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    Tissue Engineering

    Optimizing Large Polyacrylamide Hydrogels for High-Yield Cell Culture

    (K-412) Optimizing Large Polyacrylamide Hydrogels for High-Yield Cell Culture

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row K - Poster # 412
    Introduction::
    The extracellular environment plays an important role in regulating cell behavior. Hydrogels are biomaterials with tunable mechanical properties and biocompatibility that can recapitulate native cellular environment1. Hydrogels are three-dimensional networks of polymers that mimic the extracellular matrices, have similar mechanical properties to soft tissues, and can support cell adhesion and protein sequestration1. Polyacrylamide (PA) hydrogels are the most widely used substrates for cell culture due to characteristics like changeable elasticity, resistance to degradation, and controllable surface biochemistry2. These features enable the investigation of cellular behaviors in a physiologically relevant setting. While PA hydrogels have been extensively utilized in small-scale cell culture studies, their application in large-batch cell culture remains limited. The traditional method to prepare PA substrates, the glass-sandwich protocol, is tedious and challenging to conduct experiments that require million-level cell yield2. This study aimed to develop a scalable method to fabricate large-format PA hydrogels for high-yield cell culture.


    Materials and Methods:: Large PA hydrogel was prepared between two glass surfaces of a 10 cm petri dish. The pre-gel solution was made using acrylamide, bis-acrylamide, and deionized water, with the addition of Tetramethylethylenediamine (TEMED) and Ammonium Persulfate (APS) catalysts. The petri dish lid with the opening side facing up was treated with 5% (3-Aminopropyl)triethoxysilane (APTES) and 5% Glutaraldehyde. The bottom half of the petri dish was treated with Rain-X. The polymerized hydrogel in the petri dish lid was covered with parafilm. Using a syringe, 55% Aqueous Hydrazine Hydrate (HH) was inserted through the parafilm onto the hydrogel, followed by Glacial Acetic Acid (AA) wash. A protein solution containing Fibronectin, Sodium Periodate, and Phosphate-buffered saline (PBS) was added to polydimethylsiloxane (PDMS) stamps for patterns to be printed on the polymerized hydrogel for cell culture.

    Results, Conclusions, and Discussions::

    To optimize the polymerization process, large PA hydrogel was prepared between two glass surfaces of a 10 cm petri dish. The pre-gel solution was pipetted into the petri dish lid, with the opening side facing up, and the bottom half of the petri dish was placed on top of the pre-gel solution with the opening side facing up. The desired gel stiffness was made using formulations of acrylamide, bis-acrylamide, and deionized water. The pre-gel solution contained the desired gel stiffness solution and the addition of TEMED and APS catalysts. The petri dish lid with the opening side facing up was treated with 5% APTES and 5% Glutaraldehyde to provide a hydrophilic region of strong adhesion between the hydrogel and the glass surface. The bottom half of the petri dish was treated with Rain-X, establishing a hydrophobic region that allowed for easy removal from the surface of the hydrogel after polymerization. The polymerized hydrogel in the petri dish lid was covered with parafilm, followed by HH and AA washes to functionalize the gels. The gels were stamped using protein-covered PDMS stamps.
    The large-format PA hydrogels of 10 kPa and 40 kPa stiffnesses were produced using this optimized method. Under a light microscope, it was observed that the large-format hydrogel was able to support the adhesion and growth of cells. Faults in the stamping technique were attributed to cells not adhering to the specific stamped patterns. The observed results from using this protocol indicated that PA hydrogel production can be scalable and easily replicated. This optimized approach provides a less tedious and more cost-effective method to produce hydrogels than traditional methods. The hydrogels were able to support the adhesion and growth of cell cultures. The ability to culture large numbers of cells will enable the study of cell behavior in unprecedented detail. Specifically, this method could be used to study the effects of different mechanical stimuli on cell behavior, to develop new cell-based therapies for diseases such as cancer and heart disease, and to improve the efficiency of drug discovery.



    Acknowledgements (Optional): : This work was conducted at UMass Lowell’s REU Site: Inclusion and Innovation in Medical Devices, funded by the National Science Foundation (Award# 2243722).

    References (Optional): :

    1. Caliari, Steven R, and Jason A Burdick. “A practical guide to hydrogels for cell culture.” Nature methods vol. 13,5 (2016): 405-14. doi:10.1038/nmeth.3839


    2. Shi, Xuechen, and Paul A Janmey. “Large Polyacrylamide Hydrogels for Large-Batch Cell Culture and Mechanobiological Studies.” Macromolecular bioscience, e2300042. 2 May. 2023, doi:10.1002/mabi.202300042