Hypothermically stored alginate encapsulated mesenchymal stromal cells enhance corneal wound healing via paracrine action

Abstract

Purpose : Previously we have shown that alginate hydrogels are capable of retaining adipose derived mesenchymal stromal cells (ASCs) in a non-proliferative but viable state during hypothermic storage. Thus, we hypothesized that such entrapped, and previously stored, cells could act as reservoir of secretory factors that enable an enhanced corneal stromal wound repair in the form of a temporary stem cell bandage.

Methods : Human ASCs (Invitrogen, UK) were expanded until 70% confluent, detached and re-suspended (1x10⁶ cells/mL) in alginate discs (Atelerix Ltd, UK) then stored at 4 or 15^oC for 72 hours in airtight container. Following storage, the alginate discs (+/- ASCs) were tested for their effect as a stem cell bandage in vitro using a scratch assay and then in vivo using a mouse corneal burn model. The scratch assay comprised of corneal stromal cells extracted from human donor limbal rings. At confluence the cells were serum starved for 3 days and a 1mm scratch performed across the bottom of 6-well plates. Scratch healing was followed over two days in the presence of alginate (+/- ASCs) n=7 . RNA was extracted from ASCs and soluble growth factors measured using a cytokine array (R&D systems, UK). For the in vivo study, the left corneas of 6 mice under general anesthesia, were treated with 20% ethanol for 3mins and washed with HBSS. Alginate 3.5 mm discs (+/- ASCs), previously stored for 3 days at 15^oC, were placed upon the corneal surface and the eye lids sutured closed for 4 days. After 7 days the animals were sacrificed, eyes enucleated and prepared for histology.

Results : Alginate encapsulated ASCs stored at 15^oC significantly improved scratch closure (p < 0.0001). Moreover, they expressed higher levels of HGF and TSG-6 (p = 0.0197 and 0.0441 respectively) at the gene level and higher levels of IL-6, ENA-78 and IL-8 (p > 0.0001) at the protein level within conditined media. Clinical evaluation and histological sections from the in vivo study showed a decrease in corneal haze and significantly reduced number of infiltrating neutrophils (p<0.5) within the treated group.

Conclusions : Alginate encapsulated ASCs were shown to maintain the ability to produce a cocktail of paracrine factors following hypothermic storage and that these soluble factors can improve corneal wound healing, in vitro and in vivo.

This is a 2020 ARVO Annual Meeting abstract.