A Model System to Analyze Cell:Cell and Cell:ECM Interactions
Authors: Worden Austin , Uline Mark , Potts Jay 
Published: April 2020

Studies of in vivo structures have been made possible through ex vivo cell culture systems. While two‐dimensional (2D) systems have long provided inexpensive means to test functionality of cells, three‐dimensional (3D) systems have been found to be more advantageous in providing accurate physiological response (i.e. in vivo tissue formation, increased differentiation, reduced proliferation, etc). One such technology that allows for a widely tunable extracellular matrix (ECM) is a hydrogel. Hydrogels have been proven useful in the advancements of drug delivery and tissue repair and engineering while also providing a solid molecular scaffold for cellular system modeling. Previously, our lab has observed a novel physiological response when cells were placed on top of a Collagen I hydrogel. Cells became long and thin while migrating on and through the hydrogel to form a ring‐like structure, a toroid. Interestingly, the developing toroid takes the shape of the well in which its formed. A toroid was not formed, however, with cells mixed into the hydrogel. In this case, there was an observed scaffold contraction with cells remaining uniformly distributed. Our recent studies expand on these findings by modeling cell:cell and cell:ECM interactions through the creation of single‐ and multi‐cellular environments within different hydrogel matrices. To date, we have used 9 formulations of matrices including three types of Collagen I (PureCol, Nutrigen, and Fibricol), Collagen III, Collagen V, Rat Tail Collagen, and three types of synthetic matrices (AlphaBioGel, Matrigel, and VitroGel3D) and used no less than 10 cell types including cancer cells, cardiac fibroblasts (NHFs), microvascular endothelial cells, and several types of stem cells (ADSCs).

Each hydrogel was prepared using a different, optimized protocol for the specific matrices used. Matrix solution was placed in a 96 well plate (100μL/well) and 50,000 cells in media were placed on top of each stabilized gel. Gels were incubated at 37 °C for 24 hours. They were then fixed in 2% Paraformaldehyde (PFA) prior to immunofluorescence staining.

A toroid is a structure created by cells cultured on top of a hydrogel. In our studies, all combinations of cell:cell:gel produced toroids except for various cancer cell lines (cells stayed spread through the hydrogel). When cells were cultured on Collagen V, no toroids were formed and the matrix was remodeled by cells, and VitroGel3D, carbohydrate matrix produced a scaffold containing cell clumps. The size of the toroids varied depending on the matrix which cells were cultured on. This suggests the possibility for optimizing the application for various protocols. Interestingly, when NHFs and ADSCs were co‐cultured on the same PureCol hydrogel, the ADSCs created florets within the toroid which was not observed in other multi‐cellular toroids. Confocal imaging showed cellular interactions in response to other cells and the changing ECM of hydrogels. Using this model of cellular interactions and programming, we can examine and explore [1] the understanding of early embryonic development and [2] the optimization of hydrogels for in vivo applications.

Support or Funding Information

SPARC graduate research grant, Cook Biotech, FirstString Research Inc, NIH 2 P20‐RR016434‐06, NIH INBRE grant for South Carolina P20GM103499, R01 HL126747.