Simulated Microgravity Increases the Permeability of HUVEC Monolayer through Up-Regulation of Rap1GAP and Decreased Rap2 Activation

Space microgravity condition has great physiological influence on astronauts’ health. The interaction of endothelial cells, which control vascular permeability and immune responses, is sensitive to mechanical stress. However, whether microgravity has significant effects on the physiological function of the endothelium has not been investigated. In order to address such a question, a clinostat-based culture model with a HUVEC monolayer being inside the culture vessel under the simulated microgravity (SMG) was established. The transmittance of FITC-tagged dextran was used to estimate the change of integrity of the adherens junction of the HUVEC monolayer. Firstly, we found that the permeability of the HUVEC monolayer was largely increased after SMG treatment. To elucidate the mechanism of the increased permeability of the HUVEC monolayer under SMG, the levels of total expression and activated protein levels of Rap1 and Rap2 in HUVEC cells, which regulate the adherens junction of endothelial cells, were detected by WB and GST pull-down after SMG. As the activation of both Rap1 and Rap2 was significantly decreased under SMG, the expression of Rap1GEF1 (C3G) and Rap1GAP in HUVECs, which regulate the activation of them, was further determined. The results indicate that both C3G and Rap1GAP showed a time-dependent increase with the expression of Rap1GAP being dominant at 48 h after SMG. The down-regulation of the expression of junctional proteins, VE-cadherin and β-catenin, in HUVEC cells was also confirmed by WB and immunofluorescence after SMG. To clarify whether up-regulation of Rap1GAP is necessary for the increased permeability of the HUVEC monolayer after SMG, the expression of Rap1GAP was knocked down by Rap1GAP-shRNA, and the change of permeability of the HUVEC monolayer was detected. The results indicate that knock-down of Rap1GAP reduced SMG-induced leaking of the HUVEC monolayer in a time-dependent manner. In total, our results indicate that the Rap1GAP-Rap signal axis was necessary for the increased permeability of the HUVEC monolayer along with the down-regulation of junctional molecules including VE-cadherin and β-catenin.

Biotization of tissue culture banana plantlets with Methylobacterium salsuginis to enhance the survival and growth under greenhouse and open environment condition

Aim: This study aimed to investigate the impact of M. salsuginis TNMB03 biotization on tissue culture banana cv. Grande Naine plantlets growth and survival under greenhouse and open environmental condition (exposed to direct sunlight). Methodology: Banana plantlets were transferred from culture flasks to protray and maintained under the greenhouse and open environmental condition for 30 days with or without M. salsuginis TNMB03 treatment. After 30 days, plant growth parameters like pseudostem height, girth, number of leaves, leaf area, fresh and dry biomass, root parameters, plantlet survival, chlorophyll a and b, total chlorophyll, carotenoids and soluble protein and Methylobacterium population in upper and lower surface of leaf, as well as endophytic population were assessed. Results: This study showed that the plantlets biotized with M. salsuginis TNMB03 had better acclimatization response under both the experimental condition than that of uninoculated plantlets. Positive influence on the survival and growth of M. salsuginis TNMB03 biotized plantlets was observed when transferred directly to greenhouse and open environmental condition. Inoculation of M. salsuginis TNMB03 increased the plant height, girth and number of leaves, root length, lateral root and biomass in comparison to the uninoculated plantlets in greenhouse and open environment. Uninoculated plantlets kept under open environment had lower chlorophyll content and sun scorching damages compared to M. salsuginis TNMB03 inoculated plants, which had dark green leaves and increased chlorophyll content. Interpretation: This study shows a new potential technique of using M. salsuginis TNMB03 in tissue culture plantlets, which can help in enhancing the growth of plantlets transferred from culture vessel to greenhouse or open environmental condition without undergoing the routine acclimatization procedure.

