Abstract
Heterogeneity is inherent to biology, thus it is imperative to realize methods capable of obtaining spatially-resolved genomic and transcriptomic profiles of heterogeneous biological samples. Here, we present a new method for local lysis of live adherent cells for nucleic acid analyses. This method addresses bottlenecks in current approaches, such as dilution of analytes, one-sample-one-test, and incompatibility to adherent cells. We make use of a scanning probe technology - a microfluidic probe - and implement hierarchical hydrodynamic flow confinement (hHFC) to localize multiple biochemicals on a biological substrate in a non-contact, non-destructive manner. hHFC enables rapid recovery of nucleic acids by coupling cell lysis and lysate collection. We locally lysed ~300 cells with chemical systems adapted for DNA or RNA and obtained lysates of ~70 cells/μL for DNA analysis and ~15 cells/μL for mRNA analysis. The lysates were introduced into PCR-based workflows for genomic and transcriptomic analysis. This strategy further enabled selective local lysis of subpopulations in a co-culture of MCF7 and MDA-MB-231 cells, validated by characteristic E-cadherin gene expression in individually extracted cell types. The developed strategy can be applied to study cell-cell, cell-matrix interactions locally, with implications in understanding growth, progression and drug response of a tumor.
Highlights
Liquid localization is implemented by simultaneous injection and aspiration of a processing liquid; a principle termed hydrodynamic flow confinement (HFC)
This approach of selective sampling using the hHFC is strategic for concentrated lysates free of contaminants and a rapid overall workflow for nucleic acid analysis
To address the above requirements, we investigated interaction of hHFC with cells, chemical systems for local lysis and the downstream analysis of nucleic acids in the context of using the MFP for local lysis
Summary
The locally-confined processing liquid interacts with the biological substrate without the probe physically contacting the substrate, making this approach non-destructive to the surrounding sample. Real-time visual feedback of the substrate processing allows the user to adapt operating conditions during MFP operation, e.g., the probe-to-surface distance, coordinates of the probe on the substrate, and flow rates. This provides the user a high degree of control and flexibility while operating on delicate biological substrates. To the best of our knowledge, this work is the first demonstration of spatially-resolved sampling from live co-cultures for analysis of DNA and mRNA
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