Stem Cell Therapy for Corneal Epithelium Regeneration following Good Manufacturing and Clinical Procedures

Beatriz E Ramírez, Ana Sánchez, José M Herreras, Itziar Fernández, Javier García-Sancho, Teresa Nieto-Miguel, Margarita Calonge, Beatriz E Ramírez, Ana Sánchez, José M Herreras, Itziar Fernández, Javier García-Sancho, Teresa Nieto-Miguel, Margarita Calonge

Abstract

Objective: To evaluate outcomes of cultivated limbal epithelial transplantation (CLET) for management of ocular surface failure due to limbal stem cell deficiency (LSCD).

Design: Prospective, noncomparative, interventional case series and extensive comparison with recent similar studies.

Participants: Twenty eyes with LSCD underwent CLET (11 autologous; 9 allogeneic) and were followed up for 3 years. Etiologies were divided into 3 prognostic categories: Group 1, chemical injuries (7 eyes); Group 2, immune-based inflammation (4 eyes); and Group 3, noninflammatory diseases (9 eyes). Intervention. Autologous and allogeneic limbal epithelial cells were cultivated on amniotic membranes and transplanted. Evaluations were based on clinical parameters, survival analysis, and in vivo confocal microscopy (IVCM). European Union Tissues/Cells Directive and good manufacturing procedures were followed.

Main outcome measures: Improved clinical parameters, absence of epithelial defects, and improved central corneal epithelial phenotype.

Results: Success rate was 80% at 1-2 years and 75% at 3 years. Autografts and allografts had similar survival. Success rate was significantly lower in prognostic Group 1 (42.9%) than in Groups 2-3 (100% each). All clinical parameters improved substantially. By IVCM, 80% of cases improved in epithelial status.

Conclusions: CLET improved corneal epithelium quality, with subsequent improvement in symptoms, quality of life, and vision. These results confirm that CLET is a valid therapy for ocular surface failure.

Figures

Figure 1
Figure 1
Limbal biopsy, limbal epithelial cultivation, and cultivated limbal epithelial transplantation (CLET). (a) Healthy donor eye 24 hr. after a 2 × 2 mm limbal biopsy; (b) the biopsy tissue was placed in an Eppendorf tube with culture medium (b1); (c) the biopsy was processed in a good manufacturing practice-cell processing unit within the next 4 hr. and for the next 4-5 weeks; and (d) the explant was placed on denuded human amniotic membrane, as viewed by contrast phase microscopy. Limbal epithelial cells began outgrowth from the explant at 1-2 weeks (d1). The explant was then removed, and the outgrowth was maintained until reaching confluence at which time it contained approximately 250,000 cells. The cell product was then sent to the medical center for CLET (d2). (e) Superficial keratectomy in the diseased contralateral eye (Case 1); (e1) human amniotic membrane with epithelial limbal cells confluent on top is removed from culture dish; (e2) the complex of amniotic membrane-limbal stem cells is placed on top of the previously denuded corneal and sclerolimbal surface; the amniotic membrane limit is observed (black arrow) with cells facing down and sutured (white arrows). A scleral lens is then applied.
Figure 2
Figure 2
Kaplan-Meier survival (success) curves of the 20 cases undergoing cultivated limbal epithelial transplantation (CLET) according to follow-up time (maximum of 36 months) (a) and separated by the origin of cells, allogeneic CLET (b) and autologous CLET (c). Shaded areas represent the confidence bands. Survival analysis showed a probability of success at 1-2 years and at 3 years after CLET of 0.80 (confidence interval [CI] 95%, 0.643–0.996) and 0.75 (CI 95%, 0.582–0.966), respectively, for all cases (a). Allogeneic CLET had a 1-2-year and a 3-year survival probability of 0.667 (CI 95%, 0.420–1) and 0.556 (CI 95%, 0.31–1), respectively (b). The survival probability for autologous CLET was 0.9091 (CI 95%, 0.0867–0.7541) after 1, 2, or 3 years (c). The difference in survival between autografts and allografts was not significant (log-rank test, p value: 0.0949).
Figure 3
Figure 3
Case 17 (Table 1) before and after cultivated limbal epithelial transplantation (CLET). This 52-year-old male suffered a chemical injury in his right eye 5 years earlier. He developed total limbal stem cell deficiency and showed at the initial visit an opaque and vascularized cornea (a) with a conjunctival-like phenotype at in vivo confocal microscopy (IVCM) in central cornea (star), with goblet cells (horizontal arrow), inflammatory cells (vertical arrow), and Langerhans cell (circle), (a1). He received an allogeneic cultivated limbal epithelial transplantation (CLET). (b) After 12 months and although his symptoms and ciliary hyperemia had improved, this case was considered a CLET failure as his epithelial phenotype in central cornea (b1) was still conjunctival-like (star), with inflammatory cells (vertical arrow), evaluated by IVCM.
Figure 4
Figure 4
Case 8 (Table 1) before and after cultivated limbal epithelial transplantation (CLET). This 36-year-old man had limbal stem cell deficiency due to a unilateral chemical burn, with vascular pannus invading the visual axis (a). (a1) In vivo confocal microscopy (IVCM) shows a typical conjunctival-like epithelial phenotype in his central cornea (star), with goblet cells (arrow) and fibrosis (black arrowhead). This case was graded preoperatively in terms of visual prognosis as Grade 1, meaning that only cultivated epithelial transplantation (CLET) would be required for visual rehabilitation. After 12 months, this case was considered successful as all clinical signs improved (b) and symptoms decreased and IVCM showed an epithelial corneal phenotype (b1).
Figure 5
Figure 5
Case 11 (Table 1) before and after cultivated limbal epithelial transplantation (CLET). This 27-year-old male had a total limbal stem cell deficiency due to an early failed penetrating keratoplasty 7 years before. It was performed 5 years after a contact lens-related Acanthamoeba keratitis (a). (a1) In vivo confocal microscopy (IVCM) in the central cornea showed intense fibrosis (black arrows) and a conjunctival epithelial phenotype. (a2) Limbal cells for cultivated limbal epithelial transplantation (CLET) were obtained from his contralateral healthy eye, the biopsy site of which is shown 3 months after biopsy. (b) Twelve months after autologous CLET, corneal neovascularization had almost vanished and IVCM showed a mixed epithelium phenotype (b1), conjunctival phenotype (star), and corneal phenotype (square). (b2) Limbal donor site 12 months after biopsy. (c) Fourteen months after CLET, a penetrating keratoplasty and cataract removal were performed, followed 12 months later with a compact and clear graft. IVCM showed a corneal phenotype (c1). (c2) The corneal transplant was still successful after 2 years (3 years after CLET) although an Ahmed valve was implanted 10 months after corneal transplant to treat his elevated intraocular pressure.

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