Granular Matrigel: Restructuring a Trusted Scaffolding Material to Improve Matrix Permeability

Matrigel is a commercialized extracellular matrix (ECM) protein mixture widely used for cell-culture applications. Since its discovery in 1975, Matrigel has grown to become the most widely used scaffolding material for three-dimensional cell culture. Matrigel serves important roles in supporting cell viability, adhesion, and differentiation, as well as tissue organization and tissue explant growth. Despite its widespread use, the permeability of Matrigel to certain molecules and cells is limited. For example, heparin sulfate, a component of Matrigel binds strongly to proteins and limits their diffusion through the matrix. Matrigel has also been shown to physically limit the diffusion of colloidal particles and migration of biological cells. To overcome this limitation, here we present a method for structuring Matrigel into a three-dimensional porous scaffold composed of concentrated Matrigel microgels. The porous scaffold provides the mechanical integrity required for cell culture, but offers enhanced permeability for molecules, colloidal particles, and biological cells. Porous scaffolds composed of synthetic microgels are referred to as granular hydrogels, so we refer to our scaffolds as granular Matrigel.

To form these scaffolds, monodisperse Matrigel drops are created using drop-based microfluidics, and concentrated in well-plates using centrifugation. We analyze the structure of the resulting scaffolds using fluorescent and backscattered-light confocal microscopy and find that scaffolds with high or low porosity can be obtained by controlling centrifugation speed. To evaluate their permeability, we image the infiltration of fluorescent microsphere particles (diameter of 1 µm) and migration human dendritic cells (DCs) into the scaffolds using time-lapse confocal microscopy. Our imaging shows that particles disperse through the scaffold in a matter of hours. Similarly, DCs, which we show cannot migrate through Matrigel, migrate freely through granular Matrigel. We also observe that DCs migrate through granular Matrigel in response to a cytokine (CXCL1) gradient. To evaluate granular Matrigel for 3D cell culture applications, we incorporate spherical human gastric organoids, image the growth and viability of the organoids over eight days, and follow the migration of DCs to the organoids after their addition to the media. These studies highlight the potential of granular Matrigel for long-term and 3D cell culture applications. Our coculture experiments indicate that granular Matrigel provides a sufficient mechanical integrity to support organoid growth, while simultaneously facilitating the migration of DCs through the scaffold (see Figure below).

In conclusion, here we present granular Matrigel as a permeable alternative to bulk Matrigel. Granular Matrigel is flexible to be used for coculture applications where a scaffold is required to support the cell structure and interconnected pores are required to facilitate the permeation of drugs, nutrients, or cells. The permeability of these scaffolds can be tuned using centrifugation. We expect these scaffolds will enable a wide variety of experiments in tissue engineering, cell-to-cell interaction and signaling studies, therapeutic-cell manufacturing, physiology and tissue development, injectable scaffolds, and tumor studies.

Figure. Confocal laser scanning micrograph images support the use of granular Matrigel as a scaffold for coculture studies. (A) Max projection micrograph of human gastric organoids (stained with CellTracker green (C7025)) on day four of culture in granular Matrigel. (B) Max projection micrograph of human dendritic cells (stained with CellTracker Deep Red (C34565)) reveal their migration through the granular Matrigel structure, 30 hours after addition. (C) 3D micrograph of dendritic cell movement tracked with IMARIS software. Scale bar represents 200 µm for all images. Micrographs constructed from 340 µm z-stacks.