METODOLOGÍA

The disparity in translation observed between in vitro (2D and 3D model systems), preclinical trials (animal models) and the results observed in clinical trials is attributed, in part, to the unrealistic synthetic systems that are the current

standard in cancer research approaches.

In cancer research, treatments have been discovered that enable a drug compound to be directed to a specific type of cell. Often, these approaches are developed by using model cell-line screening processes or in vivo xenograft models that identify unique genetic biomarkers (usually because of a single mutation).1 _{However, these models have poor clinical translation because of a} systematic inability to mimic key pathophysiological features. For example, 2D tissue culture conditions are entirely unrealistic in that tissue culture media (concentrations of nutrients such as glucose, glutamine, and other essential amino acids)2,3 _{as well as incubator conditions (oxygen levels in particular) are designed} to sustain mammalian cells on a dish rather than to mimic the physiologically restrictive conditions of an in vivo primary tumor. In addition, 2D cultures lack cellular, biochemical, and metabolic heterogeneity as well as the complexity of a 3D tissue microenvironment4_{, which directly influences the translatability of in vitro} drug screening. Xenograft models suffer from the limitations of the biology of the mouse itself. In comparison to the human systems it is designed to mimic, the mouse has a higher metabolic rate, is inbred, has a short lifespan, is reared in

sterilized environments, and in immunodeficient mice, transplantable tumors grow to treatment size over weeks (not years as is typical in humans).5 _{In comparison} to 2D cultures, xenograft models lack parametric control for mechanism of action investigations, and in many cases the animal system is actually too complex for meaningful controls. In addition, the use of monogenic human tumor cell lines in xenograft models is lacking the genomic instability of a primary cancer and is one of the main reasons why xenotransplantation data fails to translate to human clinical results.6 _{Although recapitulating all of the pathophysiological aspects of an} in vivo tumor may never be fully realistic, there is a need to define a new paradigm in tissue culture practices in order to implement new assay formats that mimic more of the essential aspects of heterogeneity in human tumors.

The multicellular tumor spheroid (MTS) model is a 3D in vitro cell culture system containing growth kinetics, cellular heterogeneity, nutrient and waste gradients, hypoxia, acidosis, drug penetration, drug response/resistance, and metabolic interactions mimicking that of an in vivo tumorigenic tissue.7,8 _This imitation of key pathophysiological conditions makes 3D cell culture an improvement when compared to overly simplistic 2D tissue culture or overly complex xenograft models. In fact, it is common opinion that intelligently designed 3D culture systems will bridge the gap between 2D and animal models.9 _However, there are practical drawbacks currently preventing 3D model systems from being widely implemented between standard 2D cell culture and xenograft experimental model systems. Conventional aggregation-based (or scaffold-free) spheroid

distribution, are low yield because of multiple factors (size selection to obtain a population with the same sized spheroids, number of wells on a plate to form spheroids etc.), are characterized by an inability to control cell ratios in co-culture populations, and have a marked lack of standardized assays for evaluation (proliferation tracking, viability, correlation to local biochemical microenvironments etc.). Cell encapsulation by droplet generation to produce MTS templates (scaffold-based using biomaterials) has been attempted using both in-air and microfluidic platforms10-12_{, but generally falls short in terms of overall cost,} simplicity of device fabrication, high throughput operation, monodispersity, biocompatibility, and recovery of cells for downstream assays.

The results presented in Chapters 4 and 5 have demonstrated droplet generation with biocompatible chemistries by microfluidic (Chapter 4) or centrifugal (Chapter 5) synthesis methods to produce uniform cell encapsulating droplets. The studies shown in Chapter 4 specifically validated a qualitative approach to cell encapsulation in droplets and growth over 9 days using spinner flask bioreactor culture and verified that the method of encapsulation was not cytotoxic using a LIVE/DEAD flow cytometric assay. These validation studies for 3D cell culture are a good step in progress towards developing standardized evaluation methods. However, as previously discussed, the microfluidics approach to cell encapsulation is still technologically limited in certain aspects, especially throughput. In contrast, the results in Chapter 5 demonstrate the use of centrifugal generation methods to produce droplets with encapsulated cells in a relatively high throughput manner and with excellent reproducibility. Therefore, in this Chapter centrifugal droplet

generation is used to begin the process of demonstrating (1) the formation of spherical constructs for cell proliferation tracking with the goal being comparison to results observed in 2D monolayer cultures; (2) the generation and culture over time of scaffold-supported spheroids and instant MTSs (iMTS); and (3) the culturing of cells in encapsulating droplets in altered chemical environments (Figure 6.1). The work here uses mouse and human cancer cell lines with a wildtype +HIF-1α phenotype (WT) and knockout -HIF-1α phenotype (KO), HKO3- TR and A549 respectively, as well as human MEL28. The use of these cell lines is relevant to the biomedical hypothesis posed in Chapter 1 so that discussion of the methods in progress are ultimately tied to a significant question concerning the interaction of pH, oxygen, and HIF-1α.

Figure 6.1 Droplet-based Bioassay Applications. Summary of the

approaches in development for the standardization of evaluation methods used to measure proliferation of droplet encapsulated cells, culturing of scaffold supported templates for spheroid formation, and the use of cell encapsulating droplets in altered environment experimental designs.