Current Biotechnology - Volume 2, Issue 3, 2013

Space exploration encounters numerous hurdles partially because of the need to adapt technologies originally designed for earth applications. The integration of space conditions and weightlessness provides opportunities for the development of new technologies and the adaptation of existing but space-friendly approaches for physical and biological processes. Improvements of Solid Phase Gene Extraction (SPGE) provide ideal conditions for sampling of biological material under space conditions where liquid handling is challenging and thus best avoided. SPGE is capable of fine-tuning gene expression studies without the need to sacrifice the biological material and can be used in a self-referencing mode. The technology can be combined with microfluidics systems to enable on-orbit assessment of gene expression studies that take advantage of qPCR, minimize fluid handling, and retain fluids in a controlled environment.

The last decade has seen an impressive expansion of our understanding of the role of osteocytes in skeletal homeostasis. These amazing cells, deeply embedded into the mineralized matrix, are the key regulators of bone homeostasis and skeletal mechano-sensation and transduction. They are the cells that can sense mechanical forces applied to the bone and then translate these forces into biological responses. They are also ideally positioned to detect and respond to hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts through the production and secretion of molecules such as Sclerostin and RANKL. How osteocytes perceive mechanical forces and translate them into biological responses in still an active area of investigation. Novel “in vitro” models as well the opportunity to study these cells under microgravity condition, will allow a closer look at the molecular and cellular mechanisms of mechano-transduction. This article highlights investigations on osteocytes and discusses their significance in our understanding of skeletal mechano-transduction.

Spaceflight presents many challenges and opportunities for understanding human physiology and for administering healthcare in a harsh, remote and resource-limited environment. Technologies and countermeasures developed for space must be portable, minimize invasiveness, and be easy to use and maintain. Medical devices must also be robust and preferably require low power and consumables. Solutions meeting these standards for spaceflight have potential commercial advantages over similar technologies developed only for Earth markets. Many technologies for space have Earth-based spin-offs. In medicine, recent advances from space enhancing terrestrial healthcare delivery include, but are not limited to, (1) new training methods, and advanced diagnostic and therapeutic applications for ultrasound, (2) near-infrared spectroscopy for portable brain imaging, and (3) non-invasive assessment of intracranial pressure. With the assistance of the National Space Biomedical Research Institute (NSBRI), funding for biomedical innovation and applications for space includes a program aimed at small companies, with commercialization support competitively awarded to transition technologies from bench to market.

Scientists and technicians have been innovative to develop experimental platforms in order to achieve functional weightless conditions in their Earth-bound laboratories. As a result, various experimental platforms are available in order to perform studies with molecules or single cells up to humans and to study gravity-related mechanisms. These ground-based simulators of microgravity are not only tools to prepare spaceflight experiments, but they have been established as stand-alone facilities for gravitational research. This review provides an overview of some of the most frequently used microgravity simulators, most of which are in use at DLR´s Institute of Aerospace Medicine at Cologne, Germany. Their individual capacities but also experimental limitations, especially regarding their range of applicability for biological specimens, are exemplarily reviewed here. Overall, it is necessary to compare data achieved by using simulators with the data obtained in real microgravity. Furthermore, it should be carefully considered which kind of simulation might be the optimum for a given model organism or cell.

The BIOLAB is a multi-user facility of the European Space Agency ESA, accommodated in the European COLUMBUS Module of the International Space Station. The BIOLAB flight facility enables biological and biomedical experiments investigating the effects of weightlessness and/or space radiation on microorganisms, cell cultures of various origins, lower organisms and small plants and animals. The proposals for Life Sciences experiments are selected by an ESA peer group. The BIOLAB facility consists of an automated left part and a right part which is manually operated by the astronauts on orbit. The biological and biomedical samples are accommodated in experiment-specific containers, dedicated to each individual experiment. Sample preparation can be performed in the Bioglovebox, storage of biosamples in a cooler/freezer. On-orbit analyses can be performed by means of a microscope. The incubator as the heart piece of BIOLAB includes a life support system and two rotor platforms. Furthermore the video camera system mounted on the rotor platforms enables a regular control of the biosamples via telescience from/to ground. The BIOLAB facility is controlled and monitored by the Microgravity User Support Center (MUSC) as Facility Responsible Center (FRC) at DLR, Cologne, Germany, on behalf of the European Space Agency. MUSC is interfacing with the astronauts on orbit and the partners of the ISS ground segment, space hardware developers and the COLUMBUS Control Center. Main MUSC tasks for BIOLAB are increment preparation, experiment optimization and flight qualification, on-orbit operations via telescience, as well as post-increment evaluation. BIOLAB ground models - complemented by hyper-g and µg simulation devices - are available for experiments. DLR MUSC is supported by the specific expertise of BIOTESC (Lucerne, Switzerland). This review encompasses a description of the BIOLAB facility and exemplarily some experiments hitherto flown.

