Human primary corneal fibroblasts synthesize and deposit proteoglycans in long‐term 3‐D cultures

Abstract

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2–4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue–stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.

Effects of Detergent-Based Protocols on Decellularization of Corneas With Sclerocorneal Limbus. Evaluation of Regional Differences

“…[31][32][33][34] Proteoglycans and glycoproteins are located between collagen fibers leading to establish a well-defined distance between collagen fibers that generates the lamellar pattern. 29,31,35 Therefore, to preserve both kinds of molecules is necessary to maintain the normal structural characteristics of the native cornea.…”

Design and Performance of an Optically Accessible, Low-Volume, Mechanobioreactor for Long-Term Study of Living Constructs

“…Having attained satisfactory mechanical and thermal control, the environmental qualities were investigated to validate that the device is capable of providing an aseptic/sterile chamber, able to support a living cell culture system for extended periods of time. To perform the complete functionality testing, we transferred our established human primary corneal fibroblast (HPCF) tissue engineering construct system 46,47 into the bioreactor. Briefly, HPCFs from a human donor were expanded in culture, released in the medium, and seeded into the bioreactor under sterile conditions in a laminar flow hood.…”

Bioengineering Organized, Multilamellar Human Corneal Stromal Tissue by Growth Factor Supplementation on Highly Aligned Synthetic Substrates

“…Zieske and collaborators have shown TGF-b to induce stromal tissue secretion by primary human corneal fibroblasts. [13][14][15][16] These constructs were up to 60 mm thick and multilayered. The TGF-b3 was most effective to induce the stromal matrix without expression of fibrotic markers.…”