NASA's Cell Culture Unit Brings Space Station Research Down to Earth

A profusion of present-day bioreactor and fermentor systems offers remarkably diverse capabilities, ranging from microfluidics to bulk production vats, simple petri dishes to complex artificial organ cultivators, and suspension, adhesion, perfusion, and many other culture management methods. Each of these systems is well suited to address specific research problems, but few are widely adaptable to diverse experiment demands - such as those conducted in space.

Figure 1. This close-up cutaway view of the CCU Prototype Experiment Carousel shows the fluid handling system configured to support six culture chambers.

NASA's Cell Culture Unit (CCU) is being developed by Payload Systems to offer customizable culture management capabilities supporting a very broad range of small-volume culture applications in space and on Earth.

The CCU (Figures 1 and 2) features a triad of attributes which offer a new type of controlled environment for cell culture experiments. First, multiple parameters (for example, temperature, O₂ concentration, and pH) can be sensed and independently controlled in parallel; second, all culture management functions - such as fluid flow, gas exchange, additive delivery, sampling, and waste removal - are provided online and can be automated; and third, the design is modular, accommodating multiple, simultaneous cultures (up to 18 mutually isolated perfusion loops) with individual control. Investigators may choose from 3-, 10-, or 30-mL growth chambers, compatible with attachment, suspension, and tissue cultures. The CCU also offers online video microscopy to simplify observations of cultures at mid-experiment and to minimize the environmental-control disruptions normally associated with temporarily removing active cultures for microscopic viewing.

Figure 2. The CCU System Configuration: (from left to right) the enclosure, a cutaway view of the internal assembly, and an experiment carousel with nine culture chambers installed on the top face.

In recognition of the widespread misconception that space bioreactor systems are precious devices lacking the necessary rigor for serious experimentation, the CCU team is conducting extensive ground studies proving it can support first-rate research both in space and on the ground.

Recent investigations have tested the CCU system design using diverse cell types and experimental conditions, including fluid flow, gas exchange, and other critical environment-control factors. Six reference cell types (Figure 3) were selected to represent the variety of possible cell biology studies performed aboard the International Space Station or undertaken in terrestrial laboratories: suspension cultures of yeast (Saccharomyces cerevisiae) and tobacco cells (BY2), an aquatic photosynthetic protist (Euglena gracilis), monolayers of murine muscle cells (C2C12 cell line) and human dermal fibroblast cells (primary), and three dimensional skeletal muscle "organoids" (based on embryonic chick skeletal myoblasts).¹ Cultures under nominal laboratory conditions have been used as controls to evaluate CCU hardware performance,² to define proper flow environments and optimal life support environments, and to exercise the various CCU functions. Brief reports on the results of these tests follow.

Figure 3. Six Reference Cell Types. Reference specimens were selected for CCU development testing in support of research on-board the International Space Station.

The C2C12 myoblast cell line, a well-established model to study mammalian myogenesis, was chosen as a model cell system for in vitro cultivation of mammalian adherent cells in CCU. The main requirements of in vitro cultivation for this cell type include cell attachment and growth in monolayers, cell fusion into multinucleated myotubes, and myogenesis (on a molecular level, the expression of muscle-specific proteins and on a cellular level, the formation of three-dimensional contractile myotubes). The conditions required to support C2C12 culture involve efficient transport of nutrients, gases, and metabolites in conjunction with minimal hydrodynamic shear. The flow conditions were selected to maintain set levels of oxygen (80 to 160 mm Hg) and pH (7.2 to 7.4) with an average hydrodynamic shear of 0.02 dynes/cm² acting on the cells. C2C12 cells cultured in three CCU bioreactors (n = 3) were comparable to static well-plate control cells in attachment, proliferation, and morphology. Cell viability was >95% in both CCU bioreactors and controls. Overall expression of structural proteins (for example, tropomyosin) was comparable to controls (Figure 4).

Figure 4. Tropomyosin staining of C2C12 myotubes with DAPI counter stain after ten days of culture in CCU bioreactors (n = 3) and static well plate controls (n = 3); image magnification = 100x.

