Method for measuring extracellular flux from intact polarized epithelial monolayers

Area of Science:

• Cell Biology
• Metabolic Assays
• Biotechnology

Background:

• The Seahorse XFp platform is crucial for cellular metabolic assessment.
• Standard protocols necessitate cell replating, posing challenges for sensitive cell types like primary human fetal RPE (hfRPE) cells requiring extended differentiation.
• This limitation hinders comprehensive metabolic analysis of such cells.

Purpose of the Study:

• To develop a novel method for assaying intact, differentiated hfRPE cell monolayers on the Seahorse XFp platform.
• To eliminate the need for cell replating, thereby preserving cell integrity and polarization.
• To enable robust metabolic assessment of cells cultured on filter supports.

Main Methods:

• Primary hfRPE cells were differentiated on filter inserts.
• Triangular filter sections with attached cells were excised and transferred to XFp miniplate wells.
• Cells underwent mitochondrial stress tests, with comparisons to replated controls and cells stimulated with photoreceptor outer segments (POS).

Main Results:

• Assaying intact monolayers via filter excision yielded higher maximal respiration and spare respiratory capacity compared to replated cells.
• Extracellular acidification rate (ECAR) was also increased in intact monolayers.
• hfRPE cells stimulated with POS showed enhanced maximal respiration and spare capacity, with no significant ECAR change.

Conclusions:

• A reproducible method for assaying intact, polarized hfRPE cell monolayers with the Seahorse XFp platform has been established.
• This technique provides more robust metabolic data than standard methods.
• The approach is suitable for evaluating metabolic consequences of prolonged cell culture and perturbations, benefiting studies on filter-cultured adherent cells.