尼罗罗非鱼肠单细胞悬液制备用于单细胞测序
1State Key Laboratory of Marine Resource Utilization in South China Sea,Hainan University, 2Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, College of Fishery,Guangdong Ocean University, 3Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of Marine Science,Hainan University, 4Department of Animal Wealth Development, Faculty of Veterinary Medicine,Suez Canal University
Summary
在这里，我们演示了用于单细胞测序的高质量罗非鱼肠单细胞悬液的制备。
Abstract
尼罗罗非鱼是全世界最常见的养殖淡水鱼种之一，是水产养殖鱼类研究中广泛使用的研究模式。高质量单细胞悬液的制备对于单细胞水平研究（如单细胞RNA或基因组测序）至关重要。然而，对于水产养殖鱼类，特别是罗非鱼肠道，没有现成的规程。有效的解离酶因组织类型而异。因此，通过选择合适的酶或酶组合来优化组织解离方案，以最小的损伤获得足够的活细胞至关重要。本研究说明了一种优化的方案，以制备来自尼罗罗非鱼肠的高质量单细胞悬液，并具有胶原酶/分散酶的酶组合。这种组合对于解离非常有效，利用牛血清白蛋白和脱氧核糖核酸酶来减少消化后的细胞聚集。细胞输出满足单细胞测序要求，细胞活力为90%，细胞浓度高。该协议也可以修改以从其他鱼类的肠道制备单细胞悬浮液。这项研究提供了一个有效的参考方案，并减少了在制备水产养殖鱼类单细胞悬浮液方面进行额外试验的需要。
Introduction
细胞是生物体的基本单位。与大体积组织研究相比，单细胞水平研究可以反映细胞异质性并提供更高分辨率的信息1。近年来，研究人员在哺乳动物、斑马鱼和其他模式生物的单细胞水平上应用单细胞测序技术进行基因组、转录组、表观基因组或多组学研究，并报告了重大突破2，3，4，5，6，7.虽然大多数研究都集中在模式生物上，但很少有用于经济鱼类单细胞测序的参考方案或商业解离试剂盒，这限制了单细胞测序在水产养殖研究中的应用。因此，开发能够产生具有高细胞活力和核酸完整性的高质量单细胞悬液的组织解离方案至关重要。
使用适当的酶或酶组合优化组织解离方案以最小的损伤获得足够的活细胞至关重要。组织解离最有效的酶因组织类型而异。在哺乳动物中，几种酶已被用于制备哺乳动物实体组织的单细胞悬浮液，包括胶原酶、分散酶、胰蛋白酶、木瓜蛋白酶、弹性蛋白酶、透明质酸酶、自由酶、accutase 和 trypLE8，9。胰蛋白酶消化结合机械破碎通常用于解离组织以进行鱼的细胞培养10，11，12，13，14。胰蛋白酶也被使用或添加到消化混合物中，用于大鼠肠道15和斑马鱼鳃组织16中的组织解离。然而，由于多种原因，胰蛋白酶不是单细胞测序的最佳选择。单独使用胰蛋白酶通常对组织解离无效。此外，胰蛋白酶诱导DNA链断裂17，18和RNA降解19。
木瓜蛋白酶降解构成细胞之间紧密连接的蛋白质。在哺乳动物神经和平滑肌细胞中，木瓜蛋白酶比其他蛋白酶更有效，破坏性更小20，21。然而，与胰蛋白酶一样，由于酶消化过程中发生的细胞裂解，木瓜蛋白酶导致游离 DNA 诱导的细胞聚集9.弹性蛋白酶分解弹性蛋白，弹性蛋白通常存在于皮肤、肺、韧带、肌腱和血管组织中22.它通常与胶原酶、分散酶或胰蛋白酶联合使用，用于解离肺组织8.透明质酸酶切割透明质酸的糖苷键，有助于消化各种结缔组织和皮肤中的细胞外基质9，23。
一般来说，胶原酶和分散酶是细胞外基质分解的良好选择。它们已被用于解离人、小鼠和斑马鱼肠道24，25，26，27。胶原酶破坏胶原蛋白中的肽键，促进细胞外基质的消化，并将细胞释放成悬浮液，因此，胶原酶通常用于人和小鼠的实体组织解离，包括肝脏28，29，脾脏30，胰腺31和肠道25.分散酶是一种蛋白酶，可水解非极性氨基酸残基的N端肽键，比胶原酶温和。它切割细胞外基质成分，如纤连蛋白、IV 型胶原蛋白，并在较小程度上切割 I 型胶原蛋白，而不影响细胞间连接。分散酶单独使用或与其他酶一起用于组织解离，例如用于肠25，32，脑33，肝脏34等。此外，市售的消化混合物，包括解放酶、阿克库酶和胰蛋白LE，也是实体组织解离的良好替代品，特别是对于皮肤、肝脏和肾脏8，9。
尼罗罗非鱼（Oreochromis niloticus）属于鲈形目慈鲷科。它是热带和亚热带地区养殖最多的淡水鱼种之一，2022年年产量为450万吨35。它是研究得最好的水产养殖鱼类之一，具有很好的基因组注释。尼罗罗非鱼具有生成时间短、易于养殖、适应各种养殖环境等优点，是水产养殖鱼类的理想研究模式。肠道是营养消化吸收、代谢和粘膜免疫的器官，因此具有很大的研究意义。肠道是微生物种群的栖息地，是必需的免疫组织36。由于存在多种免疫细胞类型，包括巨噬细胞、B 细胞、粒细胞和 T 细胞，它具有免疫活性。
在目前的研究中，我们开发了一种从尼罗罗非鱼肠道制备高质量单细胞悬液的方案，以促进水产养殖鱼类的单细胞水平研究。根据这些组织特异性酶的特性和初步工作，胶原酶/分散酶适合于解离罗非鱼肠道组织。制备单细胞悬液时要考虑的最终酶类型是DNase-I，它通过降解酶消化过程中通过死细胞裂解释放的游离DNA来防止细胞聚集，而无需启动凋亡途径9 ，并提高活细胞产量36。此外，将牛血清白蛋白（BSA）添加到洗涤缓冲液中，以减少细胞结块并提高细胞活力。一些试剂公司将BSA描述为酶稳定剂。向PBS（磷酸盐缓冲盐水）中添加0.04%-1%BSA已被用于开发用于制备无不良反应的单细胞测序悬浮液的洗涤溶液38。添加低比例的BSA有助于维持细胞活力，并避免由于细胞裂解而导致的游离DNA诱导的细胞聚集。该协议还可以为开发来自其他水产养殖鱼类肠道的细胞解离方案提供有价值的参考。
Protocol
本研究期间的所有动物方案均已获得海南大学机构动物护理和使用委员会的批准（实验方案编号：HNUAUCC-2022-00063;批准日期：2022-03-03）。本实验中使用的设备和用品清单可以在 材料表中找到。当前协议的摘要如图 1所示。 1. 鱼的准备 从可靠来源获得平均体重为 100 克的 6 个月大的尼罗罗非鱼。选择没有任何疾病迹象的鱼?…
