Research into diseases of the conjunctiva and the development of potential treatments, such as isolation and expansion of conjunctival stem cells, is impeded by the absence of intact sheets of human conjunctival tissue. Impression and brush cytology techniques only enable collection of the superficial layers of epithelia and disturb the normal anatomy (Tsubota et al. 1990; Calonge et al. 2004). Although small samples of full-thickness conjunctiva may be obtained from surgical specimens (Ang et al. 2004), or cadaveric donors (Cook et al. 1998), many studies would benefit from assessment of the tissue as a whole enabling comparison of different areas. To meet such needs, we have developed and described a surgical technique for retrieval of whole human cadaveric conjunctiva for ex vivo and in vitro research purposes.
Ethical approval was obtained from the appropriate ethics committee, and the study was performed in accordance to the standards laid down in the 1964 Declaration of Helsinki. Informed consent was obtained from donor relatives prior to their inclusion in the study. Conjunctival tissue was obtained from human cadaveric donors within 24 hr of death. The eyes and surrounding tissue were cleaned with sterile water followed by a 5% (w/v) solution of povidone iodine. The surgical technique is demonstrated in Fig. 1. Using an aseptic technique, both eyelids were split medially to laterally along the grey line using a number 15 blade and Moorfields forceps. The dissection was continued along this surgical plane until beyond the level of the fornices. Great care was taken not to touch or handle the conjunctiva with any instruments, as histological analysis of initial samples revealed corresponding tarsal epithelial loss. The medial and lateral canthi were similarly dissected to adjoin the upper and lower lid dissections. A 360° limbal peritomy was then performed with Westcott scissors as close to the limbus as possible, followed by a full blunt dissection below Tenon’s capsule in the episcleral space around the surface of the globe. This dissection was similarly continued until past the level of the fornices. The fatty tissue and Tenon’s capsule behind the conjunctiva were then divided until the anterior and posterior planes met, as demonstrated in Fig. 1I. It was found that it was easier to perform this dissection from the anterior plane using scissors to cut down onto the globe, taking care to do so beyond the fornices, so as not to breach the conjunctiva. This was completed circumferentially allowing the conjunctiva to be excised as a whole specimen. Eye sockets were reconstructed using cotton wool and plastic eye shields that hold the eyelids in a closed position in accordance with the guidance standards of the Royal College of Ophthalmologists (2008) with good aesthetic results.
Using this technique, whole conjunctival tissue was successfully obtained from 16 human cadaveric donors (32 eyes). Conjunctival tissue was subsequently wax embedded, sectioned and examined histologically confirming the presence of tarsal to limbal conjunctiva (Fig. 1J). Conjunctival epithelial cells were also harvested using trypsin disaggregation and co-cultured on an inactivated J23T3 mouse fibroblast feeder layer. Cultured cells were fixed in methanol and immunofluorescently stained for the conjunctival marker cytokeratin 19 (R. M. K. Stewart unpublished data).
We have developed a technique to retrieve whole human cadaveric conjunctiva. This will further enable future ex vivo and in vitro analyses of the whole human conjunctiva, which require a continuum of conjunctival tissue. This is of particular benefit to studies assessing the distribution and characterization of conjunctival stem cells to aid propagation and transplantation; similarly for inflammatory cells and vasculature to improve knowledge of ocular surface immunity and potential for therapeutic agents.