A Droplet Microfluidic Platform Used to Encapsulate Single Pre-formed Cancer Spheroids in Hydrogel Microenvironments

Abstract

Cancer has been a leading cause of death around the world for many years. Even with the emerging technologies seen in these times, there is still a lack of truly personalized treatments since cancer tumors are not fully understood, especially between different cancer patients. 3D models known as patient derived tumor organoids (PDTOs) have been gaining traction as in vitro models to mimic a patient’s tumor outside of their body. Grown from the patients' cells into cell clusters (spheroids) or organoids (mini parts of the tumor tissue), these models can serve as precise avenues for personalized drug discovery or studying tumor complexity. However, there are still limitations to these 3D models due to conventional fabrication methods. In order to achieve personalized care, the models being tested on should be consistent through each batch, reliable, and affordable. Traditionally, organoid development from cells aggregating in hanging droplets or rotating in flasks for maintained cell-aggregate suspension, can cause batch-to-batch inconsistencies (lack of uniformity between samples), low throughput, and often require high volume of reagents. To recreate a 3D suspension for cells in a more native environment, hydrogels have become popular biocompatible scaffolds capable of sustaining cell growth. Hydrogels themselves have been widely studied for ideal cell environments, yet common methods of cell encapsulation into these hydrogels (e.g. manually pipetting) do not address the mentioned limitations, but rather introduce inconsistencies. Droplet microfluidics (DM), with the inclusion of hydrogels, has become a technology that has assisted in organoid fabrication, addressing conventional limitations. DM can create uniform aqueous droplets at high frequencies through controlled emulsion, using microchannels. These uniform droplets allow for controlled cell encapsulation with minimal reagent usage, thus addressing the drawbacks of traditional organoid formation and cell encapsulation techniques. Yet, the downside to using DM for cell encapsulation (whether it is multi- or single- cell encapsulation) is the high initial concentration of cells used at the inlet reservoir (millions), compared to the typical sample size obtained from patient biopsies (thousands). To still leverage the advantages of DM, thoughts of encapsulating pre-formed cell clusters or spheroids (instead of forming them with the DM devices) can aim to lower the number of initial cells used, and aid in growing spheroids into organoids. Thus, the proposal of this thesis was to employ DM and defined hydrogels to encapsulate pre-formed spheroids into their own microenvironment, for the goal of supporting the growth of primary cancer patient cells into PDTOs in a robust and uniform iv manner. This thesis intends to give a better understanding of the need of using DM for pre-formed spheroid encapsulation, and the ways to achieve a robust system for this purpose. The first chapter of this thesis provides a deeper background of the motivation to the overarching goal and a further breakdown of the project tasks. Specifically, projects were designed around two aims: (1) optimizing portions of the system and (2) validating the system. In addition to this background information, more context is given through a literature review found in Chapter 2. This literature review further justifies the rationale of the work by providing more insight into each component. After outlining protocols used in each project (Chapter 3), the optimization work began, starting with finding the best way to form spheroids that would enter the DM device (Chapter 4). It was important that the mechanism of forming these spheroids was also robust and uniform to maintain the advantage over conventional spheroid fabrication techniques. A well-established micropattern technology known as the AggreWellTM was implemented due to its capabilities of forming uniform spheroids in a high throughput manner with a low quantity of cells. Using PDMS replicas of the AggreWellTM (referred to as PDMS AWs) caused for adjustments to typical protocols followed when using the original AggrewellTM. Parameters such as surface treatment, cell culture conditions, and collection methods, were studied to find the most appropriate outcome of spheroids for the intended application. Continuing in the avenue of optimization, the other portion of Chapter 4 focused on exploring DM devices and fabrication techniques. After comparing parameters such as mold fabrication through 3D printing and soft lithography, chip design and geometry (T-junction versus double flow focusing (DFF)), and system tubing size, the selected chip design was a DFF junction, similar to one used in previous work of single cell encapsulation. The other factors ensured that uniform droplet formation and single pre-formed spheroid encapsulation could be achieved, in part of making the system robust. Using the decisions made in the optimization work, validation of hydrogel selection, encapsulation efficiency, and spheroid viability were assessed in the main chapter (Chapter 5). This thesis work aimed to demonstrate the capabilities of the DFF DM device with the use of stratified flow. A hydrodynamic focusing stream of either Gelatin Methacrylate (GelMA) or sodium alginate hydrogel precursors helped to gather spheroids into their own droplets. Depending on the flow conditions, the width of the flow focusing stream varied, affecting the encapsulation efficiency. Along with focusing width, the concentration of spheroids at the inlet reservoir also affected the encapsulation efficiency. This study was able to show spheroid encapsulation with a range of spheroid quantities; from 1000 to 7000 total spheroids in 500 μL of hydrogel precursor. Lastly, the spheroid laden hydrogel droplets were crosslinked and assessed for stability in cell culture conditions over time. With this, the spheroid viability was also tested to ensure the overall system was not too harsh on them. As most spheroids showed a high viability, this was enough to satisfy the proposed objective and conclude that this system has potential to be further explored. In conclusion, the overall system showed success in robustly encapsulating pre-formed spheroids, in hopes of being applied to patient derived samples for uniform growth into relevant 3D models. As this work is preliminary, Chapter 6 outlines recommendations and suggestions to future work, to guide this project towards the overarching goal.