Representative Results
该协议描述了用于单细胞测序的高质量尼罗罗非鱼肠单细胞悬液的制备（图1）。这项研究表明，胶原酶/分散酶混合物具有良好的解离效果，对肠道组织温和。选择最佳消化酶对于制备高质量的单细胞悬液至关重要。在初步工作中，比较了几种常用酶的解离效率，结果列于 表1中。经验证，胶原酶/分散酶混合物的解离效果比单独的胶原酶、分散酶或胰蛋白酶以及…
Discussion
该协议描述了尼罗罗非鱼肠高质量单细胞悬液的制备。在解离之前，有必要从肠道中去除脂肪和肠系膜，特别是对于脂肪含量高的肉食性鱼肠。使用注射器而不是硬刮去肠内容物可减少对细胞的机械损伤。为了确保细胞活力，对于组织解剖和冲洗步骤，还必须将温度保持在20°C或以下。洗涤液在冰上冷却，并提前将离心机温度调节至4°C。最重要的是，应为所研究的组织选择温和有效的细胞解离酶?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者希望感谢海南省自然科学基金（编号：320QN211）和广东省水生动物疾病防治与健康文化重点实验室（NO. PBEA2021ZD01）的研究基金计划。
Materials
| 0.22-μm Sterile Filter | Solarbio Life Sciences | SLGV033RB | It is used to filter and sterilize the enzyme solution. |
| 40-μm Cell Strainer | Solarbio Life Sciences | F8200 | Cell Strainer is applied to eliminate undigested tissue pieces. |
| Bovine serum albumin (BSA) | Sigma-Aldrich | SRE0098 | Powder; dilute 0.04 g BSA with 100 mL 1× DPBS to prepare 0.04% BSA-DPBS washing bffer. Store at 2 – 8 °C. |
| Collagenase II | Sangon Biotech | A004202 | Dilute with PBS to a final concentration of 1 mg/mL. |
| Collagenase/dispase | Roche | 10269638-001 | Dilute with PBS to a final concentration of 1 mg/mL. |
| Dispase | Sigma-Aldrich | D4818 | Dilute with PBS to a final concentration of 1 mg/mL. |
| DNase I | Sigma-Aldrich | AMPD1 | DNase I helps reduce cell clumping. |
| Dulbecco's phosphate-buffered saline (DPBS), Ca2+/Mg2+-free | Solarbio Life Sciences | E607009-0500 | Store at room temperature. |
| Elastase | Sangon Biotech | A600438 | Dilute with PBS to a final concentration of 0.5 mg/mL. |
| Fetal bovine serum (FBS) | Gibco | 16000-044 | Serum, used at volume of 5% in digetstion solution. |
| Inverted Microscope | Leica | qTOWER3G | It is used to examine cell viability. |
| Liberase | Roche | 5401119001 | Dilute with PBS to a final concentration of 0.25 mg/mL. |
| Nile tilpia (Oreochromis niloticus) | ProGift Aquaculture Technology Co. Ltd. | NA | Healthy fish with no disease signs (Mean body weight: 100 g). |
| Phosphate-buffered saline (PBS) | Solarbio Life Sciences | P1020 | Store at room temperature. |
| Refrigerated Centrifuge | Eppendorf | 5424 | It is used to spin down the tissue and cell petet. |
| RNase inhibitor | NEB | M0314L | Inhibit RNase activity |
| Solid-phase RNase-Be-Gone Reagent | Sangon Biotech | B644201-0050 | It is used to remove the RNase from tools such as dissecting scissors and glass pipettes. Store at room temperature. |
| Tricaine methanesulfonate (MS-222) | Sigma-Aldrich | E10521 | For fish euthanasia. |
| Trypan Blue | Invitrogen | C0040 | It is used for staining dead cells. |
| Trypsin | Sangon Biotech | E607001 | Dilute with PBS to a final concentration of 1 mg/mL. |
References
- Tang, X., Huang, Y., Lei, J., Luo, H., Zhu, X. The single-cell sequencing: new developments and medical applications. Cell and Bioscience. 9 (1), (2019).
