Biomolecules & Therapeutics  https://doi.org/10.4062/biomolther.2020.175
Late Passage Cultivation Induces Aged Astrocyte Phenotypes in Rat Primary Cultured Cells
Minji Bang, Edson Luck Gonzales, Chan Young Shin and Kyoung Ja Kwon*
Department of Neuroscience, School of Medicine and Center for Neuroscience Research, Konkuk University, Seoul 05029, Republic of Korea
*E-mail: neuro11@kku.ac.kr
Tel: +82-2-454-5630, Fax: +82-2-2030-7899
Received: October 6, 2020; Revised: November 4, 2020; Accepted: November 6, 2020; Published online: December 2, 2020.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Cultureconditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.
Keywords: Astrocytes, Late passage cultivation, Cellular senescence, Neuro-inflammatory response, Phagocytosis, Wound healing

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