Preservation of Ocular Epithelial Limbal Stem Cells: The New Frontier in Regenerative Medicine

Abstract

Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as "medicinal products". Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.

Identification of the regulatory circuit governing corneal epithelial fate determination and disease

“…To characterize gene expression patterns that define the cell fate difference between human cornea LSCs and human skin KCs ( Fig 1A ), we used cultured LSCs established from post mortem limbal biopsies and basal KCs from skin donors. Both cultured cells have the capacity to regenerate stratified epithelial tissues in vitro [ 41 , 42 ] and have high p63 expression ( S1 Fig ), thus exhibiting the progenitor cell state. We performed comparative RNA-seq analyses from bulk and pseudobulk RNA-seq data (aggregated from scRNA-seq using cultured LSCs and KCs) experiments and incorporated our data with publicly available RNA-seq data for robustness ( S1 Table ) ( S1 Fig ) [ 16 , 32 ].…”

Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease

“…To characterize gene expression patterns that define the cell fate difference between human cornea limbal stem cells (LSCs) and human skin keratinocytes (KCs) (figure 1A), we used LSCs established from limbal biopsies taken from post-mortem cornea and basal KCs from skin donors. Both cultured cells have the capacity to re-generate stratified epithelial tissues in vitro 40, 41 , and have high p63 expression (supplementary figure 1F), thus exhibiting the progenitor cell state. We performed RNA-seq analyses on.…”

Defining the optimal cryoprotectant and concentration for cryopreservation of limbal stem cells

“…To circumvent this risk, small limbal biopsies may be taken from the healthy limbus and cultured in vitro [ 11 ]. Two strategies are employed: one is to expand the limbal biopsies on amniotic membrane while the other is expansion in suspension culture [ 8 ]. Few cells are required to seed suspension cultures and the cells are transferred to a scaffold to facilitate epithelium layer formation.…”

“…Few cells are required to seed suspension cultures and the cells are transferred to a scaffold to facilitate epithelium layer formation. This represents an autologous transplant and is conducted under good manufacturing practice (GMP) conditions in several clinical facilities worldwide [ 5 , 8 ]. In the UK, few centres can offer both GMP culturing facilities and surgical expertise.…”

“…In the UK, few centres can offer both GMP culturing facilities and surgical expertise. Therefore, dissemination of cultured material will be required, both for research and clinical transplantation [ 8 ]. Furthermore, cryopreservation of limbal suspension cultures is beneficial as it obviates the need for further biopsies in the case of graft failure [ 5 ].…”