Introduction word count (does not include references): 559Discussion word count (does not include references). 1333
Expanding whole blood sample collection for transcriptome analysis beyond traditional phlebotomy clinics will open new frontiers for remote immune research and telemedicine. Determining the stability of RNA in blood samples exposed to high ambient temperatures (>30°C) is necessary for deploying home-sampling in settings with elevated temperatures (e.g., studying physiological response to natural disasters that occur in warm locations or in the summer). Recently, we have developed homeRNA, a technology that allows for self-blood sampling and RNA stabilization remotely. homeRNA consists of a lancet-based blood collection device, the Tasso-SST™ which collects up to 0.5 ml of blood from the upper arm, and a custom-built stabilization transfer tube containing RNAlater™. In this study, we investigated the robustness of our homeRNA kit in high temperature settings via two small pilot studies in Doha, Qatar (no. participants = 8), and the Western and South Central USA during the summer of 2021, which included a heatwave of unusually high temperatures in some locations (no. participants = 11). Samples collected from participants in Doha were subjected to rapid external temperature fluctuations from being moved to and from air-conditioned areas and extreme heat environments (up to 41°C external temperature during brief temperature spikes). In the USA pilot study, regions varied in outdoor temperature highs (between 25°C and 43.4°C). All samples that returned a RNA integrity number (RIN) value from the Doha, Qatar group had a RIN ≥7.0, a typical integrity threshold for downstream transcriptomics analysis. RIN values for the Western and South Central USA samples (n = 12 samples) ranged from 6.9–8.7 with 9 out of 12 samples reporting RINs ≥7.0. Overall, our pilot data suggest that homeRNA can be used in some regions that experience elevated temperatures, opening up new geographical frontiers in disseminated transcriptome analysis for applications critical to telemedicine, global health, and expanded clinical research. Further studies, including our ongoing work in Qatar, USA, and Thailand, will continue to test the robustness of homeRNA.
Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.
Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.
Expanding whole blood sample collection for transcriptome analysis beyond traditional phlebotomy clinics will open new frontiers for remote immune research and telemedicine. Determining the stability of RNA in blood samples exposed to high ambient temperatures (>30°C) is necessary for deploying home-sampling in settings with elevated temperatures (e.g., studying physiological response to natural disasters that occur in warm locations or in the summer). Recently, we have developed homeRNA, a technology that allows for self-blood sampling and RNA stabilization remotely. homeRNA consists of a lancet-based blood collection device, the Tasso-SST™ which collects up to 0.5 mL of blood from the upper arm, and a custom-built stabilization transfer tube containing RNAlater™. In this study, we investigated the robustness of our homeRNA kit in high temperature settings via two small pilot studies in Doha, Qatar (no. participants = 8), and the Western USA during the summer of 2021, which included a heatwave of unusually high temperatures in some locations (no. participants = 11). Samples collected from participants in Doha were subjected to rapid external temperature fluctuations from being moved to and from air-conditioned areas and extreme heat environments (up to 41°C external temperature during brief temperature spikes). In the USA pilot study, regions varied in outdoor temperature highs (between 25°C and 43.4°C). All samples that returned a RNA integrity number (RIN) value from the Doha, Qatar group had a RIN ≥7.0, a typical integrity threshold for downstream transcriptomics analysis. RIN values for the Western USA samples (n=12 samples) ranged from 6.9–8.7 with 9 out of 12 samples reporting RINs ≥7.0. Overall, our pilot data suggest that homeRNA can be used in some regions that experience elevated temperatures, opening up new geographical frontiers in disseminated transcriptome analysis for applications critical to telemedicine, global health, and expanded clinical research. Further studies, including our ongoing work in Qatar, USA, and Thailand, will continue to test the robustness of homeRNA.
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