A new standardized immunofluorescence method for potency quantification (SMPQ) of human conjunctival cell cultures

AbstractThe aim of this study is to set up a standardized and reproducible method to determine the potency (=  stem cell content) of human conjunctival cell cultures by means of immunofluorescence-based analyses. This will help the development of new Advanced Therapy Medicinal Products (ATMPs) to use in future cell therapy clinical studies when fewer cells are available to perform the quality controls. To achieve this purpose, a reference standard was investigated and the expression levels of ΔNp63α (considered as a marker of conjunctival stem cells) was correlated to cell size. The limbal hTERT cells were used as reference standard to define the expression value of ΔNp63α. The mean intensity va lue of limbal hTERT cells ranging between 15 and 20 µm in diameter was used to distinguish between ΔNp63α bright and not bright cells. As ΔNp63α bright expression was mainly seen in the smaller cell size group (10–15 µm), we defined as conjunctival stem cells (= potency) those cells whic h were bright and with sizes between 10 and 15 µm. Assays on cells from clonal analyses were used to validate the method, as they do allow to observe a decrease in potency (Holoclones >  Meroclones >  Paraclones). The stem cell content of conjunctival grafts was found to be 11.3% ± 5.0 compared to 21.9% ± 0.6, 9.0% ± 8.1 and 0% from Holoclones, Meroclones and Paraclones, respectively. This new method, here named as Standardized Method for Potency Quanti...
Source: Cell and Tissue Banking - Category: Stem Cells Source Type: research