Saccharomyces cerevisiae was selected as a model of fast-growing microorganisms and as a well-established model for studies of molecular mechanisms of cell growth and function and genetic manipulations. In this study, a mutated yeast strain (genotype of MATa ura3-52 his3-Δ200 leu2-3 trp1-1 ade2-Δbar1) was tested in CCU bioreactors with conventional shaker flasks as controls. The culture process development focused on two aspects: selecting optimal cell culture parameters (principally, proper gas exchange to match fast-growing yeast, uniform cell suspension with micromagnetic stirring, and a gentle hydrodynamic shear environment for cell phenotype maintenance) and characterizing the performance of CCU bioreactors compared to shaker flasks through evaluation of three bioreactors and four subcultures. A moderate perfusion rate (0.42 to 2.50 mL/min) was optimized with flow direction variation (forward and reverse), flow rate in one direction (1.15 to 6.40 mL/min), and period of flow (2 to 5 sec) in conjunction with stirring (90 rpm) that changed direction every 60 seconds. Our assessment of cultures in CCU bioreactors and shaker flasks showed comparable cell-growth kinetics (Figure 5) and final cell concentration, normal cell morphology, uniformity of cell suspension, adequate gas exchange, and maintenance of cell phenotype.

Figure 5. Representative yeast growth curves are comparable in CCU bioreactors (n = 3) and shaker flasks (n = 3).

Tobacco cells(BY2) were selected to challenge the performance of the CCU in at least three respects: by the complex requirements for maintenance of uniform cell suspension, by the profuse extracellular matrix that could compromise chamber membranes, and by pronounced sensitivity of the cells to hydrodynamic shear and biochemical factors.2 Flow visualization and mixing were studied to characterize flow patterns of fluid and suspended cells. Tobacco cells were cultured in four subcultures (each with a 100-fold increase in cell concentration) using shaker flask cultures as controls. All cultures were performed in triplicate. As shown in Figure 6, CCU bioreactors and control flasks yielded comparable cell growth rates (0.687 ± 0.032 and 0.709 ± 0.025 per day, respectively), as well as normal cell viability and morphology. In addition, the CCU achieved a high uniformity of cell suspension (80 to 100%), chamber membranes remained unblocked, and the pH and oxygen levels maintained were indistinguishable from those in shaker flasks.

Figure 6. Representative tobacco growth curves are comparable in CCU bioreactors (n = 3) and shaker flasks (n = 3).

In summary, CCU perfusion-based cultures are wholly comparable to nominal laboratory cultures. Furthermore, in contrast to nominal laboratory techniques, CCU experiments can be conducted manually and under automated control with much greater ability to independently and concurrently control multiple environment-control parameters.

The results summarized above and related ground-evaluation results indicate the CCU's significant value as a rigorous space biology research apparatus, beginning with its maiden flight planned for 2006. Given the environmental control performance and wide diversity of experiment management options the CCU offers, terrestrial researchers may soon be putting the CCU to wider use right here on Earth.

References

1. Vunjak-Novakovic G, Preda C, Bordonaro J, Pellis N, de Luis J, Freed LE. Microgravity studies on cells and tissues: from Mir to the ISS. Proceedings of the Space Technology and Applications International Forum; 1999 Jan; Albuquerque, NM. New York: American Institute of Physics; 1999; p. 442-452.

2. Searby ND, Vandendriesche D, Havens C, Donovan F, de Luis J, Pretorius S, Lagaz J, Berzin I, Sun L, Kundakovic L, Preda C, Vunjak-Novakovic G. Life support from the cellular perspective. Proceedings of the 31st International Conference on Environmental Systems; 2001 Jul; Orlando, FL.

3. de Luis J, Vunjak-Novakovik G, Searby N. Design and testing of the ISS cell culture unit. IAF/IAA-00-G.4.06; Proceedings of the 51st International Astronautical Congress; 2000 Oct; Rio de Janeiro, Brazil.