Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

IBM Research - Zurich

Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications.

The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide-pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ.

Chondrocyte culture in three dimensional alginate sulfate hydrogels promotes proliferation while maintaining expression of chondrogenic markers.

We devised, implemented, and tested a new concept for efficient local surface chemistry that we call hierarchical hydrodynamic flow confinement (hierarchical HFC). This concept leverages the hydrodynamic shaping of multiple layers of liquid to address challenges inherent to microscale surface chemistry, such as minimal dilution, economical consumption of reagent, and fast liquid switching. We illustrate two modes of hierarchical HFC, nested and pinched, by locally denaturing and recovering a 26 bp DNA with as little as 2% dilution and by efficiently patterning an antibody on a surface, with a 5 μm resolution and a 100-fold decrease of reagent consumption compared to microcontact printing. In addition, valveless switching between nanoliter volumes of liquids was achieved within 20 ms. We believe hierarchical HFC will have broad utility for chemistry on surfaces at the microscale.

Hierarchical hydrodynamic flow confinement: efficient use and retrieval of chemicals for microscale chemistry on surfaces.

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.

Aditya Kashyap

IBM Research - Zurich

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe