Poster E6 - Optimization and Characterization of Alginate Micro-beads to quantify Intra-tumoral Stress

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Erica Rios Hernandez, BS

Student
San Jose State University
Concord, California, United States

Co-Author(s)

BT

Bryce Thompson, BS

Graduate Student
University of Louisville, United States
ZF

Zachary Fowler

Visiting Researcher
University of Louisville
Prospect, Kentucky, United States

Primary Investigator(s)

Joseph Chen, PhD

Principal Investigator
University of Louisville, United States

Introduction: Cancer progression is highlighted by the emergence of aberrant mechanical features in the tumor microenvironment that present pathological signals to drive tumor malignancy. Alterations to extrinsic signals such as tumor ECM stiffness and composition have been well described; however, the development of solid stresses within the tumor and its effect on tumor evolution is poorly understood. Recent reports have suggested that intra-tumoral stress guides cancer cell escape from tumors, highlighting an emergent mechanobiological driver of cancer progression; however, these aspects are difficult to investigate with standard in vitro tools and requires the development of advanced biophysical tools. To address this, we have developed a microfluidic platform that generates deformable alginate microbeads that are able to quantify compressive stresses generated within a growing glioblastoma (GBM) tumorsphere.

Materials and

Methods: Devices were fabricated using an SU-8 mold patterned with our device, which was designed using AutoCAD. Design consists of oil inlet (i), alginate inlet (ii) and collection outlet (iii) (Figure 1A). A 1:10 ratio of curing agent to Sylgard 184 elastomer was mixed, cured PDMS was bonded to glass slide. The design featured a Water-in-Oil emulsion junction that generates microbeads using a flow-based mechanism. Flow-based parameters were set using syringe pumps and 0.5% w/t alginate solution (MP Biomedicals) served as the dispersed phase and soybean oil (Thermo-Fisher) with Span-80 served as the continuous phase (Figure 1B-C). Once formed, microbeads were collected in 1% CaCl2 solution (Thermo Scientific) via collection outlet. Further characterization of beads involved particle geometry analysis using ImageJ (FIJI) and Atomic Force Microscopy (AFM) to revealing alginate bead stiffness.

Results, Conclusions, and Discussions: To optimize bead fabrication parameters, we iteratively modulated alginate flow rates and characterized bead formation rate and size. Oil flow was a control variable kept at 200 μL/hr, alginate flow rate was tested at 5 µL/hr, 10 µL/hr and 15µL/hr. Most success was found using 10 µL/hr whilst others experienced issues with blockage and undesirable flow behavior. Using set parameters, beads were monodisperse and spherical with a radius of 3-4 μm, as evidenced by shape analysis via ImageJ processing (Figure 1D). We then characterized the mechanical properties of alginate via AFM by scanning 3 10X10 µm regions and found a stiffness of ~ 2 kPa (Figure 1E). Lastly, we successfully embedded the force probe into GBM tumorspheres (Figure 1F). Ongoing work will quantify bead deformation with compressive stress through finite element analysis. Here, we present the development of a force probe platform capable of quantifying compressive stress in a 3D context. We have demonstrated an approach that generates monodisperse and mechanically uniform force probes in a high throughput manner. Additionally, this platform opens the door for new inquiries of the impact of compressive stress in other contexts, including developmental biology, stem cell biology, and fibrotic disease.

Acknowledgements (Optional):