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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Jennifer
Liford (Jill Blackmer, Dept. of Veterinary Clinical Sciences,
LSU School of Veterinary Medicine) Development
of three-dimensional tissue assemblies of equine tracheal epithelial
cells under microgravity conditions
A three-dimensional in vitro equine cell model was developed
in microgravity conditions using a rotating-wall vessel(RWV),
known as a bioreactor (Synthecon, Inc.). The cells, which were
from an established cell line derived from equine respiratory
tissue, were grown in standard tissue culture conditions and
in microgravity conditions. Cells were grown in monolayers to
establish a growth curve in order to predict the time of cell
confluency and to obtain the desired concentration of cells
for initiation into the RWV. Plastic collagen-coated microcarrier
beads were used as the substrate for the cells to colonize.
Ten million cells and 250mg of beads were seeded in 50mL of
MEM growth medium containing sodium bicarbonate, HEPES buffer,
fetal bovine serum, and antibiotics to achieve a ratio of 10
cells/bead. The RWV was rotated at approximately 20 rpm in a
5% CO2 incubator set at 37C. Culture media was changed daily
for 20 days.
The bead/cell assemblies visually increased in size over the
course of the culture period and resembled grains of sand suspended
in the culture medium. Tissue assemblies were harvested on days
4,5,11, and 20 for light and/or electron microscopic examination.
Light microscopic examination on days 4 and 5 showed a layer
of cells covering most of the beads. By day 11, multiple layers
of cells had coated the beads with some beads forming aggregates.
Electron microscopy on day 5 showed a coating of cells around
the beads several nuclei thick. Also, mucus production was evident
around the outside layer of cells. Scanning electron microscopy
on day 20 showed aggregates of cells on beads with microvilli
coating the surface. Paraffin sections stained with hematoxalyn
and eosin on day 20 showed polygonal epithelial cells with rounded,
healthy nuclei.
Equine respiratory epithelial cells were successfully grown
in microgravity conditions using a bioreactor. Three-dimensional
tissue assemblies were developed and exhibited features of respiratory
tissue, such as microvilli and mucus production. This novel
in vitro technique will hopefully be used in future research
of equine respiratory disease, as it simulates in vivo conditions
more accurately than current in vitro cultures such as, monolayers.
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