- He, H., et al. Single-cell transcriptome analysis of human skin identifies novel fibroblast subpopulation and enrichment of immune subsets in atopic dermatitis. The Journal of Allergy and Clinical Immunology. 145 (6), 1615-1628 (2020).
- Carmona, S. J., et al. Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types. Genome Research. 27 (3), 451-461 (2017).
- Wen, L., Tang, F. C. Single cell epigenome sequencing technologies. Molecular Aspects of Medicine. 59, 62-69 (2018).
- Xu, R., et al. Single cell sequencing coupled with bioinformatics reveals PHYH as a potential biomarker in kidney ischemia reperfusion injury. Biochemical and Biophysical Research Communications. 602, 156-162 (2022).
- Potter, S. S. Single-cell RNA sequencing for the study of development, physiology and disease. Nature Reviews Nephrology. 14 (8), 479-492 (2018).
- Andrews, T. S., Hemberg, M. Identifying cell populations with scRNASeq. Molecular Aspects of Medicine. 59, 114-122 (2018).
- Lafzi, A., Moutinho, C., Picelli, S., Heyn, H. Tutorial: Guidelines for the experimental design of single-cell RNA sequencing studies. Nature Protocols. 13 (12), 2742-2757 (2018).
- Reichard, A., Asosingh, K. Best practices for preparing a single cell suspension from solid tissues for flow cytometry. Cytometry Part A. 95 (2), 219-226 (2019).
- Sathiyanarayanan, A., Goswami, M., Nagpure, N., Babu, P. G., Das, D. K. Development and characterization of a new gill cell line from the striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878). Fish Physiology and Biochemistry. 48 (2), 367-380 (2022).
- Ager-Wick, E., et al. Preparation of a high-quality primary cell culture from fish pituitaries. Journal of Visualized Experiments. (138), e58159 (2018).
- Kumar, R., et al. Establishment and characterization of a caudal fin-derived cell line, AOF, from the Oscar, Astronotus ocellatus. FishPhysiology and Biochemistry. 45 (1), 123-131 (2019).
- Schnell, S., et al. Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia. Nature Protocols. 11 (3), 490-498 (2016).
- Xu, S. H., Cooke, I. M. Voltage-gated currents of tilapia prolactin cells. General and Comparative Endocrinology. 150 (2), 219-232 (2007).
- Ayyaz, A., et al. Single-cell transcriptomes of the regenerating intestine reveal a revival stem cell. Nature. 569 (7754), 121-125 (2019).
- Pan, W., et al. Single-cell transcriptomic analysis of neuroepithelial cells and other cell types of the gills of zebrafish (Danio rerio) exposed to hypoxia. Scientific Reports. 12, 10144 (2022).
- Huang, H. L., et al. Trypsin-induced proteome alteration during cell subculture in mammalian cells. Journal of Biomedical Science. 17 (1), 36 (2010).