The great variety of possible configurations of Biological Life Support Systems (BLSS) and regimes for their operation necessitates a “top-down” approach to BLSS development and design. This top-down approach cannot be effectively performed without the criterion of LSS efficiency. In the paper, different criteria for comparing LSS and selecting the most appropriate one are discussed. The most general criterion of success of a space mission inevitably includes reliability or crew safety. Here, the necessity of using the criterion of “integral reliability” of a space mission as a whole is discussed. This criterion incorporates three main indices: reliability, mass, and quality of life. Possible ways of converting mass and quality of life into reliability are considered via examples of LSS launch missions and lunar base scenarios. The unique characteristic of the components of BLSS compared with physicochemical LSS is their ability to self-restore. Accounting for this property may enable ultra-reliable BLSS to be established. The problems hindering the development of technology for building reliable LSS are also considered. Among these, the problems of measuring the reliability of experimental BLSS prototypes and outlining the permissible range of their operation are of key significance.

Presented work focuses on the response of osteogenic, osteoblastic and mesenchymal stem cells to real and simulated microgravity. Mesenchymal stem cells are the residents of adult bone marrow stroma. These cells are capable of differentiating towards osteogenic, adipogenic and chondrogenic lineages. The advantage of using MSCs in space-related research is that these cells are relevant tool to develop bone tissue. Osteogenic potential of MSCs allows investigating osteogenesis from the most uncommitted cells while still maintaining all main characteristics of bone differentiation (osteogenic gene expression, osteogenic markers, matrix maturation and mineralization). Recent studies have demonstrated that MSCs are targeted by microgravity and accordingly can be involved in space flight-induced osteopenia. These findings determine the necessity of further study of human MSC biology in real and simulated microgravity conditions.

Saccharomyces cerevisiae is an eukaryotic model organism that has been used for space biology research. Microgravity is a tool to study yeast mechanobiology by removing the gravitational force on the cells. Yeast cells possess mechanosensors that can sense mechanical forces. The cells transduce a mechanical stimulus into a specific cellular response by activating intracellular signaling pathways that can ultimately lead to an altered function. Microgravity is “sensed” by yeast cells as a stress condition and several mitogen-activated protein kinases (MAPK) signaling pathways are activated, including the cell wall integrity (CWI)/protein kinase C (PKC), the high osmolarity glycerol (HOG) and the target of rapamycin (TOR) pathways. One of the indicators of morphological changes is an increase at random bud scar profile. Microgravity influences on the growth rate of yeast cells have been observed. The colony growth rate of the agar invasive S. cerevisiae ∑1278b strain was reduced as well as its agar invasiveness. Post-flight growth experiments of a brewer’s top yeast strain showed an increase in G2/M and a decrease in Sub-G1 cell population; an increased viability, a decreased lipid peroxidation level, increased glycogen content, and changes in carbohydrate metabolic enzyme activities were also observed. Using the S. cerevisiae BY4741 deletion collection, genes that provide a survival advantage in space, were identified in a batch growth experiment; no difference in growth rate was observed. Freeze-dried strains showed significant changes in the cell wall thickness. Spaceflight unique gene expression changes were observed in stress response element (STRE) genes with transcription regulation involving Sfp1 (which is involved in the TOR pathway) and Msn4. Some of the components of the ribosome biogenesis (which is under the control of Sfp1) as well as components of the proteasome were down regulated in microgravity. Recent results indicate that microgravity imposes a “microgravity” stress on the cells, which has the characteristics of an osmotic stress. Cellular energy is directed towards protective measures such as cell wall biosynthesis (CWI pathway activation) and the production of compounds (glycerol, trehalose) that increase the osmotolerancy (HOG pathway).

The cultivation of plants in space and on the Moon and Mars implies exposure to physical factors that are different from Earth: radiation, gravity levels and magnetic fields. As part of the MELiSSA (Micro-Ecological Life Support System Alternative) project where higher plants represent one of the compartments of an enclosed life support system, literature has been reviewed to assemble the relevant knowledge within space plant research. This review has particularly focused on the effect of the physical factors mentioned above on higher plant morphology, anatomy, gene regulation and genetic damage. Radiation studies on ground have shown increased damages to plant cells, and magnetic field studies have indicated some stress responses and altered growth rates, although not conclusive. Both in space and on ground (clinostat experiments) there are inconsistent results regarding gene expression and plant morphology. Anatomical flaws on the cellular level such as a reduced proliferation, displaced statoliths, ovoid chloroplasts, progressive vacuolization, and altered cell walls have been observed in several experiments. A normal plant reproduction process in weightlessness is feasible with a properly designed hardware. The same can be expected with low gravity levels, like those on Moon and Mars. In conclusion, the published results show that plants will grow and reproduce in low Earth orbit, but they might experience genotoxic stress and anatomic changes. Longer space studies, in particular outside the geomagnetic field and its radiation belt, are needed in order to investigate potential adaption responses since many studies show that plants can adapt in one generation to extreme environments.

