Establishing Primary and Stable Cell Lines from Frozen Wing Biopsies for Cellular, Physiological, and Genetic Studies in Bats

Abstract

AbstractBats stand out among mammalian species for their exceptional traits, including the capacity to navigate through flight and echolocation, conserve energy through torpor/hibernation, harbor a multitude of viruses, exhibit resistance to disease, survive harsh environmental conditions, and demonstrate exceptional longevity compared to other mammals of similar size.In vivostudies of bats can be challenging for several reasons such as ability to locate and capture them in their natural environments, limited accessibility, low sample size, environmental variation, long lifespans, slow reproductive rates, zoonotic disease risks, species protection, and ethical concerns. Thus, establishing alternative laboratory models is crucial for investigating the diverse physiological adaptations observed in bats. Obtaining quality cells from tissues is a critical first step for successful primary cell derivation. However, it is often impractical to collect fresh tissue and process the samples immediately for cell culture due to the resources required for isolating and expanding cells. As a result, frozen tissue is typically the starting resource for bat primary cell derivation. Yet, cells in frozen tissue are usually damaged and represent low integrity and viability. As a result, isolating primary cells from frozen tissues poses a significant challenge. Herein, we present a successfully developed protocol for isolating primary dermal fibroblasts from frozen bat wing biopsies. This protocol marks a significant milestone, as this the first protocol specially focused on fibroblasts isolation from bat frozen tissue. We also describe methods for primary cell characterization, genetic manipulation of primary cells through lentivirus transduction, and the development of stable cell lines.Basic Protocol 1:Bat wing biopsy collection and preservationSupport Protocol 1:Blood collection from bat-venipunctureBasic Protocol 2:Isolation of primary fibroblasts from adult bat frozen wing biopsySupport Protocol 2:Maintenance of primary fibroblastsSupport Protocol 3:Cell banking and thawing of primary fibroblastsSupport Protocol 4:Growth curve and doubling timeSupport Protocol 5:Lentiviral transduction of bat primary fibroblastsBasic Protocol 3:Bat stable fibroblasts cell lines developmentSupport Protocol 6:Bat fibroblasts validation by immunofluorescence stainingSupport Protocol 7:Chromosome counting