title: A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts.
abstract: Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.
authors: Department of Science and Medical-Surgical Biotechnologies, Faculty of Pharmacy and Medicine, University of Rome Sapienza, C.so della Repubblica 79, 04100, Latina, Italy. elena.defalco@uniroma1.it
authors: Elena
authors: E
authors: De Falco
authors: Gaia
authors: G
authors: Scafetta
authors: Chiara
authors: C
authors: Napoletano
authors: Rosa
authors: R
authors: Puca
authors: Enzo Maria
authors: EM
authors: Vingolo
authors: Giuseppe
authors: G
authors: Ragona
authors: Olga
authors: O
authors: Iorio
authors: Giacomo
authors: G
authors: Frati
mesh_terms: D000367:Age Factors; D001706:Biopsy; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D064987:Cell- and Tissue-Based Therapy; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D020405:Dermis / Q000187:drug effects / Q000473:pathology; D005128:Eye Diseases / Q000628:therapy; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D066298:In Vitro Techniques; D012737:Sex Factors; D058475:Tenon Capsule / Q000473:pathology*; D014015:Tissue Banks
chemical_list: D017077:Culture Media, Conditioned