A new Apparatus to Simulate the Tumor Environment and Reproduce Organs using an Interactive and Dynamic System: The ASTEROIDS Technology
In order to better understand organ’s physiology and identify new therapeutic targets and drugs, there is a need to evolve from standard cell culture methods to more complex systems1 able to reproduce the three-dimensional (3D) structure2, the intercellular and extracellular matrix interactions and the dynamic fluid compartment of a specific tissue. This project aims to demonstrate the proof-of-concept of a new Apparatus to Simulate the Tumor Environment and Reproduce Organs using an Interactive and Dynamic System, referred to as ASTEROIDS. The ASTEROIDS technology is comprised of two perfused chambers separated from a central piece containing six separated round-bottom well by porous membranes, thus allowing simultaneous culture of 3D cellular structures loaded into the well with cells seeded on membranes and submitted to fluid stress. In addition, in order to provide a user-friendly device, the ASTEROIDS is designed to be compatible with multiple bioassays and can be easily dismounted or directly monitored by microscopy or standard plate reader. To establish the feasibility of such technology, the objectives for this project were to demonstrate immune cells infiltration, assess cell attachment and growth on the membrane, and to monitor spheroid growth within the device in order to reproduce ovarian tumor microenvironment. Assessment of different porous polycarbonate (PC) membranes were tested for endothelial cell attachment and immune cell infiltration. PC 3 and 8 µm membranes were attached on an insert, coated with collagen, and HUVECs were seeded and allowed to attach for 1h. Jurkat T-cells were then injected in the presence or not of SDF-1α at the bottom of the well and counted after 3 hours incubation. The results showed that the 3 µm membrane prevented Jurkat T-cells infiltration and that the number of Jurkat T-cells crossing the membrane decreased in the presence of HUVECs. Consequently, an 8 µm membrane was mounted in the ASTEROIDS to study HUVECs behavior within the device under flow. The ASTEROIDS device was assembled and sterilized by X-Ray irradiation before collagen coating and cells injecting. After one-hour incubation, ASTEROIDS platform was run with a flow rate of 25 µL/min (Shear stress = 0.14 dyne/cm2). The results showed that HUVECs are attached from one hour and aligned with the flow from 24 hours. Finally, SKOV3 cells were seeded in 96-well round bottom low-affinity plate and after 5 days, spheroids were transferred into the device where they were directly visible under microscopy. Consequently, spheroid growth, after external stimulus (i.e. 2Gy X-ray irradiation) was compared between culture on the plate and ASTEROIDS. As a result, irradiated spheroids cultured in the plate showed a decreased growth rate. However, spheroids in the device were not visible within the next two days due to potential toxicity or leaking in the first design of ASTEROIDS prototyping. In conclusion, an 8 µm membrane was determined most efficient for HUVECs attachment and T-cells infiltration. Further, HUVECs were able to attach within ASTEROIDS in a dynamic environment and spheroids can be seeded within ASTEROIDS and be visualized directly under the microscope. This study highlights the promising potential of the ASTEROIDS to propose an alternative of standard model that could lead to a more physiologically relevant platform for biomedical research.