Poster M17 - Microgel assembly via cell adhesion and migration for biohybrid spheroid formation

Introduction: The fabrication of functional tissues in vitro is pivotal for advancing tissue engineering, regenerative medicine, and drug testing. Traditional two-dimensional (2D) cell culture fails to replicate the structural complexity and spatial organization of native tissues, which feature diverse cell types embedded within an extracellular matrix (ECM). Three-dimensional (3D) spheroids, which foster enhanced cell-cell interactions and adhesion, provide a more physiological representation of tissue architecture and function. However, spheroids created by conventional methods often form dense structures that can restrict oxygen and nutrient diffusion, leading to hypoxia and reduced cell viability. To address these challenges, we introduce a novel platform called biohybrid spheroids (BHS), developed via the self-assembly of adherent cells with microscale hydrogel particles (microgels) as ECM substitutes, resembling a cellular snowballing process. BHS allows for the construction of porous spheroids that maintain high cell viability even at the millimeter scale. This method may advance biofabrication by enabling the creation of large-scale, physiologically relevant tissue constructs without the need for complex vascularization strategies.

Materials and

Methods: Gelatin methacryloyl (GelMA) was synthesized by dissolving 200 mg of gelatin in 400 mL of Dulbecco’s phosphate buffered saline (DPBS) at 50 °C, followed by the dropwise addition of 16 mL of methacrylic anhydride. The reaction was quenched via dilution with an excess amount of DPBS (800 mL), then the product was dialyzed against Ultra-pure MilliQ water, and lyophilized. GelMA droplets were produced using high-throughput step-emulsification microfluidic devices. The aqueous phase consisted of 5% w/v GelMA with 0.1% w/v photoinitiator in DPBS, while the oil phase included a biocompatible surfactant (PicoSurf 2% v/v) in Novec7500 engineered fluid. Microgels were crosslinked by exposing the droplets to light (wavelength 395-400 nm, 15 mW/cm2 intensity for 2 min. NIH/3T3 fibroblast cells, cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% antibiotics, were used for BHS formation. BHS and microgel-free cellular spheroids (CS) were then formed by mixing different sizes/amounts of microgels and cells. Assemblies were formed on (1) flat, untreated surfaces (unconstrained), or (2) in untreated U-bottom well plates (constrained). The resulting aggregates were characterized for morphology, porosity, cell metabolic activity, and viability using various analytical techniques such as image processing, scanning electron microscopy (SEM), and confocal microscopy. These aggregates serve as building blocks for the formation of larger tissue-like structures, which are further analyzed for their potential in tissue engineering applications.

Results, Conclusions, and Discussions: Three distinct sizes of microgels were fabricated, 28 ± 2 μm (small), 76 ± 6 μm (medium), and 152 ± 10 μm (large) in diameter (Figure 1A). Cell-cell interactions were predominantly governed by integrin-ECM and cadherin-cadherin homophilic interactions, while cell-microgel interactions were primarily facilitated through integrin-RGD binding (Figure 1B). These interactions led to the formation of BHS or CS, which can happen in both flat, planar (Figure 1C) or U-bottom well-plates (Figure 1D), with varying kinetics and structural characteristics depending on the microgel size. Formed BHS with small (BHS-S), medium (BHS-M), large (BHS-L), or CS (control) underwent various morphological and biological assessments, namely scanning electron microscopy (SEM) (Figure 1E), confocal microscopy (Figure 1F), lightsheet microscopy, metabolic activity (Figure 1G), and real-time brightfield/fluorescence microscopy for morphological characterization, porosity measurement, circularity, and cell viability. BHS-M and BHS-L had higher porosity and metabolic activity compared with BHS-S and CS controls, as a result of higher compactness and lower porosity. However, BHS-S formed faster, in 2 days, whereas BHS-M and BHS-L took 3 and 4 days, respectively. BHS aggregates, owing to their fusibility, served as building blocks for creating larger mm-scale constructs (Figure 1H). BHS fusion was assessed using various types and configurations of BHS/CS, showing the superiority of BHS over conventionally used CS as building blocks for tissue like structures. Our study represents a novel approach for addressing the limitations of traditional spheroids by using BHS, which enables the construction of larger, more complex tissue structures with improved nutrient and oxygen diffusion. By orchestrating microgels cell-mediated assembly, this method not only mirrors the complexity of native tissues but also ensures enhanced viability and metabolic function. This technique offers scalable platforms for creating physiologically relevant tissue models.

Acknowledgements (Optional): Research reported in this publication was partially supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number R01HL167939 (A.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Part of the Figure were created using BioRender.com.