Single-cell biopsies enable non-destructive temporal single cell analysis.
Cellular heterogeneity poses a formidable challenge for researchers across various disciplines, including developmental biology, cellular reprogramming, immuno-oncology, and cellular disease modeling. Advances in single-cell extraction methods have begun to unravel the intricate and dynamic nature of cellular heterogeneity. Gaining a comprehensive understanding of single-cell dynamics requires extracting target molecules from individual cells to analyze and uncover novel transcripts, proteoforms, and post-transcriptional and -translational modifications. However, many current techniques require removing cells from their natural habitats and lysing them, leading to post-lysis analyte alterations and loss of vital contextual information. In response to these limitations, scientists have proposed the use of minimally invasive sampling devices, such as atomic force microscopy (AFM) tips and micro/nanopipettes.
A significant breakthrough has been with the development of the single-cell biopsy workflow based on the FluidFM technology, which allows researchers to delve into the contents of living cells. Imagine the single-cell biopsy workflow as a delicate procedure that collects a small portion of a cell's cytoplasm, skillfully extracted without inflicting any harm on the cell itself. This innovative approach promises to revolutionize our understanding of cellular heterogeneity and propel the field of cellular research to new heights.
History of single cell biopsies
The term “single-cell biopsy” was used for the first time by Actis P. et al. (2012) that employed nanopipettes to aspirate cytoplasmic material from individual cells without compromising cell viability. [1] Throughout the years, nano- / micropipettes were associated with scanning microscope and electro-wetting [1], scanning ion conductance microscopy. [2], robotic surgery [3], and cantilever probe technology [4].
On the left hand-side: SEM image of a typical single barrel with tip size of 100 nm. On the right hand-side:chematic representation of nano-pipetting procedure. Source: A. Sahota, T. Monteza Cabrejos, Z. Kwan, B. P. Nadappuram, A. P. Ivanov and J. Edel, Chem. Commun., 2023, DOI: 10.1039/D3CC00573A [22].
One popular method for extracting cytoplasmic fluid is through the use of electro-wetted nanopipettes. Imagine a tiny, specialized pipette filled with an organic solvent called 1,2-dichloroethane. When an electric field is applied, the interfacial tension between the solvent and the cell changes, allowing the cytoplasmic fluid to be gently drawn into the pipette through the force of interfacial electric stress. [2,4,5] However, this technique does come with a caveat: to effectively extract the cytosol, the nanopipette must penetrate at least 3 μm beneath the cell membrane. This constraint can make the approach ill-suited for thin, flat cell types. A non-destructive alternative to nanopipettes: nano-straws. These remarkable structures efficiently penetrate cell membranes, granting direct fluidic access to the inner workings of live cells. [6-13] One standout feature of nano-straws is its ability to support the direct culturing of cells on the nano-straw-infused substrate. While nano-straws offer immense versatility for a range of biological applications, their invasive nature is underscored by the findings of Seong et al. and Palankar et al. that revealed that nano-straws can impact cellular morphology,[14] gene expression, [15] cellular alignment [7] and poration of cell membranes [12].
SEM image of the 150 nm diameter Nano-straws. Source: A. Sahota, T. Monteza Cabrejos, Z. Kwan, B. P. Nadappuram, A. P. Ivanov and J. Edel, Chem. Commun., 2023, DOI: 10.1039/D3CC00573A [22].
An additional alternative approach to studying cells: dielectrophoretic systems, including nanopipette-based DEP nano-tweezers [16] and Dielectrophoretic Nano-tweezers (DENT) [17]. Nadappuram et al. (2019) introduced the concept of a dielectrophoretic nano-tweezer for the non-destructive concentration and extraction of biomolecules at the subcellular level. [16] Remarkably, these techniques are cost-effective and straightforward to perform. The nano-tweezer's unique ability to isolate polarizable molecules directly from specific cell regions eliminates the need for cytoplasmic fluid removal, which could otherwise cause local changes or stress responses. [16,18]
As a result, this method may be less invasive than aspirating techniques and allows for greater control over the location of desired material within the cell, enhancing spatial resolution. However, despite recent advancements in surface functionalization, the quantity of subcellular material extracted by the nano-tweezer may still be limited for downstream analysis, such as RNA-seq.
Illustration of Nano-tweezers working principle. Source: A. Sahota, T. Monteza Cabrejos, Z. Kwan, B. P. Nadappuram, A. P. Ivanov and J. Edel, Chem. Commun., 2023, DOI: 10.1039/D3CC00573A [22].
Overall, both nanopipette-based extractions methods and the FluidFM-based single-cell biopsies involve the withdrawal of cytoplasmic material from living cells. Yet, unlike previous approaches, in 2016, Guillaume-Gentil et al., proposed a FluidFM-based single-cell biopsy approach to operate non-destructive and quantitative extractions from single cells with spatiotemporal control. [19]
A single cell sampling method that does not kill the cell.
This study has introduced a groundbreaking workflow that aims to extract intracellular fluid from living cells and employ it to perform different downstream analytical techniques. Using fluidic force microscopy (FluidFM), a protocol was successfully established for inserting, stabilizing, and withdrawing a micro-channeled probe inside a cell, allowing extraction with minimal invasiveness. [19]
Different volumes of GFP-containing extracts collected from single-cell biopsies. Source: Guillaume-Gentil, Orane, et al. "Tunable single-cell extraction for molecular analyses." Cell 166.2 (2016): 506-516. [19]
To demonstrate the efficacy of this novel method, researchers utilized cultures of HeLa cells expressing green fluorescent protein (GFP) in a series of experiments. With precise optical control and force spectroscopy, the researchers were able to guide the FluidFM probe towards the intended compartment, navigating through the cell's membrane and carefully extracting the desired cellular content via the application of under-pressure and for different volumes of extracts.
Schematic of the FluidFM-based extraction procedure. Source: Guillaume-Gentil, Orane, et al. "Tunable single-cell extraction for molecular analyses." Cell 166.2 (2016): 506-516. [19]
The exploration of cellular research methods takes an important stride forward with the single-cell biopsy workflow, as showcased by the two primary findings from this study. The first notable result highlights the non-destructive nature of the extraction process. After removing up to 4.0 pl of cytoplasm, a remarkable 82% of cells remained viable, indicating their resilience to such substantial cytoplasmic loss. Assessing cellular viability post-extraction revealed that collecting up to 4.0 pl of cytoplasmic and 0.6 pl of nucleoplasmic samples posed a minimal risk to cell survival. [19] By carefully controlling the volumetric extraction, researchers managed to avoid unintended damage to the cells while obtaining biologically relevant material, such as metabolites, transcripts, and proteins, for subsequent downstream analysis.
The study underscores the single-cell biopsy workflow's efficiency, selectivity, controlled volumetric extraction, and non-destructive nature, emphasizing its strong potential for diverse research applications. As the journey continues, this innovative method promises to advance our understanding of the complex world of cells.
Conclusions
The results achieved by this groundbreaking research have set the stage for a revolution in the field of cell biology research. This breakthrough technology has uncovered the ability of cells to withstand the extraction of up to several picoliters, paving the way for the study of cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. With the single-cell biopsy approach, researchers have been able to analyze cellular function and response to external stimuli without isolating or killing the cells, as was previously required. [20,21]
The temporal dimension this technology brings to single-cell analysis is truly remarkable, and the application of collecting sub-cellular amounts of RNA has opened up a plethora of new possibilities in the field. The gene expression profiles obtained through single-cell biopsies have been shown to be accurate representations of lysed cell transcriptomes, making this approach an invaluable tool in promoting non-destructive cell dynamics analysis for a broad range of biological applications.