Poster G10 - Engineering the Pancreatic Ductal Adenocarcinoma Microenvironment in a microfluidic 3D PEGDA hydrogel

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sydney Allen

Student
University of Tennessee, Knoxville
Knoxville, Tennessee, United States

Co-Author(s)

SA

Samuel Agro

Graduate Student
University of Virginia, United States

Last Author(s)

LT

Lakeshia J. Taite

Professor
University of Virginia, United States

Introduction: Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), is challenging due to its aggressive nature and poor prognosis. Traditional 2D cell culture models fail to replicate the complex tumor microenvironment (TME), limiting our understanding of cancer biology and hindering the development of effective treatments. Microfluidic devices, which allow for precise manipulation of fluid flows, offer a promising solution. When used in conjunction with bioactive poly(ethylene glycol) diacrylate (PEGDA hydrogels, which provide the necessary support and environment for three-dimensional cell culture, these devices can more accurately mimic in vivo conditions. The hydrogel can be tailored to present the microenvironmental cues, both biochemical and biomechanical, while the device geometry enables precise control and distribution of nutrients and signaling molecules.1 This study focuses on the design of a microfluidic device-encapsulated hydrogel microenvironment that can be utilized for TME studies of the PDAC microenvironment, aiming to develop a more physiologically relevant in vitro model. By optimizing the microfluidic design and hydrogel composition, we seek to enhance the study of cell behavior, viability, and interactions within a controlled environment. This approach not only provides a more accurate representation of the tumor microenvironment but also facilitates long-term monitoring of cellular responses and interactions. This research can connect traditional 2D cell cultures with more physiologically relevant 3D studies, providing an improved in vivo culture system to study pancreatic cancer.

Materials and

Methods: The device mold was designed in Autodesk Fusion 360 and produced by Protolabs. Polydimethylsiloxane was mixed with a curing agent (10:1), poured into the mold and vacuum desiccated for 60 minutes (or until all bubbles were gone). The mold was then cured in an oven at 90°C 1 hr. The device was then cut from the mold and inlet and outlet ports were made by punching through the PDMS with a biopsy punch (1mm and 2mm). The devices were then plasma bonded to a glass slide and incubated again in the oven for another hour to ensure bondage. All devices were sterilized by exposure to UV light before cell seeding and encapsulation.
The peptide GGGPQGIWGQGK (PQ) and the cell-adhesive peptide RGDS were purchased from Genscript (Piscataway, NJ). Peptides were conjugated with acryloyl-PEG-succinyl valeric acid (PEG-SVA; Laysan Bio, Arab, AL) to produce mono- and diacrylate-PEG-peptides and acrylate-PEG-proteins capable of photo crosslinking into degradable hydrogel matrices. 5% (w/v) PEG-PQ and 3.5 mM PEG-RGDS were dissolved in HEPES (pH 7.4) with 1% (v/v) photo initiator (2,2-dimethoxy-2-phenylacetophnenone in N,N-vinylpyrrolidone; DMAP) to create the hydrogel precursor solution. HPAF-II cells (ATCC, Manassas, VA) were suspended in the gel precursor and cell culture media. The hydrogel precursor was then injected into the gel channel of the microfluidic device and exposed to UV light (365 nm, 10 mW/cm2) for 30 s to form a device-encapsulated hydrogel. Cells were cultured in the device overnight, then stained with a fluorescent Live/Dead stain to visualize cell viability within the device.

Results, Conclusions, and Discussions:

Results: The microfluidic devices successfully encapsulated HPAF cells in PEG-PQ and PEG-RGDS hydrogels, demonstrating high uniformity. The encapsulation process involved precise control over the flow rates of the cell suspension and the hydrogel precursors within the microfluidic channels, ensuring consistent encapsulation. The device's design, featuring a series of narrow constrictions, facilitated the formation of uniformly sized hydrogel droplets encapsulating single cells or small cell clusters (Figure 1A). The crosslinking of PEG-PQ and PEG-RGDS hydrogels was achieved in situ, leveraging the rapid mixing and confinement provided by the microfluidic system. This method not only ensured high encapsulation efficiency but also maintained cell viability and functionality within the hydrogels. Live/Dead staining revealed that cell viability was maintained. (Figure 1B) The percent of live and dead cells was quantified by counting the number of cells fluorescing green (live cells stained with Calcein AM) and the number of cells fluorescing red (dead cells stained with ethidium homodimer) divided by the total number of cells observed in each device. From our image analysis, the viability within the devices was approximately 67.74% ± 1.84% live cells and 37.25% ± 2.22%.

Discussion/

Conclusions: These findings suggest that the tested hydrogel environments are suitable for the culture of HPAF cells. By providing a more accurate representation of the tumor microenvironment, these hydrogels could improve our understanding of tumor biology, particularly the complex interactions between cancer cells and their surroundings. This advancement has the potential to enhance the development of therapeutic strategies by providing a more relevant in vitro model for drug testing and cancer research. Future studies should focus on fine-tuning these hydrogel compositions to better monitor cell-cell and cell-material interactions. Additionally, incorporating stromal and immune cells will provide a more comprehensive model of the tumor microenvironment.

Figure 1. A) Overview of the microfluidic device used in the study; features intricate channel networks designed for precise fluid control and manipulation. B) Encapsulated HPAF cells. The image shows high-resolution overlay microscopy of HPAF cells encapsulated within the polymer matrix, highlighting the structural integrity and distribution of the cells. This encapsulation technique is crucial for studying cell behavior and interactions.

Acknowledgements (Optional): We would like to extend our sincere gratitude to Dr. Candice Hovell of InVitri LLC for her invaluable contribution to the design and testing of our device.