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The design of the space bioreactor stems from three considerations. First, and foremost, it must sustain cells in microgravity. Closely related is the ability to take advantage of the weightlessness and microgravity. Lastly, it should fit into a bioprocess. The design of the space bioreactor is described in view of these considerations. A flow chart of the bioreactor is presented and discussed.
The purpose is to review some of the physical/metabolic factors which must be considered in the development of an operating strategy for a mammalian cell bioreactor. Emphasis is placed on the dissolved oxygen and carbon dioxide requirements of growing mammalian epithelial cells. Literature reviews concerning oxygen and carbon dioxide requirements are discussed. A preliminary, dynamic model which encompasses the current features of the NASA bioreactor is presented. The implications of the literature survey and modeling effort on the design and operation of the NASA bioreactor are discussed.
We conducted a series of experiments using mouse immune-precursor cells, and observed that bioreactor culturing results in the loss of antigen-specific cytotoxic T lymphocyte (CTL) function. The reason for the abrogation of CTL function is microgravity conditions in the bioreactor, but not the antigen per se or its MHC restriction. Similarly, we observed that allostimulation of human PBMC in the bioreactor, but not in the T flask, resulted in the blunting of both allo-CTL function and the NK activity, indicating that the microgravity-associated functional defects are not unique to the mouse system. These results provide further confirmation to the microgravity-associated immune dysfunction, and constitute ground-based confirmatory data for those related to space-travel.
The Hydrodynamic Focusing Bioreactor-Space (HFB-S) is being developed as a possible replacement for the Rotating Wall Perfused Vessel (RWPV) bioreactor currently planned for use on the International Space Station (ISS). The HFB-S is being developed with the ability to remove gas bubbles that may inadvertently enter the system during long duration experiments (approx. 1-3 months). The RWPV has been used in the past with great success on Shuttle flights and Mir missions, but has occasionally experienced problems with gas bubbles entering the fluid-filled vessel. These bubbles are harmful to the cell science, and bubble removal in the RWPV is problematic. The HFB-S has an access port on the rotation axis that allows for bubble removal under specific operating conditions without detrimentally affecting the cell tissue. Experiments on bubble removal with the HFB-S were conducted in the microgravity environment on NASA's KC-135 Reduced Gravity Aircraft. The first set of flights provided useful data on bubble trajectories that are validating computational predictions. The second set of flights free-floated the apparatus and tested the most recent configuration of the bioreactor while focusing on the bubble removal process itself. These experiments have shown that gas bubbles can successfully be driven to the removal port and purged in microgravity. The last day's experiments had an excellent microgravity environment due to calm air, and the experience gained in previous flights allowed successful bubble removal 18 out of 35 tries, remarkable given the microgravity time constraints and g-jitter on the KC-135.
Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.
A sensor system was proposed that would monitor spaceflight bioreactor parameters. Not only will this technology be invaluable in the space program for which it was developed, it will find applications in medical science and industrial laboratories as well. Using frequency-domain-based fluorescence lifetime technology, the sensor system will be able to detect changes in fluorescence lifetime quenching that results from displacement of fluorophorelabeled receptors bound to target ligands. This device will be used to monitor and regulate bioreactor parameters including glucose, pH, oxygen pressure (pO2), and carbon dioxide pressure (pCO2). Moreover, these biosensor fluorophore receptor-quenching complexes can be designed to further detect and monitor for potential biohazards, bioproducts, or bioimpurities. Biosensors used to detect biological fluid constituents have already been developed that employ a number of strategies, including invasive microelectrodes (e.g., dark electrodes), optical techniques including fluorescence, and membrane permeable systems based on osmotic pressure. Yet the longevity of any of these sensors does not meet the demands of extended use in spacecraft habitat or bioreactor monitoring. It was therefore necessary to develop a sensor platform that could determine not only fluid variables such as glucose concentration, pO2, pCO2, and pH but can also regulate these fluid variables with controlled feedback loop.
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.
