Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.
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.
Ren
R
R
Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Hutcheon
A E K
AE
Guo
X Q
XQ
Saeidi
N
N
Melotti
S A
SA
Ruberti
J W
JW
Zieske
J D
JD
Trinkaus-Randall
V
V
D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D011509:Proteoglycans / Q000096:biosynthesis*
D011509:Proteoglycans