Abstract
Chronic conditions requiring long-term rehabilitation therapies, such as hypertension, stroke, or cancer, involve complex interactions between various systems/organs of the body and mutual influences, thus implicating a multiorgan approach. The dual-flow IVTech LiveBox2 bioreactor is a recently developed inter-connected dynamic cell culture model able to mimic organ crosstalk, since cells belonging to different organs can be connected and grown under flow conditions in a more physiological environment. This study aims to setup for the first time a 2-way connected culture of human neuroblastoma cells, SH-SY5Y, and Human Coronary Artery Smooth Muscle Cells, HCASMC through a dual-flow IVTech LiveBox2 bioreactor, in order to represent a simplified model of nervous-cardiovascular systems crosstalk, possibly relevant for the above-mentioned diseases. The system was tested by treating the cells with 10nM angiotensin II (AngII) inducing PKCβII/HuR/VEGF pathway activation, since AngII and PKCβII/HuR/VEGF pathway are relevant in cardiovascular and neuroscience research. Three different conditions were applied: 1- HCASMC and SH-SY5Y separately seeded in petri dishes (static condition); 2- the two cell lines separately seeded under flow (dynamic condition); 3- the two lines, seeded in dynamic conditions, connected, each maintaining its own medium, with a membrane as interface for biohumoral changes between the two mediums, and then treated. We detected that only in condition 3 there was a synergic AngII-dependent VEGF production in SH-SY5Y cells coupled to an AngII-dependent PKCβII/HuR/VEGF pathway activation in HCASMC, consistent with the observed physiological response in vivo. HCASMC response to AngII seems therefore to be generated by/derived from the reciprocal cell crosstalk under the dynamic inter-connection ensured by the dual flow LiveBox 2 bioreactor. This system can represent a useful tool for studying the crosstalk between organs, helpful for instance in rehabilitation research or when investigating chronic diseases; further, it offers the advantageous opportunity of cultivating each cell line in its own medium, thus mimicking, at least in part, distinct tissue milieu.
Highlights
Biomedical molecular research aimed to the study of complex relationships between various tissues, as it happens in chronic diseases or when investigating resilience from a traumatic event, such as for example in rehabilitation or during aging, needs translationally relevant experimental models
The cell viability was studied after exposing both SH-SY5Y and Human Coronary Artery Smooth Muscle Cells (HCASMC) cell types for 6, 24 and 48 hours to increasing concentrations of angiotensin II (AngII) (1nM, 10nM and 100nM)
After 48 hours treatment, consistent with previously published data [8], a significant decrease in SH-SY5Y viability was observed at all AngII concentrations (85.5%±2.1, 78.1%±2.1 and 74.9% ±1.9 at 1nM, 10nM and 100nM respectively)
Summary
Biomedical molecular research aimed to the study of complex relationships between various tissues, as it happens in chronic diseases or when investigating resilience from a traumatic event, such as for example in rehabilitation or during aging, needs translationally relevant experimental models. The recent development of Next-Generation In Vitro Testing Tools, engineered within the 3Rs research, opens a new scenario to explore living systems providing a bridge between the use of cultured cells on a petri dish and in vivo/ex vivo experiments on small laboratory animals. An example of such a tool are IVTech Bioreactors [1], where, within the tissue engineering field, a bioreactor is defined as a device able to simulate a physiological environment allowing cell or tissue growth. It is designed to be consistent with plates or transwells, allowing the use of standard protocols for in vitro procedures
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