Human Corneal Endothelial Cell Cultivation From Old Donor Corneas With Forced Attachment

Abstract

Human corneal endothelial cells (HCEnCs) are responsible for maintaining the transparency of the cornea. Damaged or diseased HCEnCs may cause blindness. Replacement of the diseased cells with a healthy donor endothelium is the only currently available treatment. Tissue-engineering can serve as an alternative to conventional donor corneal transplantation. Due to the global shortage of donor corneas, a wide interest in the development of cultured graft substitutes and artificial corneas has increased. Availability of the old donor corneas is higher especially for research. Although it can be proposed as a valuable source for cell culture, its less proliferative capability emerges a challenge for the researchers. This article describes the use of hyaluronic acid (HA) in combination with Rho-kinase inhibitor (ROCK) Y-27632 for the cultivation of HCEnCs from older donor corneas (age > 60 years). Four conditions including and excluding HA + ROCK and its effect on early attachment rates and proliferation was studied on forty-eight corneas. It was observed that HCEnCs reach confluence within 10–15 days when cultured with HA + ROCK. This approach improves the efficiency of cell adhesion due to force attachment. HCEnCs from old donor corneas can be cultured using this method which may further lead to cell-based therapy for treating corneal endothelial dysfunction.

Fish Scale-Derived Scaffolds for Culturing Human Corneal Endothelial Cells

“…HCEnCs were isolated from research grade donor corneas using a peel-and-digest method similar to previously published methods, [ 7 , 13 – 15 ] with limited modifications. Firstly, the corneas [ n = 30] were washed in sterile PBS and Descemet's membrane with endothelium was dissected with a fine forceps, similar to the stripping technique used for Descemet's membrane endothelial keratoplasty (DMEK).…”

“…In order to overcome this scarcity, alternative therapeutic approaches such as ex vivo expansion of HCEnCs are under investigation to enable HCEnCs transplantation as cell sheets or cell suspension [ 4 – 7 ]. Once HCEnCs from one donor eye can successfully be expanded, we would finally be able to overcome the current 1 : 1 ratio where one donor cornea is used to treat a single patient.…”

Effects of corneal preservation conditions on human corneal endothelial cell culture

“…Varying factors such as health of the donor before death, the cause of death, as well as the duration from death to enucleation and the time from preservation to the establishment of culture (Peh et al, 2011A, 2011B;Beck et al, 1999;Miyata et al, 2001; Endothelial cell culture from preserved corneas Parekh et al, 2017) have been noted to affect the cell culture. However, we did not find any significant deviation in establishing the culture of HCEnCs due to the abovementioned factors.…”

“…For ZO-1, the area was selected and using pre-defined commands in Macros for converting the image to overlay masks, the total number of cells was automatically counted whereas the hexagonal cells and polymorphic cells were counted based on the cell structure in the particular area (with 6 borders). The same Macros were used as previously described (Parekh et al, 2017) to obtain results by simply inserting the algorithm in the ImageJ analysis.…”

“…However, as most of the young donor corneas are usually suitable for transplantation, it is difficult to find such tissues for cellular expansion. If HCEnCs can be cultured from old donor corneas by modulating cell adhesion then it may potentially increase the donor pool for culturing HCEnCs (Parekh et al, 2017), Apart from donor characteristics, it is also important to evaluate the media used for corneal preservation, and its effects on culturing of the HCEnCs isolated from older donor, in a systemic donor-matched manner. If the cornea is to be preserved for a longer period of time (between 14 to 21 days), then the storage period, methodology, as well as conditions must be optimized, validated and described if the corneas are intended for subsequent cell culture and expansion.…”