52 / 2019-07-24 17:20:53

A multiplexed intracellular probing (IP) nano-chip for interrogation of myo-fibroblasts and cardiomyocytes gene in cardiac fibrosis

nano-chip,gene analysis,cardiac fibrosis

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Introduction
Heart disease remains global leading cause of mortality. In organ, most cardiac diseases are accompanied with fibrosis, a procedure that cardiac fibroblasts accumulate and over-express extracellular matrix (ECM) proteins, eventually leading to dysfunctions of cardiomyocytes. At cellular level, however, there is a lack of intracellular biomarkers of the activated myo-fibroblasts primarily due to its phenotypic heterogeneity1. Furthermore, comprehensive understanding of the mechanism of fibroblast-secreted extracellular matrix (ECM) proteins in regulating the gene expression of cardiomyocytes have been hampered due to the challenges of intracellular analysis in single living cell, which leads to inconsistent conclusions between previous reports2. Current tools remain challenging to address these two specific issues at single cell level.
Materials and methods
The silicon chip was fabricated using cleanroom techniques, mainly including projection photolithography and deep reactive ion etching (DRIE)3. Cells were positioned on IP nano-chip where each single cell is aligned with a Z-directional nanochannel. The buffers of IPs (i.e. molecular beacons (MBs)) are filled in a bottom chamber under the chip. A pulsed electric field is applied over IP nano-chip and cells between a pair of electrodes. The nanochannel focuses the electric field within a narrow area on the cell membrane, enabling safe and localized electroporation. MBs are electrophoretically driven into the cell under previse dose control.
Results
The nanochannel-assisted electroporation and electrophoretic IP delivery require that the single cell tightly connects with a nanochannel. To achieve this aim, we developed a novel on-chip microfluidics approach for moving and trapping cardio-cells on the nanochannel array, where > 10,000 cells can be massively parallel probed over a 1 cm2 single chip (Fig. 1a). Micro-well array (SU-8 photoresist), designed to capture individual cells, were photolithographically patterned around each nanochannel on the chip (Fig. 1b). For flowing cell to micro-well, microfluidic channels were fabricated on a PDMS membrane by soft-lithography. Controlling flow rate achieved precise positioning each single cell into microwell. We have designed and synthesized MBs to detect the expression of mRNAs which are hypothesized to indicate the biomarkers of myo-fibroblasts (e.g. Tcf-21, Pdgfr-α, α-SMA)2 (Fig. 1c). The expression of specific mRNAs allows to identifying the biomarkers those can exclusively “label” the activated myo-fibroblasts rather than their counterparts (e.g. epithelial fibroblasts). MBs were also applied to investigate GATA4 and HDAC2, two critical genes in cardiomyocytes in cardiac fibrosis.

Fig. 1 Micro-well and microchannel on IP nano-chip for microfluidic-assisted cardio cell trapping and single cell biomarker detection. (a) The schematics of array of micro-well array patterned around nanochannel for cell seeding. Cells flow within in microchannel manufactured on PDMS mold. (b) High density micro-well array (10 μm in diameter) has been patterned on the nanochannel array chip using photolithography. (c) Molecular beacons recognize the expression of mRNAs intracellularly in single cell. Scale bar: (b) &(c) 20 μm.

Conclusions
We herein report an advanced intracellular probing (IP) nano-chip for precise detection of gene expression within myo-fibroblasts and cardiomyocytes in cardiac fibrosis model. At single cell level, the novel IP nanochip characterized the activation of ‘myo-fibroblasts’ and provided comprehensive analysis of the biomarkers for the gene expression in cardiomyocytes in response to the activated myo-fibroblasts in cardiac fibrosis.
References

1. Krenning G, Zeisberg EM, Kalluri R. (2010) The origin of fibroblasts and mechanism of cardiac fibrosis. J. Cell. Physiol. 225: 631-637.

2. Tallquist MD, Molkentin JD. (2017) Redefining the identity of cardiac fibroblasts. Nat. Rev. Cardiol. 14: 484–491.

3. Chang L, Gallego-Perez D, Chiang CL et al. (2016) Controllable large-Scale transfection of primary mammalian cardiomyocytes on a nanochannel array platform. Small 12: 5971-5980.