Integration of 3D bioprinting and tumor-on-chip platforms for the fabrication of simple and relevant colorectal cancer models

Abstract

Cancer research has been largely limited by the use of two-dimensional culture and animal models, but in recent years, 3D cell culture platforms have emerged to alleviate the limitations of such models. Some examples of 3D cell culture platforms include spheroids and organoids, bioprinted constructs, and organ-on-chip devices. 3D bioprinting is a biofabrication technique that allows the integration of cells and hydrogels that simulate the extracellular matrix, as well as the possibility to add signaling molecules. Several cancer models have been generated by 3D bioprinting, but their size remains limited. On the other hand, Organ-on-chip, and more specifically, tumor-on-chip devices (ToC) provide dynamic conditions to current in vitro cancer models. Despite their recent broader use, ToCs found in the literature still exhibit some limitations regarding their fabrication, operation, and size. Here we propose that by combining 3D bioprinting with tumor-on-chip devices, we can provide a more accurate emulation of the tumor microenvironment (TME). This thesis is comprised of four experimental chapters, three dedicated to ToC devices and one dedicated to bioprinting. In the first experimental chapter, we describe a robust 3D-printed ToC device that can host large amounts of engineered tissue (~1 cm3) and provide adequate convective and diffusive transport. The device consists of a rhomboidal chamber, inlet, and outlet ports. The mass transport inside the ToC can be regulated with the flow rate of culture media. The possibility to regulate the flow rate of culture media also allows the perfusion of drugs to perform drug response assays. Therefore, experiments with the first version of our ToC demonstrated a better convective and diffusive transport of culture media with an internal structure comprised of Alginate/GelMA microspheres compared to an empty chamber and a monolithic block of GelMA. Caco2 cells embedded in hydrogel microspheres showed >75% survival after 10 days of culture inside the ToC. Furthermore, Caco2 cells treated with 5-FU in a similar fashion as real chemotherapeutic treatments displayed a survival below 20% and a decreased expression of VEGFA and E-cadherin. In the second experimental chapter, we used a modified version of our original ToC, which now could hold even a larger amount of biofabricated tissue (~2 cm3). This new version of the ToC was designed to hold a glucose sensor to measure glucose concentration inside the rhomboidal chamber in real time. In this case, we used different continuous culture conditions to emulate different physiological conditions, as well as an anticancer treatment. In this set of experiments with the second version of our ToC, Caco2 cells embedded in hydrogel microspheres showed a ~99% survival. A steady-state glucose consumption was observed by measuring the glucose concentration in real-time with the glucose sensor; when the flow rate was increased, the glucose consumption reached a new steady state. Observing these changes in glucose consumption is important since cancer cells change their metabolism and can metabolize glucose at a higher rate than healthy cells, and these changes in metabolism also change the expression of several markers (i.e. glucose transporters & specific transcription factors [1]). Also, observing the changes in glucose consumption in real time can provide information regarding how some of the expression of specific markers can change over time. Glucose consumption measurements with the commercial glucose sensor also show that it is possible to measure changes in the steady state when a pulse or a continuous flow of insulin is applied. Caco2 microspheres treated with 5-FU displayed changes in glucose concentration and didn’t display a significant reduction in cell survival, but the expression of markers such as E-cadherin, N-cadherin, and Ki-67 was significantly decreased. These results provide insight into the measurement of variables within in vitro cancer models, which allows us to evaluate what is happening in the biological model and obtain a deeper understanding. Measuring variables inside an in vitro cancer model gives the flexibility to simulate scenarios that are biologically relevant and can be observed in real-time, a feature that is not present when animal models are used. In the third experimental chapter we focus on bioprinting. 3D bioprinting is a great tool either to fabricate independent cancer models or to combine it with other platforms like ToCs to create more relevant and complex models, but commercial bioprinters are still quite expensive and most laboratories cannot afford to buy one. That is why we developed an affordable in-house built DiY bioprinter equipped with a printhead with temperature control. Printing experiments with GelMA and Pluronic F-127 demonstrated the importance of temperature control during bioprinting. Likewise, bioprinting with a bioink comprised of GelMA and C2C12 cells was successful as observed by Live/dead staining and Actin/DAPI staining. In the fourth experimental chapter, we briefly describe a set of experiments that provide an insight into the potential integration of vasculature to an in vitro cancer model. It is well known that endothelial cells prefer to grow on surfaces, so, for these experiments we used the first version of our ToC and two different configurations: HUVEC cells grown on hydrogel microspheres or chaotically printed hollow fibers. Our preliminary results suggest that HUVEC cells were able to attach to both microspheres and hollow fibers previously treated with fibronectin, but they remained longer inside the culture chamber when seeded on constructs that contained embedded cancer cells. This is sustained by the earlier qPCR measurements where we observed a higher expression of HIF-1α and VEGF in Caco2 cells cultured inside the ToC, markers that promote vascularization of real progressive tumors. Altogether, these results demonstrate the importance of integrating 3D cell culture techniques like bioprinting and instrumented tumor-on-chip systems to provide a wider array of possibilities for the biofabrication of more accurate and relevant in vitro cancer models that can be used for studying and understanding the cancer biology, performing drug screening, and personalized medicine.