蝙蝠以其特殊的特性在哺乳动物物种中脱颖而出,包括飞行和回声定位的导航能力,通过紧张/冬眠来节约能源,藏有大量的病毒,表现出对疾病的抵抗力,在恶劣的环境条件下生存,与其他类似大小的哺乳动物相比,表现出非凡的寿命。由于几个原因,蝙蝠的体内研究具有挑战性,例如在自然环境中难以定位和捕获它们,有限的可访问性,样本量低,环境变化,长寿,繁殖率缓慢,人畜共患疾病的风险,物种保护,和伦理问题。因此,建立替代实验室模型对于研究蝙蝠中观察到的各种生理适应至关重要。从组织中获得优质细胞是成功的原代细胞衍生的关键的第一步。然而,由于分离和扩增细胞所需的资源,收集新鲜组织并立即处理样品用于细胞培养通常是不切实际的。因此,冷冻组织通常是蝙蝠原代细胞衍生的起始资源,但是冷冻组织中的细胞通常会受损,并且完整性和生存力较低。因此,从冷冻组织中分离原代细胞提出了重大挑战。在这里,我们提出了一种成功开发的方案,用于从冷冻蝙蝠翼活检中分离原代真皮成纤维细胞。这个协议标志着一个重要的里程碑,因为这是第一个专门针对从蝙蝠冷冻组织中分离成纤维细胞的方案。我们还描述了原代细胞表征的方法,通过慢病毒转导对原代细胞进行遗传操作,和稳定细胞系的发展。©2024Wiley期刊有限责任公司。基本方案1:蝙蝠翼活检采集和保存支持方案1:蝙蝠静脉穿刺采血基本方案2:从成年蝙蝠冷冻翼活检中分离原代成纤维细胞支持方案2:原代成纤维细胞培养和继代培养支持方案3:生长曲线的测定和倍增时间支持方案4:原代成纤维细胞的细胞建库和解冻基本方案3:蝙蝠原代成纤维细胞的慢病毒转导基本方案4:蝙蝠稳定的成纤维细胞线发育支持方案5:通过成纤维细胞
Bats 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 preservation Support Protocol 1: Blood collection from bat venipuncture Basic Protocol 2: Isolation of primary fibroblasts from adult bat frozen wing biopsy Support Protocol 2: Primary fibroblast culture and subculture Support Protocol 3: Determination of growth curve and doubling time Support Protocol 4: Cell banking and thawing of primary fibroblasts Basic Protocol 3: Lentiviral transduction of bat primary fibroblasts Basic Protocol 4: Bat stable fibroblast cell line development Support Protocol 5: Bat fibroblast validation by immunofluorescence staining Basic Protocol 5: Chromosome counting.