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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)277-287
Number of pages11
JournalCell and Tissue Banking
Volume14
Issue number2
DOIs
Publication statusPublished - Jun 2013

Keywords

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

ASJC Scopus subject areas

  • Transplantation
  • Biomaterials
  • Biomedical Engineering
  • Cell Biology

Fingerprint Dive into the research topics of 'A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts'. Together they form a unique fingerprint.

Cite this