Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

Matrigel is a polymeric extracellular matrix material produced by mouse cancer cells. Over the past four decades, Matrigel has been shown to support a wide variety of two- and three-dimensional cell and tissue culture applications including organoids. Despite widespread use, transport of molecules, cells, and colloidal particles through Matrigel can be limited. These limitations restrict cell growth, viability, and function and limit Matrigel applications. A strategy to improve transport through a hydrogel without modifying the chemistry or composition of the gel is to physically restructure the material into microscopic microgels and then pack them together to form a porous material. These ‘granular’ hydrogels have been created using a variety of synthetic hydrogels, but granular hydrogels composed of Matrigel have not yet been reported. Here we present a drop-based microfluidics approach for structuring Matrigel into a three-dimensional, mesoporous material composed of packed Matrigel microgels, which we call granular Matrigel. We show that restructuring Matrigel in this manner enhances the transport of colloidal particles and human dendritic cells (DCs) through the gel while providing sufficient mechanical support for culture of human gastric organoids (HGOs) and co-culture of human DCs with HGOs.

Human articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened resulting in increased chondrocyte stress. As chondrocytes are responsible for cartilage synthesis and maintenance, it is important to understand how mechanical loads affect cellular responses of chondrocytes. Many studies have examined the chondrocyte response to in vitro mechanical loading by embedding in stiff agarose. However, these experiments are mostly performed in the absence of PCM which may obscure important responses to mechanotransduction. Here, we demonstrate that drop-based microfluidics allows culture of single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes form PCM over 10 days in these single cell microenvironments. Single cell microgels and monolayer controls were encapsulated in high stiffness agarose to mimic the cartilage PCM. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.