title: Techniques for culture and assessment of limbal stem cell grafts.
abstract: The therapeutic use of limbal cultures for the permanent regeneration of corneal epithelium in patients with limbal stem cell deficiency (LSCD) has been reported in many studies. According to the guidelines for good manufacturing practice (GMP), strictly regulated procedures and stringent quality control tests are now required to manipulate stem cells as "medicinal products" and make engraftment safer and eventually more successful. This paper describes techniques for optimal preparation of limbal stem cell grafts, including 1) a reliable impression cytology assay for the grading of LSCD, 2) culture methods that maintain high percentages of limbal stem cells, 3) the use of specific markers for the detection of corneal, conjunctival, and limbal stem cells, namely keratin 12, mucin 1, and DeltaNp63alpha, and 4) assays to assess the presence of contaminants, such as murine fibroblasts, endotoxins, mycoplasmae, and viral particles, in the cultured graft. The use of some of these assays allowed us to obtain a regenerated normal corneal epithelium in approximately 80% of 166 LSCD patients who received transplants from 2004 to 2008.
authors: Veneto Eye Bank Foundation, Venice, Italy.
authors: Enzo
authors: E
authors: Di Iorio
authors: Stefano
authors: S
authors: Ferrari
authors: Adriano
authors: A
authors: Fasolo
authors: Elisabetta
authors: E
authors: Böhm
authors: Diego
authors: D
authors: Ponzin
authors: Vanessa
authors: V
authors: Barbaro
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D003316:Corneal Diseases / Q000145:classification / Q000175:diagnosis / Q000601:surgery*; D019573:Epithelium, Corneal / Q000502:physiology*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D011786:Quality Control; D012038:Regeneration; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*
chemical_list: D015415:Biomarkers; D007633:Keratins