title: Effect of biopsy location and size on proliferative capacity of ex vivo expanded conjunctival tissue.
abstract: OBJECTIVE
  To evaluate the effect of location and size of biopsy on phenotype and proliferative capacity of cultured rat conjunctival epithelial cells.
  
  
  METHODS
  Pieces of conjunctiva were used from six areas: superior and inferior areas of bulbus, fornix, and tarsus of male Sprague-Dawley rats (n = 6). Explants were grown in RPMI 1640 with 10% fetal bovine serum on coverslips for 8 days or assayed for colony-forming efficiency (n = 9). Analysis included immunofluorescence microscopy and outgrowth measurements with ImageJ software. The Mann-Whitney test and Spearman's rank-order correlation test were used.
  
  
  RESULTS
  Superior (23.9 ± 2.9-fold growth) and inferior (22.4 ± 1.2-fold growth) forniceal tissues yielded significantly more outgrowth with respect to explant size than superior bulbar (13.4 ± 1.9-fold growth; P < 0.05 and P < 0.01, respectively), inferior bulbar (13.6 ± 1.6-fold growth; P = 0.01 and P < 0.01, respectively), and inferior tarsal tissues (14.0 ± 1.3-fold growth; P = 0.01). Outgrowth size correlated positively with explant size (r(s) = 0.54; P < 0.001), whereas explant size correlated negatively with fold growth (r(s) = 0.36; P < 0.001). Superior forniceal cells displayed higher colony-forming efficiency (3.6% ± 0.9%) than superior bulbar (1.1% ± 0.3%; P < 0.05) and inferior bulbar cells (1.6% ± 0.8%; P < 0.05). Percentage of p63+ and PCNA+ cells correlated positively with explant and outgrowth size.
  
  
  CONCLUSIONS
  Small forniceal conjunctival explants grow the most effectively; however, for transplantation purposes, the loss of p63+ and PCNA+ cells with small explants must be considered.
authors.affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114, USA. j.r.eidet@gmail.com
authors.forename: Jon R
authors.initials: JR
authors.lastname: Eidet
authors.forename: Ida G
authors.initials: IG
authors.lastname: Fostad
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Tor P
authors.initials: TP
authors.lastname: Utheim
authors.forename: Øygunn A
authors.initials: ØA
authors.lastname: Utheim
authors.forename: Sten
authors.initials: S
authors.lastname: Raeder
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D001706:Biopsy; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D003228:Conjunctiva / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology*; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley