The advent of personalized medicine has driven the development of novel approaches for obtaining detailed cellular and molecular information from clinical tissue samples. Tissue cytometry is a promising new technique that can be used to enumerate and characterize each cell in a tissue and, unlike flow cytometry and other single-cell techniques, does so in the context of the intact tissue, preserving spatial information that is frequently crucial to understanding a cell’s physiology, function, and behavior. However, the wide-scale adoption of tissue cytometry as a research tool has been limited by the fact that published examples utilize specialized techniques that are beyond the capabilities of most laboratories. Here we describe a complete and accessible pipeline, including methods of sample preparation, microscopy, image analysis, and data analysis for large-scale three-dimensional tissue cytometry of human kidney tissues. In this workflow, multiphoton microscopy of unlabeled tissue is first conducted to collect autofluorescence and second-harmonic images. The tissue is then labeled with eight fluorescent probes, and imaged using spectral confocal microscopy. The raw 16-channel images are spectrally deconvolved into 8-channel images, and analyzed using the Volumetric Tissue Exploration and Analysis (VTEA) software developed by our group. We applied this workflow to analyze millimeter-scale tissue samples obtained from human nephrectomies and from renal biopsies from individuals diagnosed with diabetic nephropathy, generating a quantitative census of tens of thousands of cells in each. Such analyses can provide useful insights that can be linked to the biology or pathology of kidney disease. The approach utilizes common laboratory techniques, is compatible with most commercially-available confocal microscope systems and all image and data analysis is conducted using the VTEA image analysis software, which is available as a plug-in for ImageJ.
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This work was supported by grants from the NIDDK: UH3DK114923, P30DK079312 and DiaComp. Microscopy was conducted at the Indiana Center for Biological Microscopy. We thank the KPMP consortium members for their significant input and support.
For the Kidney Precision Medicine Project
Richard Knight4, Stewart Lecker5, Isaac Stillman5, Gearoid Mcmahon6, Sus Waikar6, Astrid Weins6, Nir Hacohen7, Paul Hoover7, Mark Aulisio8, Leslie Cooperman9, Leal Herlitz9, John O’toole9, Emilio Poggio9, John Sedor9, Paul Appelbaum10, Jonathan Barasch10, Andrew Bomback10, Vivette D’agati10, Krzysztof Kiryluk10, Karla Mehl10, Ning (Sunny) Shang10, Chenhua Weng10, Laura Barisoni11, Theodore Alexandrov12, Tarek Ashkar13, Daria Barwinska13, Pierre Dagher13, Kenneth Dunn13, Michael Eadon13, Michael Ferkowicz13, Katherine Kelly13, Timothy Sutton13, Seth Winfree13, Steven Menez14, Chirag Parikh14, Avi Rosenberg14, Pam Villalobos14, Alison Slack15, Sylvia Rosas15, Mark Williams15, Evren Azeloglu16, Cijang (John) He16, Ravi Iyengar16, Samir Parikh17, Chris Anderton18, Ljiljana Pasa-Tolic18, Dusan Velickovic18, George (Holt) Oliver19, Joseph Ardayfio20, Jack Bebiak20, Keith Brown20, Taneisha Campbell20, Catherine Campbell20, Lynda Hayashi20, Nichole Jefferson20, Robert Koewler20, Glenda Roberts20, John Saul20, Anna Shpigel20, Edith Christine Stutzke20, Lorenda Wright20, Leslie Miegs20, Roy Pinkeney20, Rachel Sealfon21, Olga Troyanskaya21, Katherine Tuttle22, Yury Goltsev23, Blue Lake24, Kun Zhang24, Dejan Dobi25, Maria Joanes25, Zoltan Laszik25, Garry Nolan25, Andrew Schroeder25, Ulysses Balis26, Oliver He26, Jeffrey Hodgin26, Matthias Kretzler26, Laura Mariani26, Rajasree Menon26, Edgar Otto26, Jennifer Schaub26, Becky Steck26, Michele Elder27, Daniel Hall27, John Kellum27, Mary Kruth27, Raghav Murugan27, Paul Palevsky27, Parmjeet Randhawa27, Matthew Rosengart27, Sunny Sims-Lucas27, Mary Stefanick27, Stacy Stull27, Mitchell Tublin27, Charles Alpers28, Ian De Boer28, Malia Fullerton28, Jonathan Himmelfarb28, Robyn Mcclelland28, Sean Mooney28, Stuart Shankland28, Kayleen Williams28, Kristina Blank28, Ashveena Dighe28, Jonas Carson28, Frederick Dowd28, Zach Drager28, Kumar Sharma29, Guanshi Zhang29, Asra Kermani30, Simon Lee30, Christopher Lu30, Tyler Miller30, Orson Moe30, Harold Park30, Kamalanathan Sambandam30, Francisco Sanchez30, Jose Torrealba30, Toto Robert30, Miguel Vazquez30, Nancy Wang30, Joe Gaut31, Sanjay Jain31, Anitha Vijayan31, Randy Luciano32, Dennis Moledina32, Ugwuowo Ugochukwu32, Francis Perry Wilson32.
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Ferkowicz, M.J., Winfree, S., Sabo, A.R. et al. Large-scale, three-dimensional tissue cytometry of the human kidney: a complete and accessible pipeline. Lab Invest (2021). https://doi.org/10.1038/s41374-020-00518-w