High Aspect Rotating-wall Vessel Research Articles

Tri-dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction.

The high aspect rotating-wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity. We studied previously the effects of HARV cultivation on DU-145 human prostate carcinoma cells. We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods. The result was a 3-dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth. This work examines the signal transduction-second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation. We found an initial very active ceramide, a diacylglycerol increase together with increases in PI-PLC and PLA(2) a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation). There is a cross-talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down-regulation of ceramide. At this time, there is also an increase of cAMP (seen by increases in arachidonic acid). Taken together these results can explain the 3D organoid-like growth. We therefore developed a model for growth in HARV prostate cancer cells which involve temporal "switches" between second messengers, activation and cross-talk between multiplicity of signaling pathways and a central defect in PLD pathways. Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti-survival, anti-proliferation and differentiation pathways in the first days of HARV, with growth of "new" different types of prostate cancer cells which set-up for later "switch" in ceramide-PI3K to survival and proliferation.

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth.

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel.

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

Production of recombinant rotavirus VP6 from a suspension culture of transgenic tomato (Lycopersicon esculentum Mill.) cells

Recombinant rotavirus VP6 expressed in transgenic tomato cells was found primarily in the intracellular fraction and had a molecular weight of 44 kDa. In a shake flask, transgenic tomato cells produced 0.33 mg recombinant VP6 l−1 after 18 days of incubation. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the transgenic cells produced up to 0.15 mg recombinant VP6 l−1.

Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask

In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cell-cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 +/- 1% to 24 +/- 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport. These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. Copyright 1999 John Wiley & Sons, Inc.

Production of recombinant endostatin from stably transformed Drosophila melanogaster S2 cells

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Drosophila melanogaster S2 cells. Recombinant endostatin expressed in the stably transformed S2 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. The purification yield was approximately 4 μg from the medium fraction of 8 ml cultures of stably transformed S2 cells. In a T-flask, the stably transformed S2 cells produced 24 mg recombinant endostatin/l at 7 days post-induction by 0.5 mM CuSO4. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the S2 cells produced up to 13 mg recombinant endostatin/l.

Influence of simulated microgravity on the longevity of insect-cell culture

Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 × 10 7 viable cell ml −1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9–15 days in contrast to 4–5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20–90 relative to the control culture and was characterized with a death rate constant of 0.01–0.02 day −1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G1-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth. © 1996 John Wiley & Sons, Inc.

Three-dimensional modeling of T-24 human bladder carcinoma cell line: A new simulated microgravity culture vessel

We present a new device and method for culture of cell lines and primary tissues requiring high oxygen tensions. The High Aspect Rotating-Wall Vessel (HARV) described successfully propagated T-24, a human bladder transitional epithelial cell line, on Cytodex-3 microcarriers in three-dimensional cellular aggregates up to 0.5 cm in diameter. The HARV is a horizontally rotated tissue culture vessel with a large surface-area-to-volume ratio silicone membrane oxygenator. This design augments the principle of the rotating-wall vessel termed the Slow-Turning Lateral Vessel (STLV) by providing a low turbulence, low shear, cell growing environment with increased oxygen delivery capability. Comparisons of glucose metabolism, oxygen consumption, and morphology as a function of cell growth for a T-24 bladder carcinoma were performed in the HARV vs. the STLV. The HARV was superior in the culture of a variety of cell types including normal and neoplastic, anchorage-dependent and suspension cells.

Three‐dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating‐wall vessel: Morphologic and embryologic considerations

Cancer of the ovary is the leading cause of death from gynecologic malignancy. To understand better these aggressive tumors, the development of in vitro models to study human ovarian cancer is critical. However, the establishment of long-term cell lines has been difficult, due to the generalized poor survival of patient tumor cells grown in primary culture. Satisfactory culture systems for ovarian tumor cells have therefore been limited. To study cellular interactions involved in the growth and differentiation of these tumors, a cell line was established from a mixed müllerian tumor of the ovary. This cell line, designated LN1, was cultured on microcarrier beads in the high aspect rotating-wall vessel. The tumor cells grown in this vessel readily proliferated without a requirement for cocultivation with a supportive cell layer. Evaluation of cellular morphology by phase contrast light microscopy and scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-coated beads bridged together, as well as scattered aggregates of LN1 cells proliferating as spheroids free in suspension. In contrast to conventional culture systems, culture in the high aspect rotating-wall vessel facilitated the generation of multiple cell types that could be recovered. These results illustrate the ability of this culture system to provide the biological conditions necessary for pluripotent cell growth.