Cell and Tissue Banking

, Volume 14, Issue 2, pp 277–287 | Cite as

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

  • Elena De Falco
  • Gaia Scafetta
  • Chiara Napoletano
  • Rosa Puca
  • Enzo Maria Vingolo
  • Giuseppe Ragona
  • Olga Iorio
  • Giacomo Frati
Original Paper

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 cm2 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.

Keywords

GMP Standardized surgical and laboratory methodology Cell therapy for ocular disorders Tenon’s fibroblasts In vitro drug testing Cell banking 

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Elena De Falco
    • 1
  • Gaia Scafetta
    • 1
  • Chiara Napoletano
    • 2
  • Rosa Puca
    • 1
  • Enzo Maria Vingolo
    • 1
    • 4
  • Giuseppe Ragona
    • 1
  • Olga Iorio
    • 3
  • Giacomo Frati
    • 1
    • 5
  1. 1.Department of Science and Medical-Surgical Biotechnologies, Faculty of Pharmacy and MedicineUniversity of Rome SapienzaLatinaItaly
  2. 2.Department of Experimental MedicineUniversity of Rome SapienzaRomeItaly
  3. 3.Department of Surgery “P. Valdoni”University of Rome SapienzaRomeItaly
  4. 4.Ophtalmologic Centre UnitSanta Maria Goretti HospitalLatinaItaly
  5. 5.IRCCS NeuromedPozzilliItaly

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