Figure 4 - Characteristics of the 14% eSF hydrogel microfluidic platform. A) Images showing the unique mechanical properties of the developed platform after stretching (tension), torsion, and bending at days 1 and 3. B) Perfusion of blue ink through the serpentine channel and formation of a soluble ink gradient due to the hydrophilic nature of the eSF hydrogel. Scale bar: 7.3 mm. C) Representative microscopy snapshots showing the flow of microstructures along the serpentine channel.
Next, we aimed at building a very simplistic colorectal tumor model, as shown in Figure 5 A. For this, we encapsulated colorectal cancer cells within the bulk of the eSF hydrogel and HCoMECS were seeded in the walls of the serpentine microchannel. We first investigated the ability of cells to adhere and spread in the microfluidics channel. Hydrogels obtained from Bombyx mori are not utterly absent of binding sequences. Silk proteins contain a main heavy chain, considered a hydrophobic protein with a co-block design (32). Hydrophilic segments are involved in the self-assembling process, causing differences in water amount and the mechanical properties of the final hydrogel. This can elucidate the mechanisms by which a certain level of cell attachment and migration happens in Bombyx mori derived hydrogels (14). Moreover, sequences that are RGD-like are present in the N-terminal segment of heavy-chain, VTTDSDGNE and NINDFDED (32). Indeed, previous works have shown the culture of endothelial cells in eSF hydrogels with good cell adhesion and growth (11).
Figure 5 B focuses on the culture and adhesion of HCoMECS on the eSF microchannel. Cells showed normal morphology and elongation 1 day after the seeding. After 3 days, HCoMECS reached a confluent layer characteristic of native vasculature (33). Finally, due to the hydrogel’s high permeability, DAPI/Phalloidin injection also stained HCT-116 located in the bulk of the hydrogels.
We next assessed the viability of HCT-116 cells encapsulated within the 14% eSF hydrogel to determine if the cells survived the fabrication procedure. The live/dead assay performed at a random location of the device on day 1 and day 3 showed that cells were viable and proliferating (Figure 5 C-D). This experiment also showed the cells were isotropically distributed across the eSF hydrogel.
To further confirm cell viability, we quantified the levels of ATP both in 2D static and in the eSF microfluidic chip for up to 7 days. A calibration curve was used to define its relationship with bioluminescence in 2D. We performed a direct correlation using the same number of cells to correlate the results in our eSF platform. The signal obtained when testing the hydrogels shows that the cells remained highly viable (Figure 5 E). In 2D, there is a statistical difference in every day within the same timepoint due to cell proliferation in cell flasks without constrains. In 3D, there is only a statistical difference when comparing day 3 to day 1. Being encapsulated in a rather stiff hydrogel, cells proliferate at lower rates. Natural 3D hydrogels confer different characteristics to cells when compared to 2D, such as high cell viability but controlled proliferation and differentiation (34). Finally, it is worth noticing the controls, which can provide valuable information about the homeostasis of the cells in the system. This means that the chosen design allows for cell viability: feeding the system through the inlet via the serpentine is enough to provide nutrients/oxygen diffusion. A significant statistical difference was found in metabolic activity on days 1 and 3, with increased activity on day 3 (Figure 5 E). It showed the ability of the eSF hydrogel microfluidic platform to support a stable and viable culture of HCT-116 cells under perfusion.
Finally, we evaluated the effect of dynamically injecting 5 µM GEM (through the serpentine channel) on HCT-116 cells for up to 7 days to validate the model (35). GEM is now being tested as treatment of patients with advanced CRC (36). Alamar blue (cell viability) assay shows no statistical differences in the presence or absence of GEM on day 1, most probably due to the time it takes for the drug to diffuse across the hydrogel (Figure 5 F). Moreover, it was observed a slight decrease in metabolic activity for GEM between day 1 and day 3. The anti-neoplastic effect was only observed after 7 days, as corroborated by other studies that show this “prolonged” effect (37). Overall, the obtained results (AB and ATP) demonstrated that cells are viable when encapsulated in a highly concentrated mesh of eSF.
In brief, we obtained an eSF microfluidic platform that can be used for many biological applications, such as organ-on-chip and point-of-care diagnostics. As an alternative to PDMS, it can be used in the scale up of soft microfluidics devices for commercial or industrial purposes since it is cost-effective, and the stiffness can be tailored to the model of choice.