Contents lists available at ScienceDirect Tissue and Cell journal homepage: www.elsevier.com/locate/tice Correlation between valvular interstitial cell morphology and phenotypes: A novel way to detect activation Mir S. Ali a , Nandini Deb a , Xinmei Wang a , Minhazur Rahman b , Gordon F. Christopher b , Carla M.R. Lacerda a, ⁎ a Department of Chemical Engineering, Texas Tech University, Lubbock, TX, USA b Department of Mechanical Engineering, Texas Tech University, Lubbock, TX, USA ARTICLE INFO Keywords: Valvular interstitial cell Morphology distribution Phenotype Quiescent Activated Substrate stiffness Passage number ABSTRACT Valvular interstitial cells (VICs) constitute the major cell population in heart valves. Quiescent fibroblastic VICs are seen in adult healthy valves. They become activated myofibroblastic VICs during development, in diseased valves and in vitro. 2D substrate stiffness within a 5–15 kPa range along with high passage numbers promote VIC activation in vitro. In this study, we characterize VIC quiescence and activation across a 1–21 kPa range of substrate stiffness and passages. We define a cell morphology characterization system for VICs as they transform. We hypothesize that VICs show distinct morphological characteristics in different activation states and the morphology distribution varies with substrate stiffness and passage number. Four VIC morphologies - tailed, spindle, rhomboid and triangle - account for the majority of VIC in this study. Using α-smooth muscle actin (α- SMA), non-muscle myosin heavy chain B (SMemb) and transforming growth factor β (TGF-β) as activation markers for validation, we developed a system where we categorize morphology distribution of VIC cultures, to be potentially used as a non-destructive detection method of activation state. We also show that this system can be used to force stiffness-induced deactivation. The reversibility in VIC activation has important implications in in vitro research and tissue engineering. 1. Introduction Heart valve diseases are a major source of morbidity and mortality. Approximately 5 million people are burdened with heart valve disease in USA (Nkomo et al., 2006). Heart valves are passive tissues that control direction of blood flow in the heart. They have three major components: valvular interstitial cells (VICs), valvular endothelial cells (VECs) and extracellular matrix (ECM) (Donnelly, 2008). VICs con- stitute the major cell population in heart valves. Functions of VIC in- clude maintenance of structural integrity, synthesis of ECM compo- nents, homeostasis, repair and remodeling, etc. (Mulholland and Gotlieb, 1996; Taylor et al., 2003). Specific VIC functions are performed by specific phenotypes of VIC (Liu et al., 2007; Rabkin-Aikawa et al., 2004). VICs can have five phenotypes described by Liu et al. (2007) as follows: embryonic progenitor endothelial/mesenchymal cells, quies- cent VIC (qVIC), activated VIC (aVIC), progenitor VIC (pVIC) and os- teoblastic VIC (obVIC). qVICs reside in healthy adult heart valves and are responsible for physiological maintenance (Rabkin-Aikawa et al., 2004). qVICs have fibroblastic properties (Liu et al., 2007). aVICs, also known as the myofibroblastic phenotype, are activated forms of qVICs. aVICs remodel the ECM and take part in proliferation and other cellular activities (Liu et al., 2007; Rabkin et al., 2001; Walker et al., 2004). Increased population of aVIC results in abnormal valve shapes and degenerative diseases (Rabkin et al., 2001). aVICs are more abundant in developing (Hinton et al., 2006) and degenerative heart valves (Rabkin- Aikawa et al., 2004). Myofibroblastic aVICs show high expression of α- smooth muscle actin (α-SMA), non-muscle myosin heavy chain B (SMemb) and transforming growth factor β (TGF-β), typically absent from qVICs (Liu et al., 2007; Rabkin-Aikawa et al., 2004; Rabkin et al., 2001). VIC activation in vivo and in vitro occurs due to abnormal mechan- ical and chemical environments (Donnelly, 2008; David Merryman, 2010; Sacks and Yoganathan, 2007). Substrate stiffness has an im- portant effect on VIC activation or phenotype transformation (Kloxin et al., 2010; Quinlan and Billiar, 2012; Yip et al., 2009). It has been shown that VICs, in 2D in vitro conditions, become activated on sub- strate stiffness close to single digit values in kPa. Kloxin et al. (2010) showed that VICs in 2D culture become activated and deactivated above and below 15 kPa substrate stiffness. Later publications from the same research group showed VIC activation above 7 kPa substrate https://doi.org/10.1016/j.tice.2018.07.004 Received 6 June 2018; Received in revised form 23 July 2018; Accepted 27 July 2018 ⁎ Corresponding author at: Dept. of Chemical Engineering, Texas Tech University, Lubbock, TX 79409-3121, USA. E-mail address: carla.lacerda@ttu.edu (C.M.R. Lacerda). Tissue and Cell 54 (2018) 38–46 Available online 29 July 2018 0040-8166/ © 2018 Elsevier Ltd. All rights reserved. T