Abstract
AbstractWe describe the results of a phase I/II clinical trial for standardized, non‐xenogenic, cultivation and “no touch” surgical transplantation of limbal stem cell grafts. 18 eyes of 18 patients were transplanted with either autologous (n=15) or allogenic (n=3) limbo‐amnion composite grafts that were generated using a standardized culture protocol free of xenogenic culture products and transplanted using a standardized “no touch” surgical technique. In vitro cellular outgrowth and phenotype of the cultivated graft was assessed prior to transplantation. Corneal neovascularization, central corneal opacity, pain, photophobia and visual acuity were investigated pre and post transplantation. The limbal epithelial cells showed an average outgrowth of 14.2mm by day 14. The majority of the cells displayed a progenitor phenotype: p63, CK14, desmoglein, ABCG2 bright and CK3/12 dim protein expression. The transplant recipients were followed up for a mean of 22 months. 12 out of the 18 transplant recipients were graded successful, giving an overall success rate of 67%. The ocular surface photographs for pre‐ and post stem cell transplantation, showed a significant (p=0.007) reduction in the percentage area of corneal neovascularization.
Highlights
Limbal stem cell deficiency (LSCD) can result from a range of pathologies including ocular cicatricial pemphigoid, Stevens Johnson syndrome, aniridia, multiple surgeries and trauma [1]
Animal-product free culture protocol validation Progenitor cell targeted (PCT) CNT-20 media (CellnTec, Switzerland) supplemented with 1% human type AB serum was compared with supplemental hormonal epithelial medium (SHEM) consisting of DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 50 μg/ml gentamycin, 1.25 μg/ml amphotericin B, 5 μg/ml insulin-transferrinselenium growth supplement, 5% DMSO, 30 ng/ml cholera toxin, 0.5 μg/ml hydrocortisone and 2 ng/ml epidermal growth factor (Millipore, MA, USA)
Animal product-free culture validation The initial media validation experiments confirmed cellularization of the HAM with cobblestone-like appearance of limbal epithelial cell (LEC) in both SHEM and progenitor cell targeted CNT-20 medium. Both CNT-20 and SHEM showed a similar expression for ΔNp63 and extracellular matrix proteins: collagen IV, laminin and connexin 43 (Figure 2)
Summary
Limbal stem cell deficiency (LSCD) can result from a range of pathologies including ocular cicatricial pemphigoid, Stevens Johnson syndrome, aniridia, multiple surgeries and trauma [1]. The limbus is depleted of the resident epithelial stem cells permitting a vascular conjunctival membrane to grow over the cornea resulting in scarring, poor vision, pain and photophobia. These patients are a high-risk group for treatment with vision restoring therapies such as penetrating keratoplasty (PK) [2]. Transplantation may be performed either by directly implanting a kerato-limbal graft or by harvesting a biopsy, expanding the cells by tissue culture and transplanting the graft [3] The advantage of the latter is that it requires a smaller volume of donor tissue, reducing the risk of LSCD in the donor eye [4]. To determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency
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