Engineering microscale cellular niches for three-dimensional multicellular co-cultures

TLDR

Three‑dimensional extracellular matrix environments are essential for realistic tissue engineering, tumor biology, and stem cell niche studies, as cells behave markedly differently in 3D compared to 2D substrates. The authors aim to develop a microfluidic device that can pattern multiple discrete 3D cell‑laden hydrogel constructs in a simple, stepwise manner to better mimic in vivo microenvironments. The platform enables real‑time imaging of autocrine and paracrine interactions among distinct cell types embedded in collagen‑I or Matrigel ECM, with gels patterned by culturing MDA‑MBC‑231 breast cancer cells and RAW 264.1 macrophages in separate channels. Modeling and experiments showed that surface tension, hydrophobicity, and geometry confine gels within channels, permitting tight dimensional control; over seven days, RAW macrophages invaded neighboring MDA‑MBC‑231 gels but not empty ones, demonstrating the device’s versatility for studying cell–cell and cell–matrix interactions.

Abstract

Modeling the in vivo microenvironment typically involves placing cells in a three-dimensional (3D) extracellular matrix (ECM) in physiologically relevant context with respect to other cells. The mechanical and chemical features of 3D microenvironments play important roles in tissue engineering, tumor growth and metastasis, and in defining stem cell niches, and it is increasingly recognized that cells behave much differently when surrounded by a 3D ECM than when anchored to a 2D substrate. To create microenvironments that more closely mimic in vivo settings, here we describe a novel microfluidic device that allows multiple discrete constructs of 3D cell-laden hydrogels to be patterned in a sequence of simple steps. The microfluidic platform allows for real-time imaging of the interactions between multiple cell types exposed to both autocrine and paracrine signaling molecules, all within a 3D ECM environment. Detailed modeling determined that surface tension, hydrophobic interactions, and spatial geometry were important factors in containing the gels within distinct separate channels during the filling process. This allowed us to pattern multiple gel types side-by-side and pattern 3D gels spatially with tight dimensional control. Cells embedded in gels could be patterned by culturing MDA-MB-231 metastatic breast cancer cells and RAW 264.1 macrophage cells within distinct collagen type I and Matrigel ECM environments, respectively. Over a 7 day culture experiment, RAW cells invaded into neighboring gels containing MDA-MB-231 cells, but not into gels lacking cells. These studies demonstrate the versatility and potential of this new microfluidic platform to engineer 3D microscale architectures to investigate cell–cell and cell–matrix interactions.