Plastic Compressed Collagen Constructs for Ocular Cell Culture and Transplantation: A New and Improved Technique of Confined Fluid Loss

Abstract

Purpose: 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.

KEYWORDS::

• Cornea
• Collagen
• Plastic compression
• Biomimetic material
• Limbal epithelial cells
• GMP

ACKNOWLEDGMENTS

We would like to thank Dr Alex Shortt for his assistance with OCT imaging and Dr Peter Munro for his assistance with SEM and TEM imaging. Thanks to The Florida Lions Eye Institute for Transplantation and Research for assistance with procurement of research grade donor corneal tissue.

Declaration of interest: This study was supported by the Technology Strategy Board, the EPSRC (H.J.L.) and the National Institute for Health Research Biomedical Research Centre for Ophthalmology, Moorfields EyeHospital and UCL Institute of Ophthalmology (J.T.D.). R.A.L.D.: TAP Biosystems. All other authors have no conflicts of interest.