Development of accurate temperature regulation culture system with metallic culture vessel demonstrates different thermal cytotoxicity in cancer and normal cells

Hyperthermia has been studied as a noninvasive cancer treatment. Cancer cells show stronger thermal cytotoxicity than normal cells, which is exploited in hyperthermia. However, the absence of methods evaluating the thermal cytotoxicity in cells prevents the development of hyperthermia. To investigate the thermal cytotoxicity, culture temperature should be regulated. We, thus, developed a culture system regulating culture temperature immediately and accurately by employing metallic culture vessels. Michigan Cancer Foundation-7 cells and normal human dermal fibroblasts were used for models of cancer and normal cells. The findings showed cancer cells showed stronger thermal cytotoxicity than normal cells, which is quantitatively different from previous reports. This difference might be due to regulated culture temperature. The thermal stimulus condition (43 °C/30 min) was, further, focused for assays. The mRNA expression involving apoptosis changed dramatically in cancer cells, indicating the strong apoptotic trend. In contrast, the mRNA expression of heat shock protein (HSP) of normal cells upon the thermal stimulus was stronger than cancer cells. Furthermore, exclusively in normal cells, HSP localization to nucleus was confirmed. These movement of HSP confer thermotolerance to cells, which is consistent with the different thermal cytotoxicity between cancer and normal cells. In summary, our developed system can be used to develop hyperthermia treatment.

Optimized Manufacture of Lyophilized Dermal Fibroblasts for Next-Generation Off-the-Shelf Progenitor Biological Bandages in Topical Post-Burn Regenerative Medicine

Cultured fibroblast progenitor cells (FPC) have been studied in Swiss translational regenerative medicine for over two decades, wherein clinical experience was gathered for safely managing burns and refractory cutaneous ulcers. Inherent FPC advantages include high robustness, optimal adaptability to industrial manufacture, and potential for effective repair stimulation of wounded tissues. Major technical bottlenecks in cell therapy development comprise sustainability, stability, and logistics of biological material sources. Herein, we report stringently optimized and up-scaled processing (i.e., cell biobanking and stabilization by lyophilization) of dermal FPCs, with the objective of addressing potential cell source sustainability and stability issues with regard to active substance manufacturing in cutaneous regenerative medicine. Firstly, multi-tiered FPC banking was optimized in terms of overall quality and efficiency by benchmarking key reagents (e.g., medium supplement source, dissociation reagent), consumables (e.g., culture vessels), and technical specifications. Therein, fetal bovine serum batch identity and culture vessel surface were confirmed, among other parameters, to largely impact harvest cell yields. Secondly, FPC stabilization by lyophilization was undertaken and shown to maintain critical functions for devitalized cells in vitro, potentially enabling high logistical gains. Overall, this study provides the technical basis for the elaboration of next-generation off-the-shelf topical regenerative medicine therapeutic products for wound healing and post-burn care.

Microdissected Tissue vs Tissue Slices—A Comparative Study of Tumor Explant Models Cultured On-Chip and Off-Chip

Predicting patient responses to anticancer drugs is a major challenge both at the drug development stage and during cancer treatment. Tumor explant culture platforms (TECPs) preserve the native tissue architecture and are well-suited for drug response assays. However, tissue longevity in these models is relatively low. Several methodologies have been developed to address this issue, although no study has compared their efficacy in a controlled fashion. We investigated the effect of two variables in TECPs, specifically, the tissue size and culture vessel on tissue survival using micro-dissected tumor tissue (MDT) and tissue slices which were cultured in microfluidic chips and plastic well plates. Tumor models were produced from ovarian and prostate cancer cell line xenografts and were matched in terms of the specimen, total volume of tissue, and respective volume of medium in each culture system. We examined morphology, viability, and hypoxia in the various tumor models. Our observations suggest that the viability and proliferative capacity of MDTs were not affected during the time course of the experiments. In contrast, tissue slices had reduced proliferation and showed increased cell death and hypoxia under both culture conditions. Tissue slices cultured in microfluidic devices had a lower degree of hypoxia compared to those in 96-well plates. Globally, our results show that tissue slices have lower survival rates compared to MDTs due to inherent diffusion limitations, and that microfluidic devices may decrease hypoxia in tumor models.

