Perlecan - Expression - Expression in Cell Culture

Expression in Cell Culture

One of the resounding arguments against the validity of in vitro results of cell culture on 2D plastic plates is that the environment does not accurately reflect that of the cells in the organism. This problem is being dealt with by developing 3D cell cultures using a wide variety of substrates as the scaffolds or environments for the cells. In this kind of setting the expression of ECM genes has the potential to more closely resemble that of the native expression profile. 3D scaffolds, the structures on which the cultured cells grow, can be composed of other cells, i.e. cocultures, synthetic polymers mimicking the cells natural environment or purified ECM such as matrigel, and any mixture of these three components.

One such system has been developed to study skin development and basal membrane formation between keratinocytes and the stroma. This system is used to delineate the development of basement membrane between fibroblasts in the stroma (in this case fibroblasts in a type-I collagen gel) and keratinocytes grown on top of the gel. Perlecan expression and thus basement membrane maturation is dependent on nidogen crosslinking of collagen IV and laminin γ1 chain in this system. This effect also led to a lack of hemidesmosomes in the developing tissue. Another system using a disorganized hydrated collagen I gel has been used to demonstrate that primary human corneal fibroblasts will eventually invade the gel and create a matrix consisting of collagen type I and perlecan, as well as several other sulfated matrix glycoproteins. This mimics the in vivo corneal fibroblast's developmental program and response to injury.

One of the long-term goals of creating 3D cell culture systems is to engineer tissues that can be used as replacements for patients with many types of disease. In tissue engineered heart valves created by seeding myofibroblasts onto collagen type I followed by endothelial cells, heparan sulfate proteoglycan expression has been verified, although no distinction between syndecan and perlecan has been made in these tissues. Another procedure that could be made possible by tissue engineering is keratoepithelioplasty. Transplanted tissue must remain intact, which requires a pre-formed basement membrane. Collagen gels have promoted formation of a complete basement membrane by corneal epithelial cells in culture.

Perlecan also holds promise to serve as a scaffold for plating cells in culture. Human salivary gland ductal and acinar cells have been successfully grown on a bioactive peptide containing a sequence repeated in domain IV of the perlecan protein. These cells reproduce acini-like structures similar to those found in the native gland and tight junctions, along with complete basement membranes in culture.