Authors

Discussion

3D culture is essential for mimicking real tissues and organs. At present, mouse basement membrane extract (BME/Matrigel) is the standard substrate used in vitro. Major components are laminin-111, type IV collagen-121, nidogen-1 and HSPG. 

To develop a new substrate that mimics the in vivo environment, we searched for a solution which is liquid at low temperatures and gels at the temperature of the cell culture.

Our gel is based on fibrillar collagen and the E8 fragment of laminin cross-linked to hyaluronic acid with genipin.

The effects of genipin concentration, molecular weight and concentration of hyaluronic acid, and the type (acid soluble/pepsin-solubilized) and concentration of collagen were examined.

In addition to the basic formula, collagen (acid soluble, pepsin extracted; I&III, IV, V) and laminin-E8 fragment isoforms (111, 332, & 511) were used to adjust interaction with cell-specific receptors.

When 12-day mouse embryo cells isolated from kidney, colon, and liver were cultured in the gel, they differentiated and organized.

Mouse embryonic-derived renal cells cultured in the gel were monodisperse immediately after culture, but aggregation was observed after one day, and self-assembly into tissues with branching ureter was advanced by Day 7. Staining, with a marker specific for ureteral tissue, showed the branched structure.

The concept is to build physiological connective-tissue ECM including basement membrane and interstitial collagen fibrils.

This new gel can be used as an alternative to Matrigel/BME, overcoming some of the shortcomings of BME. It has more versatile properties that can regulate both mesenchymal and epithelial tissues in the body.