Purchase this article with an account.
Gary S L Peh, Benjamin L George, Khadijah Adnan, Xin Yi Seah, Heng Pei Ang, Jodhbir S Mehta; Optimizing the dual media culture system of propagating human corneal endothelial cells using GMP-certified reagents. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1166.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
For clinical translation of cell therapy, culture protocol must be standardized and adhered to strict Good Manufacturing Practices (GMPs) guidelines. The main aim of this study is to systematically optimize each GMP-certified component required in the dual media culture system used for the propagation of human corneal endothelial cells (CEnCs).
Pairs of donor corneas deemed unsuitable for transplantation were procured for this study. For the digestion of Descemet’s membrane (DM), strips of CEnCs-DM peeled from donor-matched corneas were incubated in either Collagenase (left) or Liberase (right). For all other comparative studies, CEnCs were isolated and expanded using the dual media approach to the second or third passage to obtain sufficient cell numbers. The experimental parameters evaluated were: 1) cell attachment; 2) serum used in culture; 3) single-cell dissociation; and 4) cryopreservation. Results obtained include assessment of cell morphology, overall cell yield (manual cell-counts), cell adherence (xCelligence), cell proliferation (Click-iT EdU) and cell viability (Annexin V / PI) as outlined in Figure 1.
The assessments of cGMP components substituted into each of the processes were compared to current reagent used. Specifically, digestions of DM (n=5) using Liberase was equivalent to Collagenase. Attachment of CEnCs (n=3) was significantly improved (p<0.05) when rLaminin 511 was used. Nevertheless, the use of human collagen coating was comparable to FNC coating mix. Serum comparison (n=3) revealed that cultures of CEnCs became inconsistent in the presence of human serum whereas results using Equalfetal showed comparable cell yield similar to CEnCs grown in the presence of FBS. Next, cellular dissociation of CEnCs (n=5) were comparable between TrypLE Express and TrypLE Select. Finally, for cryo-preservation of CEnCs (n=3), cells cryo-preserved in CryoScarless were significantly more viable than the three other freezing medium compared (p<0.05).
The various assessments of cGMP components substituted into each of the processes as compared to current reagent used have shown their usability towards clinical translation. Future in vivo studies are required to substantiate our current findings, an essential undertaking towards clinical cell therapy.
This PDF is available to Subscribers Only