Compact and automated sensors are desired for assessing the health of cell cultures in biotechnology experiments in microgravity. Measurement of cell culture medium allows for the optirn.jzation of culture conditions on orbit to maximize cell growth and minimize unnecessary exchange of medium. While several discrete sensors exist to measure culture health, a multi-parameter sensor would simplify the experimental apparatus. One such sensor, the Paratrend 7, consists of three optical fibers for measuring pH, dissolved oxygen (p02), dissolved carbon dioxide (pC02) , and a thermocouple to measure temperature. The sensor bundle was designed for intra-arterial placement in clinical patients, and potentially can be used in NASA's Space Shuttle and International Space Station biotechnology program bioreactors. Methods: A Paratrend 7 sensor was placed at the outlet of a rotating-wall perfused vessel bioreactor system inoculated with BHK-21 (baby hamster kidney) cells. Cell culture medium (GTSF-2, composed of 40% minimum essential medium, 60% L-15 Leibovitz medium) was manually measured using a bench top blood gas analyzer (BGA, Ciba-Corning). Results: A Paratrend 7 sensor was used over a long-term (>120 day) cell culture experiment. The sensor was able to track changes in cell medium pH, p02, and pC02 due to the consumption of nutrients by the BHK-21. When compared to manually obtained BGA measurements, the sensor had good agreement for pH, p02, and pC02 with bias [and precision] of 0.02 [0.15], 1 mm Hg [18 mm Hg], and -4.0 mm Hg [8.0 mm Hg] respectively. The Paratrend oxygen sensor was recalibrated (offset) periodically due to drift. The bias for the raw (no offset or recalibration) oxygen measurements was 42 mm Hg [38 mm Hg]. The measured response (rise) time of the sensor was 20 +/- 4s for pH, 81 +/- 53s for pC02, 51 +/- 20s for p02. For long-term cell culture measurements, these response times are more than adequate. Based on these findings , the Paratrend sensor could offer automated, continuous monitoring of cell cultures with a temporal resolution of 1 minute, which is not attainable by sampling via handheld blood analyzer (i-STAT). Conclusion: The resulting bias and precision found in these cell culture-based studies is comparable to Paratrend sensor clinical results. Although the large error in p02 measurements (+/-18 mm Hg) may be acceptable for clinical applications, where Paratrend values are periodically adjusted to a BGA measurement, the O2 sensor in this bundle may not be reliable enough for the single-calibration requirement of sensors used in NASA's bioreactors. The pH and pC02 sensors in the bundle are reliable and stable over the measurement period, and can be used without recalibration to measure cell cultures in rn.jcrogravity biotechnology experiments. Future work will test additional Paratrend sensors to provide statistical assessment of sensor performance.
Spheroids of corneal tissue about 5 mm in diameter have been grown in a bioreactor from an in vitro culture of primary rabbit corneal cells to illustrate the production of optic cells from aggregates and tissue. In comparison with corneal tissues previously grown in vitro by other techniques, this tissue approximates intact corneal tissue more closely in both size and structure. This novel three-dimensional tissue can be used to model cell structures and functions in normal and abnormal corneas. Efforts continue to refine the present in vitro method into one for producing human corneal tissue to overcome the chronic shortage of donors for corneal transplants: The method would be used to prepare corneal tissues, either from in vitro cultures of a patient s own cells or from a well-defined culture from another human donor known to be healthy. As explained in several articles in prior issues of NASA Tech Briefs, generally cylindrical horizontal rotating bioreactors have been developed to provide nutrient-solution environments conducive to the 30 NASA Tech Briefs, October 2003 growth of delicate animal cells, with gentle, low-shear flow conditions that keep the cells in suspension without damaging them. The horizontal rotating bioreactor used in this method, denoted by the acronym "HARV," was described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), NASA Tech Briefs, Vol. 16, No. 5 (May, 1992), page 150.
It was an unlikely moment for inspiration. Engineers David Wolf and Ray Schwarz stopped by their lab around midday. Wolf, of Johnson Space Center, and Schwarz, with NASA contractor Krug Life Sciences (now Wyle Laboratories Inc.), were part of a team tasked with developing a unique technology with the potential to enhance medical research. But that wasn t the focus at the moment: The pair was rounding up colleagues interested in grabbing some lunch. One of the lab s other Krug engineers, Tinh Trinh, was doing something that made Wolf forget about food. Trinh was toying with an electric drill. He had stuck the barrel of a syringe on the bit; it spun with a high-pitched whirr when he squeezed the drill s trigger. At the time, a multidisciplinary team of engineers and biologists including Wolf, Schwarz, Trinh, and project manager Charles D. Anderson, who formerly led the recovery of the Apollo capsules after splashdown and now worked for Krug was pursuing the development of a technology called a bioreactor, a cylindrical device used to culture human cells. The team s immediate goal was to grow human kidney cells to produce erythropoietin, a hormone that regulates red blood cell production and can be used to treat anemia. But there was a major barrier to the technology s success: Moving the liquid growth media to keep it from stagnating resulted in turbulent conditions that damaged the delicate cells, causing them to quickly die. The team was looking forward to testing the bioreactor in space, hoping the device would perform more effectively in microgravity. But on January 28, 1986, the Space Shuttle Challenger broke apart shortly after launch, killing its seven crewmembers. The subsequent grounding of the shuttle fleet had left researchers with no access to space, and thus no way to study the effects of microgravity on human cells. As Wolf looked from Trinh s syringe-capped drill to where the bioreactor sat on a workbench, he suddenly saw a possible solution to both problems. It dawned on me that rotating the wall of the reactor would solve one of our fundamental fluid mechanical problems, specifically by removing the velocity gradient of the tissue culture fluid media near the reactor s walls, says Wolf. It looked as though it would allow us to suspend the growing cells within the reactor without introducing turbulent fluid mechanical conditions.
We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3‐D organotypic epithelial‐stromal cultures that require an air‐liquid interface. These complex 3‐D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air‐liquid interface, these 3‐D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue‐like human epithelial‐stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air‐liquid, liquid‐liquid, and liquid‐air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add‐on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.
