ALBERT

Description: The ends of human chromosomes contain telomeres, or tandem arrays of repeating DNA sequences capped by multiple associated proteins that protect chromosomal ends from degradation. Telomeres function to preserve genomic stability by preventing natural chromosomal ends from being recognized as broken DNA double-strand breaks and triggering inappropriate DNA damage responses. Mounting evidence shows telomere length is an inherited trait that decreases with cellular division and normal aging. In addition, telomere length also appears to be influenced by other factors such as cellular oxidative stress, radiation and mechanical unloading of tissues as in microgravity. To measure these potential effects of the space environment on telomere lengths and cellular aging and regenerative potential we developed a novel telomere measurement approach based on nanopore sequencing of PCR amplified bar-coded chromosome termini. Specifically, telomeres can be directly enriched using barcode sequences ligated to the end of a free end- repaired telomere using the WetLab-2 facility SmartCycler on ISS. Prior to the ligation and amplification protocol a proteinase K digestion of capping proteins followed by a single 95-degree C heat denaturation of the protease is included. After digestion and bar-code ligation, PCR amplification will initiate with the ligated barcoded sequence, suppressing amplification of intra-genomic fragments and resulting in long read barcoded telomere amplicons including the nanopore motor protein sequences. Purified PCR amplicons are then used for nanopore sequencing library generation by simple addition of motor proteins and sequencing library is loaded into the MinION nanopore DNA-sequencer. Amplicon sequence reads from the nanopore device can be base-called quickly on ISS due to barcoding ligation and subsequent PCR amplification enhancing the telomere sequence resolution. If successfully implemented on ISS this technique will provide a novel means of measuring regenerative ability of somatic stem cells in astronauts, and of determining whether spaceflight in microgravity alters their telomere lengths and causes premature cellular aging.

Description: The NASA Bioculture System is an advanced cell culture closed-loop system containing highly automated flowpaths designed to conduct long term biology experiments on ISS with earth remote controllable medium flow, temperature, gas composition, medium exchange, cell sampling and fixation. This technology was already demonstrated with successful cardiomyocyte and osteocyte cultures experiments onboard the ISS and is now supporting NASA PI science. The Bioculture System, however, can only support 10 cassettes with disposable flowpaths, each containing a single hollow fiber bioreactor with a culture capacity of about 2ml. This constraint not only severely limits the number of investigators that can conduct experiments in space, but also subjects the experiments to limitations in the number of replicates and conditions that can be studied. To address these limitations, we sought a novel design solution to maximize the number of separate bioreactor cultures and volume that can be conducted simultaneously. To this end we designed, prototyped, and are now testing a six-Vitvo 3D Matrix 2ml bioreactor insert that replaces the conventional Bioculture System hollow fiber bioreactor. This design will allow the Bioculture System to support up to 60 different bioreactors and samples at once. Specifically, the novel gas-tight containment housing insert contains six COTS Rigenerand VITVO bioreactors stacked on each side of a heat sink powered by the existing heating element and pair of temperature sensors. Medium will be distributed into each bioreactor's cell-free chamber via its built-in Luer connector, then across the 3D matrix to the cell chamber, dissipating laminar flow and limiting fluid shear stresses that might mechanostimulate cell cultures. Gas (5% CO2 in air) will be supplied directly to the bioreactor gas-tight housing for exchange via the bioreactor flat-surface gas-permeable membranes, eliminating the need for the existing Bioculture System cassette oxygenator. If successfully implemented on ISS, this new multi-bioreactor insert for the Bioculture System has the potential to make real-time cell science experimentation in space more efficient and accessible to more investigators.