The International Space Station (ISS) offers remarkable opportunities for scientists to carry out experiments. However, the schedule for astronauts on board is tight, leaving not much time for science. This is one reason why the currently developed space hardwares such as cell culture systems and bioreactors have to demonstrate high levels of automation that save precious crew time. Life-science hardware for space applications has to provide the optimal conditions for biological samples and to ensure proper functionality under microgravity conditions. Furthermore, the hardware has to pass the stringent safety requirements for manned space flights. Before space life-science hardware is ready for the flight, numerous test runs have to be performed to verify its flawless functionality and safety. In this chapter we introduce state-of-the-art instruments as examples of currently used cell culture hardware for space applications. The “PADIAC” blood cell culture chamber was used recently in space to further investigate the behavior of T-lymphocytes to microgravity. The experiences with the “PADIAC” hardware combined with one of our previous studies such as “SACESTRE” are currently being used to develop and build a new piece of space hardware called “YEAST BIOREACTOR”. This instrument will allow yeast cultivation during an extended period of time as well as the exposure of samples periodically to other stressors in addition to microgravity. An analytical tool called “OoClamp” is also introduced to enable the measurement of the electrical properties of living cells under microgravity conditions. This tool is intended as an integrated part of future bioreactors for the on-site verification of the health status of cells, for example. Such a system would be ideal for life-science experiments in space because, without having to bring back the cells, substantial data on cellular processes could be gathered in space.

The number of astronauts involved in long-lasting missions is expected to increase in the future, increasing the chances of injury due to traumatic events or unexpected emergency surgery. In addition, in future space exploration missions, medical evacuation times to earth could become very long. Therefore, tissue repair in weightlessness has become an important topic of study. Wound healing is an intricate process, which is critical for the survival of the organism. It is based on complex interactions between cells, extracellular matrix, cytokines, growth factors, physical and topographical factors and it consists of various phases. The available literature concerning wound healing in conditions of mechanical unloading presents controversial results. However, many studies indicate an impairment of the healing processes. In this paper, we reported an overview of studies regarding the effects of weightlessness on wound healing, particularly focusing on the behavior of cells involved in the remodelling phase of repair, e.g. fibroblasts and endothelial cells. Indeed, unloading conditions can affect wound healing both indirectly, decreasing the ability of the organism to withstand injuries because of the functional alterations of many organs and systems, and directly, changing the behavior of the cells involved in inflammation, ECM remodelling and tissue regeneration. Based on the studies conducted to date, hypotheses have advanced on what might be the altered cellular and molecular mechanisms that undermine tissue repair in microgravity. Countermeasures to promote tissue repair are also proposed. However, our knowledge on tissue regeneration patterns in weightlessness is still very limited and further studies are needed to better understand how gravitational alterations affect the healing process, thus opening the way for the development of new therapeutic strategies both for counteracting delayed tissue repair in space and treating on ground chronic wounds, healing delay or failure, fibrous scars, and other pathological conditions derived from defective repair mechanisms.

A technical improvement enabling efficient and reproducible stem cell cultures is one of the most important aspects in stem cell research and cell-based therapy. For this purpose, researchers have used various cytokine cocktails and gene transfection techniques to proliferate stem cells. However, stem cell fate can reportedly be determined by physical stimuli such as gravity, electrical fields, and magnetic fields. In this regard, we have developed a novel technique utilizing a clinostat, a device capable of generating a controlled microgravity environment for robust maintenance of stem cell state. Here, we review our recent progress in expansion and differentiation of stem cells grown under simulated microgravity environment and their utility for transplantation in animal models of cartilage deficiency and neurotrauma.

Muscle atrophy is a serious concern during space flight and has been observed in vertebrates in previous space flight experiments. In the present study, proteomic analysis was performed to assess changes in protein expression due to space flight in the model species Caenorhabditis elegans. Approximately 100 proteins were identified by MALDI-TOF mass spectrometry following two-dimensional gel electrophoresis, revealing that the expression of muscle-related proteins was significantly altered in space-flown worms. In particular, the protein expression level of paramyosin, which is a core component of invertebrate thick filaments, was significantly reduced in space-flown worms. In contrast, the expression of troponin T, which binds tropomyosin and tethers the troponin complex to the thin filament, was reduced; however, phosphorylation of troponin T was increased during space flight. These proteins play important roles in the maintenance of muscle structure and function, suggesting that changes in their expression and post-translational modification may be involved in the locomotion of the worms during space flight. Furthermore, we observed that the expression level of aconitase was reduced during space flight, possibility affecting ATP generation in C. elegans muscle.