Using the core ideals of bioregenerative life support, an anaerobic–phototrophic membrane bioreactor (APMBR) has been designed and operated at NASA’s Kennedy Space Center to treat complex wastewaters with the goal of closing water and nutrient cycles on early planetary bases. The system combined the previously operated anaerobic membrane bioreactor and the phototrophic membrane bioreactor that have been detailed in presentations at previous ICES conferences. This newly combined system is able to treat complex wastewater with completely automated controls on a small footprint. The treatment of wastes in this APMBR is as follows: (1) the waste enters the anaerobic subsystem, where solids are hydrolyzed and carbon is removed via anaerobic digestion, (2) an ultrafiltration membrane is used to separate the solids and the recovered water, (3) the effluent from the anaerobic subsystem is fed to the phototrophic subsystem on command, (4) the algae–bacteria consortium aids in nitrification in the recovered water, and (5) an ultrafiltration membrane is used to separate the algae and the final recovered water. The recovered water from the APMBR is rich in nutrients, making it a sustainable source of fertilizer for downstream hydroponic systems. This conference paper will detail the design and operation of the APMBR as a bioregenerative alternative to physical–chemical systems or bag and storage systems. In this paper, data will be presented on subsystem water quality, membrane performance, and effluent quality. Overall, the APMBR has the ability to treat wastewater using a combination of biological and filtration technologies that allow for higher removal efficiencies, low consumable use, and small footprint.
The concern over greenhouse gases, methane (CH 4 ) and carbon dioxide (CO 2 ), is increasing rapidly. There have been strides to find solutions to this global issue but there is not a clear path to a successful end goal. The concentration of CH 4 and CO 2 in the atmosphere has increased significantly over the last 60 years, methane is a great source of concern due to its ability to trap a high amount of heat in the atmosphere. These greenhouse gases contribute to global warming which has caused changes in the environment, including the melting of ice caps, and altered weather patterns. Solutions for these pressing challenges have led to different avenues of methane mitigation one of which is the development of solid-state bioreactors. These reactors harness the power of biological species that have evolved to use methane as an energy source. These methanotrophs, Methylomicrobium buryatense (5GB1), have been known to produce acetate and formate as a byproduct of the methane consumption cycle. The development of Hollow Scaffold Solid State Bioreactors (HS-SSBR) utilizes these microorganisms by way of hydrogel immobilization and infiltration of 3D printed scaffolds. The scaffolds act as a substrate for methanotroph growth and methane interaction. Methane consumption was monitored by way of gas sensors which indicated a reduction in methane concentration during the first 4 hours of closed loop methane recirculation. The byproducts of formate and acetate were confirmed by way of 1 H-NMR which indicated the 5GB1 methanotrophs were producing these molecules as a result of methane consumption.
The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds. Copyright 2002 Wiley Periodicals, Inc.
Monitoring and regulating the pH of the solution in a bioprocess is one of the key steps in the success of bioreactor operation. An in-line optical pH sensor, based on the optical absorption properties of phenol red present in the medium, was developed and tested in this work for use in NASA space bioreactors based on a rotating wall-perfused vessel system supporting a baby hamster kidney (BHK-21) cell culture. The sensor was tested over three 30-day and one 124-day cell runs. The pH sensor initially was calibrated and then used during the entire cell culture interval. The pH reported by the sensor was compared to that measured by a fiber optically coupled Shimadzu spectrophotometer and a blood gas analyzer. The maximum standard error of prediction for all the four cell runs for development pH sensor against BGA was +/-0.06 pH unit and for the fiber optically coupled Shimadzu spectrophotometer against the blood gas analyzer was +/-0.05 pH unit. The pH sensor system performed well without need of recalibration for 124 days. Copyright 2002 Wiley Periodicals, Inc.
Mass transport and mixing of perfused scalar quantities in the NASA Rotating Wall Perfused Vessel bioreactor are studied using numerical models of the flow field and scalar concentration field. Operating conditions typical of both microgravity and ground-based cell cultures are studied to determine the expected vessel performance for both flight and ground-based control experiments. Results are presented for the transport of oxygen with cell densities and consumption rates typical of colon cancer cells cultured in the RWPV. The transport and mixing characteristics are first investigated with a step change in the perfusion inlet concentration by computing the time histories of the time to exceed 10% inlet concentration. The effects of a uniform cell utilization rate are then investigated with time histories of the outlet concentration, volume average concentration, and volume fraction starved. It is found that the operating conditions used in microgravity produce results that are quite different then those for ground-based conditions. Mixing times for microgravity conditions are significantly shorter than those for ground-based operation. Increasing the differential rotation rates (microgravity) increases the mixing and transport, while increasing the mean rotation rate (ground-based) suppresses both. Increasing perfusion rates enhances mass transport for both microgravity and ground-based cases, however, for the present range of operating conditions, above 5-10 cc/min there are diminishing returns as much of the inlet fluid is transported directly to the perfusion exit. The results show that exit concentration is not a good indicator of the concentration distributions in the vessel. In microgravity conditions, the NASA RWPV bioreactor with the viscous pump has been shown to provide an environment that is well mixed. Even when operated near the theoretical minimum perfusion rates, only a small fraction of the volume provides less than the required oxygen levels. 2002 Wiley Periodicals, Inc.
Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.
Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.