A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.
The bioreactor (see figure) is a thin culture chamber that has two or more inlets and two or more outlets for flows of liquids. The top face of the chamber is bounded by a membrane of porous respiratory active material that enables exchange of O2 and CO2 between the cell culture and the controlled atmosphere in which the bioreactor resides. The bottom face of the chamber can be either a second porous membrane or a microscope cover sheet, which enables microscopic imaging of cells in the chamber.
The fluid-handling subsystem includes an upstream and a downstream switching valve, flexible tubes that connect the upstream switching valve with three supply reservoirs and the tor inlets, flexible tubes that connect the downstream switching valve with the bioreactor outlets and with waste and sampling reservoirs, and a peristaltic pump. The tubes on the downstream side are draped along the roller bearings of the peristaltic pump. There are three supply reservoirs: one containing the cell-culture nutrient medium, one containing a phosphate buffer solution (PBS), and one containing trypsin.
The flow passages in the valves are arranged so as to allow only the one correct liquid to flow through a given tube at a given time. The upstream valve enables the selection of flow of either fresh nutrient medium or PBS to the inlets. Alternatively, for the purpose of effective disconnection of part of the bioreactor, the upstream valve can be set to infuse trypsin through one inlet and the nutrient medium through the other inlet. The downstream valve can be set to connect all outlets to the waste reservoir or to connect a specified outlet or all outlets to a sampling reservoir.
Because the rates of flow required to sustain cell cultures are small and the system is operated accordingly, the flow velocity in the thin culture chamber is so small that the flow can be considered to be essentially laminar and two-dimensional, so that a given infinitesimal volume of liquid can be considered to travel smoothly along a simple, well-defined path. This flow characteristic can be exploited in harvesting cells from a specific region of a culture of anchorage-dependent cells, without disturbing cells from other regions. In the case of suspension cells, harvesting is performed upon the infusion of fresh nutrient medium. Incorporated into the miniature culture system is a temperature-control system and gas-control loop. The inclusion of these two systems will enable the miniature culture system to be autonomous.
This work was done by Steve R. Gonda of Johnson Space Center and Stanley J. Kleis and Sandra K. Geffert of the University of Houston.
In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should addressed to:
Emmanuelle Schuler, Ph.D Technology Transfer Associate Office of Intellectual Property Management University of Houston
Refer to MSC-24210-1, volume and number of this NASA Tech Briefs issue, and the page number.