Optimization of culture conditions for porcine corneal endothelial cells.
OBJECTIVE
To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology.


METHODS
PCEC cultures were seeded at an initial cell density of 400 cells/cm(2) in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm(2) in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm(2) in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 microg/ml), ascorbic acid (10, 20, 40 microg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology.


RESULTS
Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium.


CONCLUSIONS
Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.
Proulx
Stéphanie
S
Laboratoire d'Organogénèse Experiméntale, Hôpital du St-Sacrement du Centre Hospitalier Affilié Universitaire de Québec and Department of Oto-Rhino-Laryngology and Ophthalmology, Université Laval, Québec, Canada. stephanie.proulx.2@ulaval.ca
Bourget
Jean-Michel
JM
Gagnon
Nicolas
N
Martel
Sophie
S
Deschambeault
Alexandre
A
Carrier
Patrick
P
Giasson
Claude J
CJ
Auger
François A
FA
Brunette
Isabelle
I
Germain
Lucie
L
D016475:3T3 Cells; D000818:Animals; D001205:Ascorbic Acid / Q000008:administration & dosage / Q000494:pharmacology; D002417:Cattle / Q000196:embryology; D002452:Cell Count; D018929:Cell Culture Techniques / Q000592:standards*; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000494:pharmacology; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects; D005312:Fetal Blood; D051379:Mice; D010902:Pituitary Gland / Q000737:chemistry; D013552:Swine; D014020:Tissue Extracts / Q000008:administration & dosage / Q000494:pharmacology
D003470:Culture Media; D014020:Tissue Extracts; D002809:Chondroitin Sulfates; D001205:Ascorbic Acid