Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

Lyndon B. Johnson Space Center, Houston, Texas

The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissue-like assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities.

Conventional bioreactors rely on agitation to suspend cells and attachment materials and to facilitate the mass transfer required for the growth of cells and tissue assemblies. However, the shear force generated by agitation can affect cell-cell interactions and degrade three-dimensional tissue development. Johnson Space Center has developed rotating-wall perfused-system (RWPS) bioreactors that create low-shear culture environments and support three-dimensional tissue development. However, their ability to control the locations of cells and tissue aggregates within vessels is limited. Moreover, air bubbles that form within the culture media in the vessels cannot be removed, although such removal is critical for operation in orbit around the Earth.

The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

Figure 1. The Hydrodynamic Focusing Bioreactor utilizes rotation and viscosity to generate hydrodynamic forces for controlling locations of cells and bubbles while maintaining low shear.

The HFB includes a rotating, dome-shaped cell-culture vessel with a centrally located sampling port and an internal rotating viscous spinner attached to a rotating base (see Figure 1). The vessel and viscous spinner can rotate at the same speed or different speeds and directions to create the desired levels of hydrodynamic force within the vessel. Both the low-shear suspension of cells and control of the locations of cells and air bubbles are effected by means of the hydrodynamic force created by the flow within the vessel and fluid drag along the surface of the viscous spinner. A gas-permeable membrane connected to the base of the vessel enables the exchange of gas between the medium in the vessel and an incubator environment in which the vessel is placed. Average sheer values of 0.001 dynes per square centimeter were estimated for a rotation rate of 10 rpm — a rate at which efforts to suspend large, three-dimensional, tissue-like assemblies have been successful.

Figure 2. Aggregates of Baby-Hamster Kidney Cells grown in the HFB during ten-day intervals were photographed at two different magnifications after histological staining.

Anchorage-dependent cells, including primary cells, transformed cells, genetically engineered cells and cell lines, and suspension hybridoma cells were successfully cultured either as mono- or co-cultures in the HFB for times as long as 2 weeks. Cultures were initiated by inoculating cells, variously with or without attachment materials, into the vessel through the sampling port, filling the vessel completely with a culture medium, and setting rotation rates to maintain suspension. Large three-dimensional, tissuelike assemblies were obtained from HFB cultures of anchorage-dependent cells (see Figure 2). In addition, critical stages in three-dimensional, tissuelike growth (cell attachment, three-dimensional aggregation, and formation of an extracellular matrix) were observed with all anchorage-dependent cells cultured in the HFB.

This work was done by Steve R. Gonda and Glenn F. Spaulding of Johnson Space Center, Yow-Min D. Tsao, Scott Flechsig and Leslie Jones of Wyle Laboratories, and Holly Soehnge of Universities Space Research Associates.

Title to this invention, covered by U.S. Patent No. 6,001,642 has been waived under the provisions of the National Aeronautics and Space Act {42 U.S.C. 2457 (f)}. Inquiries concerning licenses for its commercial development should be addressed to