(C-89) Cell Seeding Approaches to Maximize Viability and Distribution in Microporous Annealed Particle Gel

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row C - Poster # 89

Presenting Author(s)

Sloan K. Pyatt (she/her/hers)

Undergraduate Researcher
Griffin Research Group- University of Virginia Department of Biomedical Engineering
Charlottesville, Virginia, United States

Primary Investigator(s)

DG

Don Griffin

Assistant Professor
University of Virginia, United States

Co-Author(s)

CF

Clare Flanagan

PhD Candidate
Griffin Research Group- University of Virginia Department of Biomedical Engineering, United States

Introduction:: MAP gel is a versatile hydrogel composed of a (poly)ethylene glycol (PEG) backbone functionalized with maleimides and a thiol crosslinker, which participate in Michael type addition with one another. Its applications are broad, ranging from vocal fold augmentation¹ to diabetic wound healing². Microspheres of the hydrogel are formed via a microfluidic approach.³ A water-in-oil emulsion generates particles from multiple channels within a microfluidic device, which creates a high-thoroughput, monodisperse population. A photoinitiator such as Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphonate (LAP), is used to enable a secondary crosslinking reaction. When put in contact with UV light, LAP bonds the particles via a radical chain polymerization reaction, forming a bulk scaffold in situ.
Standardization of scaffold morphology is difficult to accomplish via pipetting in vitro due to high probabilities of variability. The current method, wherein cells are seeded on top of the gel following annealing (topseeding), is unable to provide thorough three-dimensional integration. The two-dimensional starting position of the cells makes spreading throughout the z-plane of the gel unfavorable. Therefore, we propose a protocol that provides a robust method for highly consistent and reproducible MAP scaffolds with consistent volume and morphology.
Sub-Track: Biomaterials for Regenerative Medicine

Materials and Methods:: Plate centrifugation was used to evenly flatten gel within a 96 well plate in order to effectively distribute it across the surface of the well, providing a consistent template for reproducible in vitro studies. However, in order to conduct studies which accurately recapitulate a three-dimensional microenvironment, cells must be suspended in the matrix prior to annealing. There are two methods by which this might be achieved.
The first consists of using a positive displacement pipette to agitate MAP gel within an Eppendorf tube that also contains a previously isolated cell pellet, effectively suspending cells within the gel. 20 ul of the suspension will then be plated into each well and spun at the appropriate condition. We named this method the “butterchurn” method.
The second consists of creating a 3:1 dilution of gel (20 ul per well) in cell solution and media (60 ul combined per well), and then spinning the plate at the appropriate condition before withdrawing the excess liquid. This will also create a three-dimensional cell-in-gel suspension, We named this method the “dilution” method.
There is a clear need for optimization of both method and centrifugation metrics to maximize the standardization of viability. Following spindown, scaffolds will be annealed with 365 nm light at 152 mA for 30 seconds. Then, cells will be incubated for 24 hours, at which point 20 uL of Invitrogen Live/Dead stain will be pipetted into each well, allowing for effective viability analysis.

Results, Conclusions, and Discussions:: A variety of speed and time conditions (see attached images and figures) utilizing both the butterchurn and dilution methods yielded insightful results. Centrifugation at a higher speed (1000 G) for shorter periods of time (10 and 30 seconds) was found to result in higher viability when the butterchurn method was used. In the case of the dilution method, centrifugation at lower speeds (300 G and 500 G) for a longer period of time (1 minute) produced the most viable result. Butterchurning was initially evaluated against the traditional topseeding method. Further studies were then conducted to evaluate comparative viability of the butterchurn and dilution methods, and it was determined that the dilution method is significantly more viable. We believe that the mechanical stress placed on cells during their physical agitation with the butterchurn method results in a lower viability. The dilution method is able to forgo this physically invasive step and at a centrifugation time of 1 minute and speed of 500 G results in the greatest viability.
In terms of three-dimensional distribution, the dilution method also produced the most uniform results qualitatively. Moving forward, we plan to utilize these results to further refine the most optimized centrifugation metrics with the dilution method and quantitatively analyze cell distribution along the z-plane.

Acknowledgements (Optional): :

References (Optional): :

1.Pruett, Lauren J., et al. “Development of a Microporous Annealed Particle Hydrogel for Long-Term Vocal Fold Augmentation.” The Laryngoscope, The American Laryngological, Rhinological, and Otological Society, Inc.,130 10.1002/28442. 10 Dec. 2019, https://doi.org/10.1002/lary.28442.

2.Pruett, Lauren J., et al. “Heparin Microislands in Microporous Annealed Particle Scaffolds for Accelerated Diabetic Wound Healing.” Advanced Functional Materials, 31 10.1002/202104337. 18 June 2021, https://doi.org/10.1002/adfm.202104337.

3.Roosa, Colleen et al. “Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold.” Journal of visualized experiments : JoVE ,184 10.3791/64119. 16 Jun. 2022, doi:10.3791/64119