an understanding of the parameters governing the survival of resident vascular smooth muscle cells. We have developed an in vitro, collagen-based 3D model of vascular media to examine the correlation of cell density, O2 requirements, and viability. Dense collagen sheets (100 micron) seeded with porcine pulmonary artery smooth muscle cells (PASMCs) at low or high (11.6 or 23.2x10(6) cells/mL) densities were spiraled around a mandrel to create tubular constructs and cultured for up to 6 days in vitro, under both static and dynamic perfusion conditions. Real-time in situ monitoring showed that within 24 hours core O2 tension dropped from 140 mmHg to 20 mmHg and 80 mmHg for high and low cell density static cultures, respectively, with no significant cell death associated with the lowest O2 tension. A significant reduction in core O2 tension to 60 mmHg was achieved by increasing the O2 diffusion distance of low cell density constructs by 33% (p<0.05). After 6 days of static, high cell density culture, viability significantly decreased in the core (55%), with little effect at the surface (75%), whereas dynamic perfusion in a re-circulating bioreactor (1 ml/min) significantly improved core viability (70%, p<0.05), largely eliminating the problem. This study has identified key parameters dictating vascular smooth muscle cell behavior in 3D engineered tissue culture.
Subject(s)
Bioreactors , Muscle, Smooth, Vascular/metabolism , Oxygen Consumption , Oxygen/metabolism , Perfusion/instrumentation , Tissue Culture Techniques/instrumentation , Tissue Engineering , Animals , Animals, Newborn , Cell Survival , Cells, Cultured , Collagen Type I/metabolism , Diffusion , Gels , Phenotype , Swine , Time FactorsABSTRACT
A novel approach was developed for the construction of biofilms with defined internal architecture using AC electrokinetics and flocculation. Artificial structured microbial consortia (ASMC) consisting of localized layered microcolonies of different cell types were formed by sequentially attracting different cell types to high field regions near microelectrodes using dielectrophoresis. Stabilization of the microbial consortia on the electrode surface was achieved by crosslinking the cells using the flocculant polyethyleneimine (PEI). Consortia of Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae were made as model systems. Also, more natural consortia were made of the bacteria Pseudomonas putida, Clavibacter michiganense, and Methylobacterium mesophilum, which are found together in consortia during biodegradation of metal-cutting waste fluids.
Subject(s)
Bacterial Adhesion/drug effects , Biofilms/growth & development , Cell Culture Techniques/methods , Electrophoresis/methods , Polyethyleneimine/pharmacology , Actinomycetales/cytology , Actinomycetales/drug effects , Actinomycetales/physiology , Bacterial Adhesion/radiation effects , Biofilms/drug effects , Biofilms/radiation effects , Dose-Response Relationship, Drug , Electromagnetic Fields , Escherichia coli/cytology , Escherichia coli/drug effects , Escherichia coli/physiology , Flocculation , Methylobacterium/cytology , Methylobacterium/drug effects , Methylobacterium/physiology , Micrococcus luteus/cytology , Micrococcus luteus/drug effects , Micrococcus luteus/physiology , Pseudomonas putida/cytology , Pseudomonas putida/drug effects , Pseudomonas putida/physiology , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/physiologySubject(s)
Biocompatible Materials/chemical synthesis , Biofilms/growth & development , Cell Division/radiation effects , Cell Movement/radiation effects , Cell Separation/methods , Electrophoresis/methods , Micromanipulation/methods , Tissue Engineering/methods , Animals , Biofilms/radiation effects , Electrophoresis/instrumentation , Humans , Micromanipulation/instrumentation , Plant Development , Plants/radiation effects , Tissue Engineering/instrumentationABSTRACT
The orientation of microbial cells may be important in cell-cell interactions within microbial consortia. As part of our research programme aimed at the construction of Artificial Structured Microbial Consortia (ASMC), we have investigated the electro-orientation of Schizosaccharomyces pombe in AC electric fields, and studied the effects of the applied frequency, voltage, and distance between the electrodes, at different medium conductivities. It is shown that the electro-orientation of S. pombe in media with conductivities similar to that of growth media is feasible using microelectrodes. Oriented growth of S. pombe can be obtained when continuously exposed to AC electric fields in growth medium over extended periods.