Plant Tissue culture and Ultra High Diluted studies: suggesting a novel model using in vitro techniques

Plant tissue culture techniques have been used to evaluate the effects of many different substances and/ or conditions in plant growth and development. It provides information of great value about problems related to basic and applied aspects of plant as well as contributed to understanding of factors responsible for growth, metabolism, synthesis of secondary compounds, stress response. Considering all this wide range of applications and as all plant tissue culture techniques are undergone under axenic and controlled conditions (culture medium composition, light and temperature, for instance), it seems to be a value model for Ultra High Diluted (UHD) studies. Lippia alba is a Brazilian plant that tissue cultures protocols and in vitro essential oil production have already been described in scientific literature. None of all scientific papers evaluated the effects of UHD substances on in vitro development or secondary metabolic production. The main goal was to evaluate the use of plant tissue culture to investigate the effects of UHD benzilaminopurine (BA) on Lippia alba shoot culture. Nodal segments obtained from plants growth in vitro was subcultured to Murashigue & Skoog semi-solid medium added with 2ml of these different solutions: BA 3µmol, BA 12CH (10-24), water 12CH and water (no dilution and succussion). Weekly 1 ml of solutions were added to cultures. The experiment was repeated twice and each one consisted in 3 culture vessel with 5 nodal segments per treatment (n=30). All plants were maintained in growth room under controlled temperature (25°C), light and photoperiod (16L/8D). The tested substances were prepared according to the method of stepwise dilution and succussion as describe in Brazilian Homeopathic Pharmacopoeia. The experiment was blinded all the time. After 60d, plantlets were evaluated for number of shoots, shoot length, rooted plants (%), callus development (%) and fresh biomass. Data were submitted to ANOVA following by Duncan’s and t-test. Plants from water 12CH and BA 12CH increased the number of new shoots and promoted the highest shoot length. By adding BA 3µmol the organogenetic response was inhibited since neither shoot nor root were developed. However, it was observed a significant basal callus development. Plant tissue culture could be adapted for UHD studies. More studies are being conducted in way to analyze other experimental conditions and biochemical/phytochemical parameters.

Plant Uptake of Lactate-Bound Metals: A Sustainable Alternative to Metal Chlorides

Global agricultural intensification has prompted investigations into biostimulants to enhance plant nutrition and soil ecosystem processes. Metal lactates are an understudied class of organic micronutrient supplement that provide both a labile carbon source and mineral nutrition for plant and microbial growth. To gain a fundamental understanding of plant responses to metal lactates, we employed a series of sterile culture-vessel experiments to compare the uptake and toxicity of five metals (Zn, Mn, Cu, Ni, and Co) supplied in lactate and chloride salt form. Additionally, primary root growth in plate-grown Arabidopsis thaliana seedlings was used to determine optimal concentrations of each metal lactate. Our results suggest that uptake and utilization of metals in wheat (Triticum aestivum L.) when supplied in lactate form is comparable to that of metal chlorides. Metal lactates also have promotional growth effects on A. thaliana seedlings with optimal concentrations identified for Zn (0.5–1.0 µM), Mn (0.5–1.0 µM), Cu (0.5 µM), Ni (1.0 µM), and Co (0.5 µM) lactate. These findings present foundational evidence to support the use of metal lactates as potential crop biostimulants due to their ability to both supply nutrients and stimulate plant growth.

In vitro determinants of e-cig aerosol condensate bioavailability and toxicity: Influence of cell culture parameters and utility of a normalized dose metric in e-cig studies

Electronic cigarettes (e-cigs) have a strong foothold in the marketplace as a product to replace tobacco cigarette usage. Despite many researchers investigating the use of e-cigs and possible health issues, there is still controversy concerning how to evaluate and use e-cig condensates. Therefore, to identify factors that influence in vitro e-cig studies, we examined parameters that can impact experimental outcomes. We generated high wattage e-cig aerosol condensate (ECAC) to determine reproducible conditions to evaluate ECAC with respect to cellular survival. Cytotoxicity of ECAC was independent of serum conditions. However, cytotoxicity of ECAC is altered by treatment duration and by physical factors, including cell seeding density and volume of ECAC used. In addition, interactions between ECAC components and cells, as well as the culture vessel surface, diminish the bioavailability of ECAC components in vitro and altered the results obtained. Moreover, the cell seeding density changes reactive oxygen species production in response to ECAC exposure. Our data indicated that normalized ECAC doses (ECAC weight per cell) better reflect the toxicity of ECAC than nominal doses (ECAC percentage). These results provide factors for researchers to consider in the design of in vitro experiments using ECAC.