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 vivo studies of bats are challenging for several reasons, such as difficulty in locating and capturing 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, but cells in frozen tissue are usually damaged and have low integrity and viability. Isolating primary cells from frozen tissues thus 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 is the first protocol specifically focused on fibroblast 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. © 2024 Wiley Periodicals LLC.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: Primary fibroblast culture and subcultureSupport Protocol 3: Determination of growth curve and doubling timeSupport Protocol 4: Cell banking and thawing of primary fibroblastsBasic Protocol 3: Lentiviral transduction of bat primary fibroblastsBasic Protocol 4: Bat stable fibroblast cell line developmentSupport Protocol 5: Bat fibroblast validation by immunofluorescence stainingBasic Protocol 5: Chromosome counting