- Kapiszewska, M., Reddy, N. M., Lange, C. S. Trypsin-induced changes in cell shape and chromatin structure result in radiosensitization of monolayer Chinese hamster V79 cells. International Journal of Radiation Biology. 60 (4), 635-646 (1991).
- Vrtačnik, P., Kos, &. #. 3. 5. 2. ;., Bustin, S. A., Marc, J., Ostanek, B. Influence of trypsinization and alternative procedures for cell preparation before RNA extraction on RNA integrity. Analytical Biochemistry. 463, 38-44 (2014).
- Huettner, J. E., Baughman, R. W. Primary culture of identified neurons from the visual cortex of postnatal rats. The Journal of Neuroscience. 6 (10), 3044-3060 (1986).
- Kinoshita, K., Sato, K., Hori, M., Ozaki, H., Karaki, H. Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-kappaB inhibitors in Crohn’s colitis model. American Journal of Physiology. Gastrointestinal and Liver Physiology. 285 (3), 483-493 (2003).
- Chung, M. I., et al. Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins. Matrix Biology. 25 (8), 492-504 (2006).
- Berry, M. N., Friend, D. S. High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. Journal of Cell Biology. 43 (3), 506-520 (1969).
- Merlos-Suárez, A., et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell. 8 (5), 511-524 (2011).
- Glass, L. L., et al. Single-cell RNA sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine. Molecular Metabolism. 6 (10), 1296-1303 (2017).
- Herring, C. A., et al. Unsupervised trajectory analysis of single-cell RNA-seq and imaging data reveals alternative tuft cell origins in the gut. Cell Systems. 6 (1), 37-51 (2018).
- Gu, W., et al. Single-cell RNA sequencing reveals size-dependent effects of polystyrene microplastics on immune and secretory cell populations from zebrafish intestines. Environmental Science & Technology. 54 (6), 3417-3427 (2020).
- Yang, W., et al. Single-cell transcriptomic analysis reveals a hepatic stellate cell-activation roadmap and myofibroblast origin during liver fibrosis in mice. Hepatology. 74 (5), 2774-2790 (2021).
- Howard, R. B., et al. The enzymatic preparation of isolated intact parenchymal cells from rat liver. Journal of Cell Biology. 35 (3), 675-684 (1967).
- Pezoldt, J., et al. Single-cell transcriptional profiling of splenic fibroblasts reveals subset-specific innate immune signatures in homeostasis and during viral infection. Communications Biology. 4 (1), 1355 (2021).
- Baron, M., et al. A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure. Cell Systems. 3 (4), 346-360 (2016).
- Barriga, F. M., et al. Mex3a Marks a slowly dividing subpopulation of Lgr5+ intestinal stem cells. Cell Stem Cell. 20 (6), 801-816 (2017).
- Volovitz, I., et al. A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells. BMC Neuroscience. 17 (1), 30 (2016).
- Chen, L., et al. Combined effects of arsenic and 2,2-dichloroacetamide on different cell populations of zebrafish liver. Science of the Total Environment. 821, 152961 (2022).
- FAO. The state of world fisheries and aquaculture 2022. Towards blue transformation. Food and Agriculture Organization of the United Nations (FAO). , (2022).
- Beck, B. H., Peatman, E. . Mucosal Health in Aquaculture. , (2015).
- Leelatian, N., et al. A Single cell analysis of human tissues and solid tumors with mass cytometry. Cytometry. Part B, Clinical Cytometry. 92 (1), 68-78 (2017).
- Lee, H., Engin, F. Preparing highly viable single-cell suspensions from mouse pancreatic islets for single-cell RNA sequencing. STAR Protocols. 1 (3), 100144 (2020).
- Bresciani, E., Broadbridge, E., Liu, P. P. An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae. MethodsX. 5, 1287-1290 (2018).
- Denisenko, E., et al. Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biology. 21 (1), 130 (2020).
- vanden Brink, S. C., et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nature Methods. 14 (10), 935-936 (2017).
- Avey, D., et al. Single-cell RNA-seq uncovers a robust transcriptional response to morphine by glia. Cell Reports. 24 (13), 3619-3629 (2018).
- Herring, C. A., et al. Unsupervised trajectory analysis of single-cell RNA-seq and imaging data reveals alternative tuft cell origins in the gut. Cell Systems. 6 (1), 37-51 (2018).
Cite This Article
Wang, P., Zhou, Y., Wang, B., Elaswad, A., Wang, S., Guo, W., Zhang, D. Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing. J. Vis. Exp. (192), e64688, doi:10.3791/64688 (2023).