Ongoing work

5.2.1 Designing a paper-based culture for flow cytometry

To adapt the PBC outlined in Chapter 4 for flow cytometric analysis, I changed the design of both the paper scaffolds and the holders (Fig. 5.1). Changes to the paper were made to accommodate the large number of cells needed to accurately measure population distributions by flow cytometry. In the assay described in Chapter 4, the nine-zone scaffolds contained 4.2x105 cells. While literature does not define the minimum number of cells needed to accurately assess population distributions by flow cytometry, protocols provided by flow cytometry facilities often recommend analyzing at least 5x104 cells and starting assays with ~2x106 cells to account for losses during sample preparation/staining. Using these recommendations, I augmented the paper scaffolds to hold 2x106 cells at the same cell density used in Chapter 4 (168,000 cells/µL). To achieve this cell number, I adjusted the diameter of the paper and removed the wax patterns. These changes allowed me to access more volume of the paper scaffolds, greatly increasing the number of cells a single scaffold can hold. It is worth noting two observations: the diameter of the paper scaffold can be changed with respect to cell density to ensure that 2x106 _{cells are} always seeded per scaffold; and wax patterns will disintegrate into particulate during post- incubation extraction and staining, which is why no new wax patterns were developed for assays described in this chapter.

Changes to paper design also required changes to the holder. For these experiments, I adopted fabrication techniques outlined in Chapter 3 and used laser-cut, biocompatible acrylic

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holders. The opening on the top acrylic holder is readily modified to match the diameter of the paper scaffolds used for a particular assay. Relative to the metal holders depicted in Chapter 1, 2, and 4, fabrication of the acrylic holders is straightforward, as it does not require equipment or expertise necessary for machining stainless steel.

5.2.2 Extracting cells from a paper-based culture

Flow cytometric analysis of PBCs requires the cells to be extracted from their paper scaffolds. To recover the cells from their Matrigel environment, I used a proprietary solution specifically developed to depolymerize Matrigel (Cell Recovery Solution, Corning). Cells were recovered by incubating the scaffolds in the recovery solution for 4 h at 4 ºC with intermittent agitation. After the extraction, cells were centrifuged, washed, and prepared for staining. The conditions for staining were recommended by the manufacturer and provided a recovery rate of 84% of the cells.

To ensure cell viability was not affected by the extraction process, HCT-116 cells were incubated in either static medium or in the recovery solution with increasing amounts of

agitation. Viability was determined by staining with a positive marker for live cells (fluorescein diacetate, FDA) and a positive marker for dead cells (7-aminoactinomycin D, 7-AAD). By using a two-dimensional analysis for viability, separation between the two populations becomes more pronounced and easier to identify. From the flow cytometric analysis of the live and dead cells, three populations were seen: cells positively stained for 7-AAD, cells positively stained for FDA, and cells negatively stained for both dyes. (Fig 5.2) The percent of unstained cells increased with agitation in recovery solution, suggesting that the recovery solution affected the metabolism of FDA. While it is not clear what inhibited this reaction, these results show that FDA should not be

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used to positively identify viable cells post extraction. Comparing the distribution of positively stained 7-AAD cells across all replicates showed there is no significant increase in dead cell populations during the extraction (Fig 5.3). These findings indicate that the extraction does not cause cell death.

5.2.3 Discerning between live, dead, and apoptotic cells

Identification of viable cell populations was performed using two fluorescent stains: an amine reactive dye (Zombie Green™, BioLegend), and annexin V labeled with pacific blue (ThermoFisher). As described in Chapter 1, amine reactive dyes covalently bond to free amines on proteins; dead/dying cells with compromised membranes allow the dye to access intracellular proteins, greatly increasing staining relative to viable cells.31 By incorporating annexin V into the staining regime, cells experiencing early-stage apoptosis with intact membranes can also be discerned from viable cells.32 _{It is important to note that amine reactive dyes are compatible with} cellular fixation and permeabilization; however, the staining regime proposed in this chapter will not require fixation or permeabilization.

After optimizing dye concentration via titrations, these dyes were used to assess relative chemosensitivity of colon carcinoma cells (HCT-116) in the PBCs described in section 5.2.1. For this work, the PBCs were assembled by stacking 9 cell-laden paper scaffolds and incubating the assembly for 24 h. After the initial incubation, the PBCs were dosed for 24 h with either a vehicle control or the active metabolite of a chemotherapeutic agent (14 nM SN-38). The assemblies were then taken apart, and the cells were extracted and stained.

Cytograms represent at least 5x104 cells per analysis (Fig. 5.4), and data extracted from three different analyses were averaged together to compare chemoresistances across scaffolds

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(Fig. 5.5). From these preliminary data, it becomes apparent that cells at the top of the stack— close to the nutrient and drug source—are sensitive to SN-38, while cells in the nutrient-poor region exhibit increased chemoresistance. While these results were similar to those seen in Chapter 4, flow cytometry was able to confirm the presence of viable cell populations in scaffolds furthest from the nutrient source.