单细胞解离、RNA测序及分离老年泪腺和唾液腺的肌上皮细胞和成纤维细胞的方案
2026年4月24日
该方案描述了一种优化方法，用于解离老鼠泪腺，以获得可用于单细胞RNA测序的活性单细胞。顺序酶促消化和温和处理保持细胞完整性，产出适合细胞类型特异性分离和泪腺群衰老相关变化的转录组分析的高质量悬浮液。
我的实验室利用流式细胞术和单细胞RNA测序，研究唾液和泪腺年龄相关的变化如何影响不同细胞类型。标准解离方法对老化唾液和泪腺脆弱的上皮细胞恢复效果不佳。我的方案提升了细胞的存活能力，并实现了可靠的单细胞分析和细胞分选。
开始时，将被安乐死的小鼠仰卧在解剖台上。用细镊子和剪刀切开眼睛和耳朵之间的皮肤。轻轻拉开皮肤，露出泪腺。
用钝尖镊子，稍微抬起泪腺。小心地通过轻轻以圆周运动的方式将周围的结缔组织分开。识别泪腺的可见边界，并将其与邻近组织（包括腮腺）分开。
然后将泪腺转移到一个35毫米的培养皿中，内含2毫升冷PBS冰镇。在解剖显微镜下，去除剩余脂肪，并小心地将结缔组织囊从泪腺中取出。将一个无菌的35毫米培养皿放在冰上。
在盘子中心加入800到1000微升冷PBS，并将一个泪腺转移到PBS中。确保组织继续浸没在PBS中。用两把无菌手术刀将泪腺细碎成小块，直到变成柔软的糊状。
将组织碎片收集到盘子一侧。抽取残留PBS，并用一毫升新鲜冷PBS清洗碎片。将洗涤后的组织碎片转移到每孔2毫升预热消化培养基的12孔板中。
将平板放入摇晃水浴中，以37摄氏度、每分钟60转的温度孵育30分钟。使用宽口径一毫升移液器尖端，轻轻搓洗组织15次。将盘子放回摇水浴中，再潜伏30分钟。
接着，使用普通一毫升移液器尖端，轻轻研磨样品10次，再次孵育30分钟。然后对样品进行轻微研磨10次，并将细胞悬浮液转移到2毫升的海豚微型离心管中。向每个凹槽加入一毫升冷阻滞培养基（BM1），收集剩余含有残留细胞的消化培养基，并将培养基冰敷，等待下一步。
将装有细胞悬浮液的管子在300克下离心，四摄氏度离心五分钟。将每管的上清液丢弃。将含有残留细胞的板中每个孔的BM1转移到相应的管子中。
接着，向每个孔加入600微升冷BM1，冲洗剩余细胞，并将洗涤液转移到相应的管子中，与细胞悬浮液混合。轻轻地重新悬挂电池悬浮液。再次以300克离心，在4摄氏度下离心五分钟，然后丢弃上清液。
向细胞颗粒中加入一毫升Accutase，并轻柔地将细胞重新悬浮。将管子以37摄氏度、每分钟60转的温度孵育5到10分钟。使用一毫升的移液器尖端，轻轻研磨样品10次。
然后向管内加入一毫升冷的BM2，通过稀释Accutase终止酶促反应。在300克下离心管，温度为4摄氏度，离心五分钟，然后丢弃上清液。将含有DNase 1的培养基加入每毫升200单位的培养基，使细胞重新悬浮并填充管顶。
在室温下孵育10分钟，5分钟后轻轻重新悬浮一次。同时，将一个70微米的细胞滤网放在一个15毫升的锥形管上，并用一毫升BM3预润滤网。孵育后，将细胞悬浮液转移到滤网上。
在原收集管中加入一毫升BM3冲洗，然后转移到同一个滤网上。用额外的5毫升BM3滤网清洗。最后，利用锥蓝染色和自动细胞计数器测定过滤细胞悬浮液的细胞浓度和存活性。
继续进行后续应用，如单细胞RNA测序进行基因表达分析，或基于流式细胞术的荧光标记细胞群分选。UMAP分析单细胞RNA测序数据，识别出泪腺中代表性较多的上皮和基质群体，包括肌上皮细胞和成纤维细胞，以及多样化的免疫细胞群体。老年泪腺含有丰富的血浆和激活表达IGHM和IGHD的B细胞。
序列流式细胞术门控使EpCAM阳性和GFP阳性肌上皮细胞与EpCAM阴性GFP阳性壁细胞清晰分离。表达PDGFR α的GFP阳性成纤维细胞为EpCAM阴性，使得与上皮细胞的区分能力强。在PDGFR α和EGFP小鼠中，所有GFP阳性细胞也表达了胶原蛋白1，确认了成纤维细胞的身份。
流式细胞术分析显示，99.6%的GFP阳性细胞对应PDGFRα免疫染色细胞。该方案旨在从泪腺分离出可存活的单细胞，用于对上皮细胞、基质和免疫群体进行分子表征。主要挑战是在脆弱老化组织解离过程中最小化机械和酶促压力，同时保持单细胞RNA测序的高细胞活性。
经过该程序，分离细胞可用于单细胞RNA测序、体质RNA测序以及特定细胞群体的分子分析。分选后的细胞也可以培养以进行下游功能性检测。
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Overview
This article presents an optimized protocol for isolating viable single cells from aged mouse lacrimal glands (LGs), enabling high-quality single-cell RNA sequencing (scRNA-seq) and flow cytometry analysis. The method addresses challenges posed by increased fibrosis, cellular fragility, and lipid accumulation in aged LGs, ensuring efficient dissociation and recovery of diverse cell types for downstream molecular profiling.
