Determining the fundamental role of gravity in vital biological systems in space is one of six science and research areas that provides the philosophical underpinning for why NASA exists. The study of cells, tissues, and microorganisms in a spaceflight environment holds the promise of answering multiple intriguing questions about how gravity affects living systems. To enable these studies, specimens must be maintained in an environment similar to that used in a laboratory. Cell culture studies under normal laboratory conditions involve maintaining a highly specialized environment with the necessary temperature, humidity control, nutrient, and gas exchange conditions. These same cell life support conditions must be provided by the International Space Station (ISS) Cell Culture Unit (CCU) in the unique environment of space. The CCU is a perfusion-based system that must function in microgravity, at unit gravity (1g) on earth, and from 0.1g up to 2g aboard the ISS centrifuge rotor. One of the biggest challenges is to optimize the flow environment within the cell specimen chamber to meet the complex and often conflicting requirements to provide uniform gas and nutrient exchange, to maintain cell suspension, and to control hydrodynamic stresses acting on the adherent or suspended cells. Other challenges include obtaining representative samples of cells and culture medium, managing any bubbles formed in the cell specimen chamber and fluid lines, preventing blockage of the cell separation membrane inside the chamber, and providing sterile containment during all experiment phases. Flight studies of cells and tissues also require the development of proper control experiments for specific parameters in the cell culture environment. Thorough ground testing is currently being carried out in development hardware called the Science Evaluation Units. We present data from our experiments using animal, plant, and microbial cells where we have optimized these life support conditions.