Cornea is the front transparent window of the eye which is responsible for optimal and clear vision. Transparency of this tissue is highly inevitable and cannot be compromised. Human cornea is made up of multiple layers out of which the posterior layer ‘endothelium’ is responsible for the transparency of the cornea. Endothelium is a monolayer of cells that allow the ions and solutes to transport from aqueous humour to the cornea and back which in turn maintains the transparency of the cornea by preserving the homeostasis between the anterior and posterior cornea. Earlier, it was observed that the endothelium had non regenerating capability however; recent studies have shown that these cells could be proliferated in vitro. Currently, the only method of treatment is the replacement of the diseased endothelium with the healthy donor endothelium. Penetrating keratoplasty which transplants a full thickness cornea was the only solution a decade ago. However, with the new advancements in the field of corneal transplants, specific surgical techniques like DMEK and DSAEK which replace only a part of the cornea have been identified. DSAEK replaces a part of the stroma along with the Descemet’s membrane and endothelium whereas DMEK only replaces the Descemet’s membrane and the endothelium and does not involve stroma. The results in terms of visual rehabilitation and outcomes have been found to be advantageous in these specific surgical procedures. However, DMEK is more challenging then DSAEK as DMEK is not yet a widespread technique, associated with steep learning curves and difficult donor tissue preparation. Despite DMEK is a challenging procedure it is becoming more popular because of the significant advantages in term of faster visual recovery, less postoperative astigmatism and reduced risk of transplant rejection, as compared to the other EK procedures. DMEK has several advantages in terms of rehabilitation rate and post-operative visual outcomes and therefore it is necessary to further refine this technique for a higher uptake of such surgeries and also considering that this is the only possible treatment for treating the patients suffering from endothelial dysfunctions. Although the corneal transplantation is well advanced, due to a limited supply of donor corneas for the transplantation purposes, alternative approaches like culturing corneal endothelium in vitro play an important role. Culturing the endothelium is not the only problem in EK but transplanting a 20 micron thick graft inside the recipient eye is another challenge. Moreover, the donor availability for culturing the corneal endothelium is less, making this strategy further more complicated. The thesis is therefore structured to highlight two significantly important issues in current scenario of endothelial keratoplasty, 1) posterior corneal transplantation or EK which is the on-going method of treatment for EK and 2) Human corneal endothelial cell culture which is the future of EK.
Chapter 1 is an introduction to the world of eye banking, its current nature and development in the modern world and as a support to the surgeons not only in terms of new techniques but also devices for selective surgeries. It also highlights the preservation of the corneal tissues which is an important element in the field of eye banking. Eye banks play a significant role in the field of corneal transplants as they collect the human corneas and process them for transplantation. The corneas that are rejected for transplantation can be used for research and therefore development of eye banking and its research can change the field of corneal transplantation. Chapter 2 introduces the field of corneal cell culture and current techniques that are followed for culturing and possible transplantation of the cultured cells.
To understand the reason and requirement of tissue engineering, it is important to study the human cornea, its extracellular matrix and its behaviour in different media. The biomechanical behaviour of the thin tissue i.e. the DM in different conditions becomes a relevant part of this study for future engineering which is studied in chapter 3. It is also important to standardize the currently available treatment options to reduce the burden of endothelial compromised patients in the future and avoid damages or tissue wastage that is currently occurring in the surgical theatres by providing standardized tissues in validated preservation medium which is studied in chapter 4. DMEK promises to become a more popular technique for the replacement of unhealthy corneal endothelium as it shows advantages like early rehabilitation rate and visual outcomes. Chapter 5 highlights the importance of new technique in rolling the DMEK tissue for easy insertion and unfolding in the recipient eye compared to the currently used technique with endothelium rolled in opposite direction. Presently, the DMEK tissues are either prepared in the surgical theatre or are stripped in the eye bank and shipped to the surgeons. However, there is no standardized procedure that could help validate a graft before surgery and provide a ready-to-use graft to the surgeons. Chapter 6 describes about a new technique of pre-loading a graft in a commercially available IOL cartridge which can be used as a preservation, transportation and transplantation device. This technique will further reduce graft wastage and will provide the surgeons a pre-validated graft further reducing the overall time in the surgical theatre and related costs. Thus different approaches for standardizing the DMEK technique were studied in the first phase of the thesis.
HCECs are currently being cultured using young donor corneas. There are two major issues, firstly, the availability of the young donor corneas is less compared to the old donor corneas and secondly, there is no standard method of culturing the HCECs obtained so far. Therefore, to reduce the global tissue demand, there is a strong need to culture the HCECs from the old donor corneas which are less proliferative and less robust in nature but with high availability of the donor source. Chapter 7 is a study on isolation of HCECs and further culture of these cells from old donor corneas. Once the protocol was obtained, a full length study was performed with high sample size to prove the consistency of this technique which is highlighted in chapter 8. Meanwhile it was also noted that cells from old donors can be cultured using ROCK inhibitor in combination with Hyaluronic Acid (HA). HA induces mechanical force to the cells attaching them forcefully on the base and allows a higher proliferation of old donor cells which was studied in chapter 9. The second part of the thesis therefore investigates the culturing technique of HCECs from old donor corneas. However, once the cells are cultured, another challenge is to transplant them in the anterior chamber of the eye. This can be performed using two strategies, first, to implant the cells as suspension in the anterior chamber which is already been proposed, but the clinical evidence is still not confirmed yet, and second, to develop a carrier to transport the cultured cells. In chapter 10, we identified fish scales as a great source of collagen and therefore have investigated it as a potential scaffold to be used for HCECs culture and transplant in the future. It is also important to understand the regulations that govern the scientific studies and its use for clinical applications. Therefore, we also identified rHSA as a source to replace FCS for preserving human corneas in chapter 11. This will also help to create a synthetic media that could be used for GMP purposes for HCECs culture in the future.
In conclusion, it was observed that pre-loading the tissues with endothelium-flapped inwards and preserved in dextran based medium could be a potential solution for providing a validated and standardized DMEK graft for the treatment of current endothelial dysfunction. Eye banks play a major role in the development of these surgical techniques and related devices which will change the face of corneal transplantation in the future. Alternatives like HCECs culture has a potential for the treatment of endothelial disorders and carriers like FSS could be used for culturing and transplanting these cells. However, the efficacy of these cells will only be validated after the clinical study. Considering the regulatory issues, synthetic medium would help both, the eye banks for preserving the corneas and its new products like pre-loaded DMEK and for cell culture in the future
