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NASA NTRS1988

Cell bioprocessing in space - Applications of analytical cytology

Cell bioprocessing experiments in space are reviewed and the development of on-board cell analytical cytology techniques that can serve such experiments is discussed. Methods and results of experiments involving the cultivation and separation of eukaryotic cells in space are presented. It is suggested that an advanced cytometer should be developed for the quantitative analysis of large numbers of specimens of suspended eukaryotic cells and bioparticles in experiments on the Space Station.

Todd, P.↗

NASA NTRS2017

Estrous Cyclicity in Mice During Simulated Weightlessness

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n=22), normally loaded HU Cage Pair-Fed controls (CPF; n=22), and Vivarium controls fed ad libitum (VIV; n=10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8+/- 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, and/or due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5% vs 3.0%, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system. Research supported by NNX15AB48G to JST.

Hindlimb Unloading↗

NASA NTRS2017

Estrous Cyclicity of Mice During Simulated Weightlessness

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n22), normally loaded HU Cage Pair-Fed controls (CPF; n22), and Vivarium controls fed ad libitum (VIV; n10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, andor due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5 vs 3.0, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system.

fertilit↗

NASA NTRS2000

Selected contribution: a three-dimensional model for assessment of in vitro toxicity in balaena mysticetus renal tissue

This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.

Whales/genetics/physiology↗

NASA NTRS1997

A quantitative assay for intercellular aggregation

In an earlier communication (Munn et al., J Immunol. Methods 166: 11-25, 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.

Non-NASA Center↗

NASA NTRS2001

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Non-NASA Center↗

NASA NTRS1995

Hair Cell Heterogeneity in the Goldfish Saccule

A set of cytological studies performed in the utricle and saccule of Astronotus ocellatus (Teleostei, Percomorphi, Cichlidae) identified two basic types of hair cells and others with some intermediate characteristics. This paper reports on applying the same techniques to the saccule of Carassius auratus (Teleostei, Otophysi, Cyprinidae) and demonstrates similar types of hair cells to those found in Astronotus. Since Carassius and Astronous are species of extreme taxonomic distance within the Euteteostei, two classes of mechanoreceptive hair cells are likely to represent the primitive condition for sensory receptors in the euteleost inner ear and perhaps in all bony fish ears.

Saidel, William M.↗