title: Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring.
abstract: Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305.
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305.
authors.forename: Martin
authors.identifier: 0000-0002-3581-4720
authors.initials: M
authors.lastname: Hjort
authors.affiliation: Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305.
authors.forename: Haodong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Stanford University Medical School, Stanford, CA 94305.
authors.forename: Fikri
authors.initials: F
authors.lastname: Birey
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305.
authors.forename: Sergio A
authors.initials: SA
authors.lastname: Leal-Ortiz
authors.affiliation: Department of Mechanical Engineering, Stanford University, Stanford, CA 94305.
authors.forename: Crystal M
authors.initials: CM
authors.lastname: Han
authors.affiliation: Department of Mechanical Engineering, Stanford University, Stanford, CA 94305.
authors.forename: Juan G
authors.initials: JG
authors.lastname: Santiago
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Stanford University Medical School, Stanford, CA 94305.
authors.forename: Sergiu P
authors.initials: SP
authors.lastname: Paşca
authors.affiliation: Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305; nmelosh@stanford.edu.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Melosh
mesh_terms: D000818:Animals; D016466:CHO Cells; D002454:Cell Differentiation / Q000187:drug effects; D058948:Cell Tracking / Q000379:methods*; D003412:Cricetulus; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000737:chemistry / Q000166:cytology; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000166:cytology; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011506:Proteins; D012333:RNA, Messenger
keywords: cellular biology; molecular biology; nanotechnology; sampling