Microgravity induces alterations in the function- ing of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux- B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reac- tive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clino- stat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this pur- pose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simu- lated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation)exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels mea- sured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity dur- ing parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the cen- trifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground- based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.
Research Containing: Clinostat
A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.
The practice of cell culture has been virtually unchanged for 100 years. Until recently, life scientists have had to content themselves with two-dimensional cell culture technology. Clearly, living creatures are not constructed in two dimensions and thus it has become widely recognized that in vitro culture systems must become three dimensional to correctly model in vivo biology. Attempts to modify conventional 2-D culture technology to accommodate 3-D cell growth such as embedding cells in extracellular matrix have demonstrated the superiority of concept. Nevertheless, there are serious drawbacks to this approach including limited mass transport and lack of scalability. Recently, a new cell culture technology developed at NASA to study the effects of microgravity on cells has emerged to solve many of the problems of 3-D cell culture. The technology, the Rotating Wall Vessel (RWV) is a single axis clinostat consisting of a fluid-filled, cylindrical, horizontally rotating culture vessel. Cells placed in this environment are suspended by the resolution of the gravitational, centrifugal and Coriolis forces with extremely low mechanical shear. These conditions, which have been called “low shear modeled microgravity”, enable cells to assemble into tissue-like aggregates with high mass transport of nutrients, oxygen and wastes. Examples of the use of the RWV for basic cell biology research and tissue engineering applications are discussed.
Influence of simulated microgravity on avian primordial germ cell migration and reproductive capacity
Fertilized eggs of chicken and quail were incubated under the simulated microgravity condition provided by a clinostat. The number of Primordial Germ Cells (PGCs) was counted in early embryogenesis, and the reproductive capacity of quail hatched following the simulated microgravity was investigated. Simulated microgravity caused significant decline of PGCs in the blood of early chicken embryos and in the gonads. The numbers of spermatogonia in the hatchling testis were also fewer than those in the control groups. Therefore, simulated microgravity may retard gonadial development and reduce the reproductive capacity. Copyright 2002 Wiley-Liss, Inc.
BACKGROUND: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. CONCLUSIONS/SIGNIFICANCE: Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.
Simulated microgravity inhibits the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (vol 40, pg 671, 2007)
OBJECTIVES: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential. MATERIALS AND METHODS: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis. RESULTS: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity. CONCLUSIONS: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.
<Go to ISI>://WOS:000253978100014
Growing evidence shows that physical microenvironments and mechanical stresses, independent of soluble factors, help influence mesenchymal-stem-cell fate. rMSCs (rat mesenchymal stem cells) present spread, spindle shape when cultured in normal gravity (NG) while in simulated microgravity (SMG) they become unspread, round shape. Here we demonstrate that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons, which might be a new strategy for the treatment of central nervous system diseases. rMSCs were cultured respectively in normal gravity and in a clinostat to simulate microgravity, followed with neuronal differentiated medium. The neuronal cells derived from rMSCs in SMG express higher microtubule-associated protein-2 (MAP-2), tyrosine hydroxylase (TH) and choline acetyltransferase (CHAT). Furthermore, as rMSCs are subjected to SMG, they excrete more neurotrophins like nerve growth factor (NGF), brain derived neurophic factor (BDNF) and ciliary neurotrophic factor (CNTF). Neuronal cells from SMG group generated more mature action potentials and displayed repetitive action potentials by comparison to cells from NG group. We conclude that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons.