Human platelet lysate (HPL) as a supplement for ex vivo expansion of human limbal epithelial cell sheet (LECS)

Abstract

Purpose : To compare the characteristics of human LECS cultivated in defined supplemented hormonal epithelium (SHEM) medium added with 1%, 5%, and 10% HPLs.

Methods : This study was approved by NTUH REC (No. 20194034RINA). Human corneoscleral rims were sent for ex vivo cultivation immediately after the surgeries. Each rim was cut into 12 pieces and immersed with defined SHEM containing 1% collagenase A for 18 hours. LECS was removed and implanted on 24mm-type 1-collagen coated Transwell. LECS from every donor were randomly and evenly distributed into defined SHEM supplemented with 1%, 5% or 10% commercialized HPL and incubated at 37°C with 5% CO₂. The culture media were changed and the LECS were recorded for cell migration and morphology every 2-3 days. Photography of cell expansion area were analyzed between day 7 to day 14.

After 2 week of culvitation, LECS was divided into parts for immunohistochemical (IHC) and immunoprecipitate exams. Four μm prarffin sections were used for H&E and IHC. SDS-PAGE was used for immunoprecipitate photography. The expression of the limbal progenitor cell marker P63α, the matured corneal epithelial marker K12, epithelial marker pancytokeratin (PCK), and the stromal/migratory epithelial marker vimentin (Vim) were quantified.

Results : LECS were harvested from 20 donors (age: 48.5±19.7 years old, post-mortum time: 10.5±2.1 days). LECS expanded faster when cultivated in 1% HPL-SHEM as compared to those in 5% HPL-SHEM and 10% HPL-SHEM in day 7, 10, and 14. (all p value <0.05) (Fig.1) .

Outgrowths of LECS contained small cuboidal epithelial-like cells in the 1% HPL-SHEM, while cells culitvated in 5% and 10% HPL-SHEM exhibited heterogeneous morphology (spindle plus cuboidal shaped) (Fig 2A). H&E stain showed that LECS in 1% HPL-SHEM can formed 3 to 7 cell layers, while sheets in the 5% and 10% HPL-SHEM had mostly 1 to 3 cell layers (Fig 2B) with loose edge and fibroblast-like morphology at periphery. Both IHC and immunoprecipitate exams showed a higher portion of P63⁺ and K12+ cells in sheets cultivated in 1% HPL-SHEM; cell sheets cultivated in all concentrated HPL-SHEM showed equivalent amount of PCK⁺ and Vim⁺ cells (Fig 2B, 2C).

Conclusions : Our study has shown HPL-SHEM being an effective medium for cultivation of human LECS. Sheets grown in low concentrated HPL (1%) showed faster expansion rates with the limbal progenitor cell markers and morphology preserved.

This is a 2020 ARVO Annual Meeting abstract.