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Biomaterials

Biomaterials - Poster Session C

Poster B8 - High-throughput Mechanical Characterization of Cell Culture Gels in 96-well plates using Needle-induced Cavitation

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  The extracellular matrix (ECM) of a cell significantly influences cell metabolism, proliferation, and consequently, tumor cell dormancy. Cancer development and progression require the extensive reorganization of the ECM. Previous studies have indicated that ECM remodeling can also impact tumor cell dormancy by altering environmental stiffness1. Understanding the mechanical changes in the tumor microenvironment could provide insights into how mechanical properties influence cell dormancy, relapse, and growth.
96-well plates are commonly used in cell culture experiments because they provide a controlled environment for in vitro cell growth and manipulation. This project aims to develop a method for measuring the mechanical properties of cell culture gels in 96-well plates and enabling high-throughput mechanical assessments by adapting cavitation rheology (needle-induced cavitation (NIC)) measurements to small, confined volumes. NIC involves inserting a needle into a sample and applying pressure into a small, localized area. The pressure is gradually increased, until an instability causes a cavity to rapidly expand at a critical pressure, Pc. (Figure 1) This critical pressure provides information about its elastic and fracture properties, such as its modulus (E)2,3.

By testing different parameters, such as needle radii and sample volume, we aim to measure the critical pressure required for cavity formation in various cell-culture gels. The critical pressure data will be analyzed to determine the elastic modulus of the gels, allowing us to understand how the different parameters influence the accuracy and consistency of modulus calculations for 96-well plates.

Materials and

Methods:  We used a well-characterized triblock copolymer organogel, poly (methyl methacrylate)-poly(n–butyl acrylate)-poly(methyl methacrylate) (PMMA-PnBA-PMMA) from Kurary Co. Ltd., as a reference model. The copolymers were dissolved in 2-ethyl-1-hexanol (from Sigma Aldrich) at 70°C overnight. Different volumes (100, 200, 350 µL) of the solution were then pipetted into Falcon 96-well Clear Microplate and cooled at room temperature at 20°C for testing. The gels are thermoreversible, so they can be heated above 60°C to repair deformation zones induced by cavitation. This allows the samples to be reused. After preliminary tests on the triblock gel, we plan to test cell culture gels such as collagen, poly (ethylene glycol) (PEG) gels and alginate gels.

For NIC measurements, a flat-tipped needle is inserted at 1.0 mm/s using TA.XTPlus Connect Texture Analyzer to a depth that is 70% of sample height, then retracted 1.0mm to 3.0mm depending upon the sample volume to create a pre-seeded fracture below the needle tip4. Air is injected at a controlled rate (2500 µL/min) using a syringe pump. When Pc is reached, the gel rapidly expands at the needle tip, followed by a drop in pressure due to the formation of a cavity. This critical pressure is used to determine the material's mechanical properties2. The needle outer diameters used were 0.3mm, 0.4mm, 0.5mm, and 0.9mm.

Results, Conclusions, and Discussions: Results and Discussion
The critical pressure recorded during NIC is plotted against 1/a (where a is the needle radius). The results show that the critical pressure increases as the needle size decreases. This trend aligns with previous studies on NIC conducted on samples with larger volumes5.
The plot also suggests that the boundary constraints in smaller samples with smaller volumes also influence the recorded critical pressure. The mechanical interactions between the sample and the base and walls of the well result in a higher critical pressure due to confinement.

Conclusions
We demonstrated that needle size and sample volume affects the critical pressure for NIC on swollen polymer gels in 96-well plates. We will develop a model and adapt existing theories to account for confinement effects in 96-well plates to enhance this methodology and enable accurate translation to biological contexts and small volumes. Additional measurements in various biocompatible cell-culture systems will be done to further examine the effects of different parameters on the mechanical properties of the samples. Cavitation rheology can determine the moduli of local heterogeneities in small, confined volumes, making it a valuable tool for characterizing cell-culture gels and understanding mechanical changes in the tumor microenvironment.

References 1. Di Martino, Cancers, 2021,13,19 4916. 2. Delbos, Soft Matter, 2012,8, 8204-8208. 3. Zimberlin, Soft Matter, 2007,3, 763-767. 4. Barney, Soft Matter, 2019,15, 7390-7397. 5. Fuentes-Caparrós, Soft Matter, 2019, 15, 6340-6347.

Acknowledgements (Optional):