title: Plastic compressed collagen constructs for ocular cell culture and transplantation: a new and improved technique of confined fluid loss.
abstract: OBJECTIVE
  Cultured limbal epithelial cell transplantation is a commonly used clinical treatment for ocular surface repair. We have previously shown that plastic compressed (PC) type I collagen constructs are a suitable substrate for human limbal epithelial cell (HLEC) culture for transplantation. For this process to achieve compliance with Good Manufacturing Practice, and therefore be suitable for therapeutic cell therapy manufacture, the original method required substantial modification.
  
  
  METHODS
  The compression method was changed from unconfined (highly variable reproducibility) to confined compression (CC) (highly reproducible manufacture) and we assessed whether this altered the physical characteristics of the substrate. We have measured transparency, assessed scanning electron microscope images of the surface and performed live/dead cell viability assays of cells within the constructs. HLECs were then cultured on the surface of both types of construct and the resulting cell phenotype characterized.
  
  
  RESULTS
  We have determined that the change in process does not alter the physical characteristics of the substrate. Furthermore, there is no change to the substrate's ability to support HLEC culture and maintenance of a mixed population of stem and differentiated cells. Additionally, cells were able to form a confluent sheet and multilayer to produce an intact epithelium.
  
  
  CONCLUSIONS
  This modification allows scaling up of the process in a well-plate format, which is essential for creation of multiple corneal epithelial models for in vitro testing. This improvement to the original plastic compression method also allows the process to be employed in custom-made cassettes, the design of which takes into consideration the manufacturing and regulatory requirements for delivery of a cell therapy.
authors.affiliation: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Bath Street, London, UK. h.levis@ucl.ac.uk
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Levis
authors.forename: Johannes
authors.initials: J
authors.lastname: Menzel-Severing
authors.forename: Rosemary A L
authors.initials: RA
authors.lastname: Drake
authors.forename: Julie T
authors.initials: JT
authors.lastname: Daniels
mesh_terms: D002102:Cadaver; D018929:Cell Culture Techniques; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D003094:Collagen / Q000737:chemistry*; D003316:Corneal Diseases / Q000601:surgery; D016039:Corneal Transplantation; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D003094:Collagen