Abstract 14580: Methodological Challenges Conducting In Vitro Ischemia and Reperfusion: A Fundamental Problem in Cell Viability Determination

Abstract

Background: Oxygen & Glucose Deprivation (OGD) and Reperfusion (OGD-R) is considered one of the best models for studying ischemia and reperfusion injury (IRI) in vitro. Cell viability (CV) experimentation can aid in estimating cell survival after IRI insults and potential benefits from therapeutic attempts. However, the different metabolites used in various CV assays may compromise the usefulness and validity of various CV tools. We conducted two independent cell viability assays (WST-8 & ATP) after OGD and OGD-R on neurons (NE) and astrocytes (AS). Methods: NE (HT-22) and AS (C8-D1A) were subjected to 6h OGD only and 6h OGD followed by 20h Reperfusion (OGD-R). OGD is based on the combination of chemical ischemia (standard culture media depleted of glucose and other energy sources) and severe hypoxia (O2 ~1.4 to 1.8%) for 6h, while 20h reperfusion consists of readdition of standard culture media and conditions (21% O2, 5% CO2, 37°C). CV was determined by WST-8 and ATP “CellTiter-Glo 2.0” assays. Results: By WST-8 assay, OGD decreased CV in NE (Fig. 1A, P value = 0.0121), but increased CV in AS (Fig. 1A, P value = 0.0079). OGD-R decreased CV in NE and AS (Fig. 2A, P value = 0.0010, 0.0146, respectively). By ATP assay, OGD decreased CV in NE (Fig. 1B, P value <0.0001) and AS (Fig. 1C, P value <0.0001). OGD-R also decreased CV in NE (Fig. 2B, P value <0.0001) and AS (Fig. 2C, P value = 0.0001). Conclusions: The current data reveals opposite CV results after OGD in AS when comparing WST-8 and ATP assays; OGD caused CV decrease in NE but increased in AS, which constitutes a paradoxical response based on previous literature. However, OGD-R caused similar injury patterns in NE and AS from WST-8 and ATP assays. It is unknown why OGD increased CV in AS through WST-8 after OGD, however, it can potentially occur due to AS hypermetabolism leading to increased NADPH+ production, which is the main substrate in this CV estimation. We conclude that CV should be carefully assessed with multiple, independent CV assays.