ASGSB 1999 Annual Meeting Abstracts

LAMINAR FLOW IN THE BIOREACTOR SHEARS CELLS FROM MEMBRANE SURFACE. J.H. Calder¹, D. Montufar-Solis¹, P.J. Duke¹ and G.P. Neitzel². ¹Dept. of Orthodontics, Dental Branch, UT Houston Health Science Center, Houston, and ²Mechanical Engineering, Georgia Institute of Technology, Atlanta.

In an attempt to grow flat disks of endochondral cartilage for implantation into skull defects, micromass cultures of embryonic (E12) mouse limb bud cells (4.362x10⁶ cells/20µl drop) were set up on Millipore membrane filters (0.8µm pore size).  Filters with cells were cultured under standard conditions for 24 hours, then transferred to the bioreactor and rotated (counter-clockwise) in order to keep them in suspension within the culture medium. The observed movement of the filters in the fluid differed from that of spherical cultures previously used in our lab.  No particular rotation rate kept all filters rotating with the medium, however 43 rpm maintained most of the filters in suspension during a 24-hour period. Then, the filters gradually moved to the bottom of the vessel along a radius at the 5 o’clock position, more or less aligned with the flow, i.e., as a weathervane.  Higher speeds would push the filters against the wall of the vessel (centrifugally), and lower ones would allow them to begin tumbling inside the vessel.  The bioreactor was stopped every 48h to change culture medium.  When rotation was restarted, the filters were redistributed and the whole cycle of movement repeated.

Scanning-electron microscopy of filters after 3 weeks of culture showed only cell debris on the filter surface; and in light microscopy, an additional layer of material, presumably remnants of secreted matrix, was seen.  This type of cell detachment has not been observed in previous cultures using spherical constructs inside the bioreactor and is presumably due to the large shear stresses experienced by cells attached to the flat surfaces while the filter is aligned with the flow.  Thus, this experiment shows the potential of having extremely high shear forces when using the bioreactor, which can be avoided by careful selection of the shape of the tissue construct being cultured.

(Supported in part by NIH/NIDR Training Grant T35 DE 07252.)

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