Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

Abstract

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

Mapping Spatial Genetic Landscapes in Tissue Sections through Microscale Integration of Sampling Methodology into Genomic Workflows

“…Co-planarity of the surfaces is ensured by threepoint adjustment of the tissue section with respect to the probe apex. A detailed description on the operation procedures can be found in Kashyap et al [53] TE buffers containing detergent and proteinase K, 50 × 10 −3 m NaOH, or Trizol were used in the inner injection (and aspiration) channel shielded by TE buffer or TE buffer containing food dye in the outer injection channel. The apex of the microfluidic chip was positioned 60 µm above the FFPE sample surface.…”

Microscale hydrodynamic confinements: shaping liquids across length scales as a toolbox in life sciences

“…Kashyap et al cultured a monolayer of MCF-7 cells on a surface and then used a cell lysate to lyse and collect cells from the surface. 122 Recently, HFCs have become popular in the field of spatial analysis of tissues to test for protein heterogeneity 123 and tumor heterogeneity. 124 As an extension to HFCs between parallel plates, Cors et al explored creating HFCs between non-parallel surfaces.…”

Biopatterning: The Art of Patterning Biomolecules on Surfaces

“…With the MFP enabling both control in spatial resolution and fluidic parameters for each aperture independently, it is possible to process single cells to hundreds of cells simultaneously to pattern and to extract their molecular characteristics (Figure a). For DNA and mRNA profiling, we obtained lysates by locally and rapidly lysing cells using the inner confinement of the hHFC, while shielding the sampled lysate from the surrounding sample using an outer confinement . This allowed minimal dilution of the collected lysate.…”

“…(c) Composite fluorescence image of breast cancer tissue with HER2 mRNA tagged using microscale RNA in situ hybridization. DAPI nuclear stains (blue) in parts b and c. Part a is adapted with permission from ref . Copyright 2016 MyJove Corp. Part b is adapted with permission from ref .…”

“…For DNA and mRNA profiling, we obtained lysates by locally and rapidly lysing cells using the inner confinement of the hHFC, while shielding the sampled lysate from the surrounding sample using an outer confinement. 87 This allowed minimal dilution of the collected lysate. For example, for DNA extraction, we demonstrated downstream PCR amplification after a continuous flow of sodium hydroxide with the MFP (NaOH, 10−50 mM), with as little as 2.3 μL of lysate and 20 s of residence time.…”