Shweta Singhal1, Geeta K Vemuganti. 1. Sudhakar and Srikant Ravi Stem Cell Biology Laboratory, L. V. Prasad Eye Institute, L. V. Prasad Marg, Banjara Hills, Hyderabad, India.
Abstract
PURPOSE: Retinal pigment epithelial (RPE) cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM) as substrate. MATERIALS AND METHODS: Human cadaver eyeballs (16) were obtained from the eye bank after corneal trephination. RPE cells were harvested by a) mechanical dissection of the inner choroid surface (10, group 1) or by b) enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2). The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students' 't' test. RESULTS: Eleven samples (68.75%) showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days) in the mechanical group, significantly earlier (P < 0.025) than in the enzymatic group (day 29-35, average 31.6 days). CONCLUSIONS: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes. Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion.
PURPOSE: Retinal pigment epithelial (RPE) cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM) as substrate. MATERIALS AND METHODS:Human cadaver eyeballs (16) were obtained from the eye bank after corneal trephination. RPE cells were harvested by a) mechanical dissection of the inner choroid surface (10, group 1) or by b) enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2). The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students' 't' test. RESULTS: Eleven samples (68.75%) showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days) in the mechanical group, significantly earlier (P < 0.025) than in the enzymatic group (day 29-35, average 31.6 days). CONCLUSIONS: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes. Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion.
Authors: Elena Daniele; Barbara Ferrari; Nicolò Rassu; Joshua Ben-Nun; Lorenzo Bosio; Vanessa Barbaro; Stefano Ferrari; Diego Ponzin Journal: BMJ Open Ophthalmol Date: 2022-09