When were human limbal cells cultivated on amniotic membrane first used in humans for corneal disease?

First Use of Human Limbal Cells Cultivated on Amniotic Membrane for Corneal Disease

Human limbal cells cultivated on amniotic membrane were first used in humans for corneal disease in 1997 by Pellegrini et al., who pioneered this technique for treating limbal stem cell deficiency.

Historical Development

The cultivation of limbal epithelial stem cells on amniotic membrane represents a significant advancement in the treatment of severe ocular surface disorders, particularly those involving limbal stem cell deficiency (LSCD). This technique evolved through several key developments:

• The recognition of limbal stem cells as the source of corneal epithelial regeneration
• The identification of amniotic membrane as an ideal biological substrate for cell cultivation
• The development of ex vivo expansion techniques for limbal epithelial cells

Cultivation Techniques

Several methods have been developed for cultivating limbal epithelial cells on amniotic membrane:

• Explant culture method: Small pieces of limbal tissue are placed on denuded human amniotic membrane (dHAM) and allowed to proliferate 1
• Cell suspension method: Limbal cells are enzymatically dissociated and then seeded onto amniotic membrane
• Culture media variations:
  • Complex media containing fetal bovine serum and growth additives
  • Simplified media using human serum as the sole growth supplement 2

The cultivation process typically involves:

1. Harvesting limbal tissue (2mm) from patients or donors
2. Placing the tissue on prepared amniotic membrane
3. Supplementing with appropriate culture medium
4. Observing cell growth for 10-14 days until adequate coverage is achieved 1

Characterization of Cultivated Cells

Cultivated limbal epithelial cells are characterized by:

• Expression of stem cell markers (p63, K19, ABCG2)
• Reduced expression of differentiation markers (K3, K12) in basal layers
• Ability to form multilayered epithelium
• Presence of slow-cycling cells (identified through BrdU labeling) 3

Clinical Applications

The primary clinical application is for treating severe ocular surface disorders characterized by limbal stem cell deficiency, including:

• Chemical or thermal burns
• Stevens-Johnson syndrome
• Ocular cicatricial pemphigoid
• Aniridia and other congenital disorders

Clinical Outcomes

Early clinical studies showed variable success rates:

• In a 2002 study, epithelial regeneration was achieved in 61.5% of cases initially, with 46.2% maintaining corneal epithelialization at final follow-up 4
• Later studies demonstrated improved outcomes with refinements in technique, with successful ocular surface reconstruction and improved visual acuity in many patients 5

Evolution of the Technique

Since its introduction, the technique has undergone significant refinements:

• Development of feeder-cell free cultivation methods
• Use of autologous serum to eliminate xenogenic components
• Improvements in amniotic membrane preparation and stabilization
• Standardization of culture conditions and quality control measures

Current Applications in Ophthalmology

Today, cultivated limbal epithelial transplantation on amniotic membrane is recognized as an effective treatment for severe ocular surface disorders. The American Academy of Ophthalmology guidelines acknowledge the role of amniotic membrane in promoting healing through anti-inflammatory, anti-angiogenic, and prohealing mediators in patients with corneal epithelial defects 6.

This technique represents a significant advancement in regenerative medicine for ophthalmology, offering hope to patients with previously untreatable corneal conditions.