Key Study Components
Area of Science
- Cellular and molecular biology
- Ophthalmology and exocrine gland research
- Single-cell transcriptomics
Background
- Aging of the lacrimal gland leads to reduced tear secretion and chronic inflammation, contributing to dry eye disease.
- Age-related changes include immune cell infiltration, cellular damage, lipid accumulation, extracellular matrix deposition, and ductal dilation.
- Single-cell RNA sequencing is a powerful tool for profiling cellular heterogeneity and transcriptional changes during aging.
- Obtaining viable single-cell suspensions from aged LGs is technically challenging due to tissue fragility and fibrosis.
Purpose of Study
- To develop a reproducible protocol for efficient dissociation of aged mouse lacrimal glands.
- To enable isolation of multiple cell types suitable for scRNA-seq and flow cytometry.
- To minimize cell damage and maximize viability during tissue processing.
Methods Used
- Careful dissection and mincing of lacrimal gland tissue from euthanized mice.
- Stepwise enzymatic digestion using Collagenase IV, Dispase II, DNase I, and Accutase.
- Use of pre-coated plasticware and controlled temperature/agitation to reduce cell stress.
- Red blood cell lysis and sequential filtration through strainers to enhance cell purity.
- Cell type-specific isolation using reporter mouse lines (Acta2GFP and PdgfraEGFP) and fluorescence-activated cell sorting (FACS) after immunostaining with EpCAM antibody.
Main Results
- The protocol yields high-quality, viable single-cell suspensions from both aged and young lacrimal glands.
- UMAP analysis of scRNA-seq data identified well-represented epithelial, stromal, and diverse immune cell populations.
- FACS enabled clear separation and identification of myoepithelial cells (EpCAM+, GFP+) and fibroblasts (PDGFRα+, EpCAM-).
- In aged LGs, abundant plasma and activated B cells expressing IGHM and IGHD were detected.
- 99.6% of GFP-positive cells corresponded to PDGFRα-immunostained fibroblasts, confirming cell identity.
Conclusions
- This protocol enables efficient and gentle dissociation of aged lacrimal glands for single-cell analysis.
- It supports high cell viability and recovery of multiple cell types, including fragile epithelial and stromal populations.
- The method is adaptable to other fibrotic or aging exocrine tissues requiring targeted cell isolation.
Frequently Asked Questions
Executive Industry Relevance
Efficient single-cell dissociation and isolation from aged lacrimal and salivary glands addresses a critical bottleneck in profiling cellular heterogeneity and transcriptional changes associated with tissue aging and fibrosis. This protocol enables high-viability recovery of fragile epithelial and stromal populations, supporting robust single-cell RNA sequencing and targeted cell sorting for discovery-stage research. The method enhances predictive confidence in cell-type specific molecular characterization, informing early target validation and mechanistic de-risking in exocrine gland disease models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of age-related cellular and molecular changes in exocrine tissues.
- Supports functional target validation by isolating viable myoepithelial cells and fibroblasts for downstream analysis.
- Facilitates mechanistic de-risking by providing high-quality single-cell suspensions for transcriptomic profiling.
- Improves predictive confidence in disease-relevant system modeling for dry eye and related disorders.
Screening & Assay Development
- Prepares validated single-cell suspensions suitable for high-throughput scRNA-seq and flow cytometry workflows.
- Standardizes dissociation and isolation steps to maximize reproducibility and quantitative output quality.
- Enables reliable cell-type specific sorting using FACS and reporter mouse lines for assay development.
- Supports scalable adaptation to other fibrotic or aging exocrine tissues with similar dissociation challenges.
Translational & Preclinical Research
- Aligns molecular profiling outputs with disease-relevant biomarkers in aged tissue models.
- Provides continuity from discovery through preclinical validation by enabling functional assays on sorted cell populations.
- Reduces biological risk in translational studies by ensuring high cell viability and purity for downstream applications.
Pipeline & Workflow Integration
This protocol integrates into the discovery-to-preclinical continuum by enabling robust single-cell analysis and targeted cell isolation from challenging aged tissues.
- Discovery Biology: Supports hypothesis testing and pathway clarification in age-related exocrine gland dysfunction.
- Screening: Delivers reproducible, high-purity single-cell suspensions for quantitative transcriptomic and cytometric assays.
- Analytics: Provides cell-type resolved molecular data and enables statistical comparison of cellular populations across age groups.
- Translational Research: Facilitates biomarker alignment and functional validation in disease-relevant aged tissue models.
- Enterprise Reuse: Adaptable protocol for diverse exocrine tissues, supporting platform scalability and cross-program standardization.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cell-type specific analyses.
- Operational Value: Enhances standardization, reproducibility, and scalability of single-cell workflows in aged and fibrotic tissues.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by enabling robust early-stage molecular profiling.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of programs targeting exocrine gland dysfunction and aging.
Implementation Considerations
- Requires expertise in tissue dissection, enzymatic dissociation, and flow cytometry-based cell sorting.
- Demands access to automated cell counters, FACS instrumentation, and single-cell RNA sequencing platforms.
- Necessitates cross-team standardization of dissociation and sorting protocols for reproducibility.
- Adaptation to other tissues may require optimization of enzyme concentrations and incubation times.
- Cell viability and yield may be limited by tissue fibrosis and cellular fragility in advanced aging models.
