Current Research Projects:
Rotating Wall Vessels

Rotating wall vessels are a specialized cell culture vessel developed initially as a microgravity simulator to mimic and model effects of microgravity on cells in earth based laboratory studies.

When cells are placed into traditional cell culture flasks, they de-differentiate and lose expression of the many tissue-specific genes and proteins that characterized their special properties. Scientists around the United states and abroad have utilized rotating wall vessels for their ability to induce re-expression of many tissue-specific proteins during cell culture. This facilitates studies on the regulation of tissue-specific gene and protein expression in a unique manner not obtainable by any other means. Tissues grown in this manner often form three-dimensional aggregates with uncanny similarities to the native tissue.

We became interested in rotating wall vessels as a culture method to induce re-expression of the receptors for common nephrotoxins in renal cell cultures. The vessels have proven very efficacious for this particular purpose.

This work expanded in several directions:

  • We are continuing our initial studies on the regulation of expression of nephrotoxic receptors.
  • Studies are maturing on the expression of production of renal hormones such as erythropoietin and the active form of vitamin D in the vessels.
  • We are investigating the genetic mechanisms by which gene and protein expression are modulated in rotating wall vessels.

    • Specific Aims

    Working hypothesis for Project 1: Simulated microgravity will enhance organ specific differentiation of renal epithelial cells in culture, and amplify analogous effects of co-culture with cortical microvascular endothelial cells. Microgravity will provide a population of cultured renal epithelial cells which express the molecular motors postulated to mediate diverse forms of tubular toxicity [gp280 and gp330].

    Specific Aim I. To compare enhancement of proliferation, and organ specific differentiation of renal epithelial cells in simulated microgravity (NASA rotating wall perfused vessels) to conventional cell culture techniques (2-D monolayer, suspension culture, beads, polarization on Millipore inserts).
    Specific Aim II. To test whether organ specific co-culture with cortical microvascular endothelial cells enhances proliferation, and organ specific differentiation of renal epithelial cells in simulated microgravity (NASA rotating wall perfused vessels) and conventional cell culture techniques.

    Working hypothesis for Project 2: Expression of 1-a-hydroxylase activity by renal epithelial cells grown in simulated microgravity will allow direct analysis of the regulation of activity, and correction of the hormone deficiencies of uremia. The specific objectives of this proposal are:

    Specific Aim I. Optimization of 1-a-hydroxylase activity in culture.

    a. To optimize 1-a-hydroxylase activity of renal epithelial cells in simulated microgravity (NASA rotating wall perfused vessels) by controlling seeding density, time in culture, split ratio and refeeding schedule, and make a comparison to conventional cell culture techniques (2-D monolayer & stirred fermentor).

    b. To test whether organ specific co-culture with the natural renal cortical mixture of cells enhances 1-a-hydroxylase activity of renal epithelial cells in simulated microgravity ( rotating wall vessels).
    Specific Aim II. Physiological regulation of 1-a-hydroxylase activity: direct studies. Maintenance of cell lines with 1-a-hydroxylase activity, including rat renal cortical epithelial cells and human embryonic kidney cells, allows for direct studies of the regulation of enzyme activity in a highly controlled setting. Proposed physiological modulators of 1-a-hydroxylase activity to be studied directly include: calcium concentration, parathyroid hormone, phosphate concentration, calcitonin, IGF-1, uric acid, purine related compounds such as theophylline, the putative low molecular weight uremic toxin guanidinosuccinic acid, and cytochrome P-450 inhibitors ketoconazole and metarypone.
    Specific Aim III. Restoration of 1-a-hydroxylase activity by cultured renal cell implantation in a rat model of uremia. To exploit the genetic identity of the Lewis strain of rats that permits tissue transplantation without rejection, to attempt restoration of 1 a-hydroxylase activity by implantation of rat renal cortical cell aggregates from rotating wall vessels into the peritoneal cavity of rats made uremic by 5/6 nephrectomy.

    Links of Interest

    Microgravity Teachers Guide

    Synthecon

    Bioreactor

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