Application of microfluidic chips in anticancer drug screening

With the continuous development of drug screening technology, new screening methodologies and technologies are constantly emerging, driving drug screening into rapid, efficient and high-throughput development. Microfluidics is a rising star in the development of innovative approaches in drug discovery. In this article, we summarize the recent years' progress of microfluidic chip technology in drug screening, including the developmental history, structural design, and applications in different aspects of microfluidic chips on drug screening. Herein, the existing microfluidic chip screening platforms are summarized from four aspects: chip structure design, sample injection and drive system, cell culture technology on a chip, and efficient remote detection technology. Furthermore, this review discusses the application and developmental prospects of using microfluidic chips in drug screening, particularly in screening natural product anticancer drugs based on chemical properties, pharmacological effects, and drug cytotoxicity.Peer reviewe

Reactive inkjet printing and functional inks : a versatile route to new programmed materials

Starting as an ink dispensing tool for documents and images, inkjet printing has emerged as an important instrument for delivering reactive fluids, into a means for creating new, programmed materials. Inkjet is a processing technology with some very unique capabilities, which allows the handling of materials in the picoliter range, and the creation of functionality in new, previously unexplored ways. In particular, drop-on-demand technology provides the chance to dispense liquids in picoliter/nanoliter quantities to very specific locations, with minimal material loss, and in a contact-free manner. This dramatic scale-down of production, not just miniaturization but "nanonization", affords materials that would be either too costly or otherwise inaccessible by other manners. As this is still an emerging technology, there remain a lot of opportunities to pioneer new applications. The underlying, unifying concept behind the chapters of this thesis has been an interest in investigating how inkjet printing, combined with reactive inks, can lead to new applications, new devices, and new materials, wherein unique functionality is imparted as a direct result of the confluence between microfluidic processing, chemistry, and life science. The ability to deliver uniform, sub-nanoliter droplets to specific locations opens up new possibilities that did not exist before. Inherent in the geometry of such droplets, the volume of liquid dispensed also offers some special utility. Based on the aforementioned diameters, drop-on-demand inkjet printing can deliver volumes in the range of approximately 0.5 to 1,000 pL; the direct writing attributes of inkjet ensure that these droplets are not only precise, but can be delivered to a specific location, giving them a "home address". This combination of precise, reproducible, small aliquots and precision deposition is especially important for preparing high-density analytical arrays, as discussed in Chapter 1 on inkjet printing of proteins. In the case of highly specialized proteins such as reactive enzymes or antibodies, where available materials are often limited, the ability to dispense precise quantities in a reproducible fashion means that small amounts of precious material can be used parsimoniously to perform thousands of experiments without compromising the quality of the data. For drop-on-demand printing, droplets normally produced by inkjet printing are commonly in the range of 10 to 125 µm in diameter, depending on the physical characteristics of the fluid, the nozzle used, and the printing conditions; taking advantage of the this aliquot size has some unique attributes that make dispensing highly suitable to materials science challenges that have gone unmet. In the second chapter of this thesis, this size domain is taken advantage of for use in tissue engineering, where it is used to create soft, cell-scale porogenic structures by the use of a reversible, rapid alginate gelation reaction to freeze droplet structures in place. By switching to a continuous inkjet device, larger volumes of beads in the size domain of 100 to 500 µm can be achieved, opening up prospects for pore sizes matching those needed for hosting capillaries. By incorporating reversible hydrogels as a motif in these applications, these controlled cell-scale dimensions can be retained during key processing steps, and then removed (or eroded) later after they have served their function. Extending the concept to the task of dispensing living cells, in Chapter 3, printed alginate structures are used for cell encapsulation. By adjusting the printing conditions to prevent jet break-up before alginate hardening, continuous, one-dimensional "living threads" can be created, which allow for cell cultures to be handled and woven into desired complex patterns. In addition to their role as basic building materials for tissue engineering scaffolds, the alginate threads provide a stable, bio-friendly environment for culturing different cell types, with cells exhibiting a high post-processing viability rate. In Chapter 4, the lower limits of single cell printing are explored, in the concept of "one cell-one well", where the attributes of inkjet printing are used to dispense individual cells. By careful selection of droplet size and accounting of cell concentrations, the statistical probability of single cell printing can be optimized, yielding spatially addressable arrays of isolated living cell cultures on a surface. Additional steps necessary to prevent cells from dehydration are also outlined, offering access to high density arrays of isolated living single cells on glass slides, where each individual droplet acts as independent nanoincubator, hosting intrinsically monoseptic cell cultures in parallel. In addition to describing the theoretical limits of single-pass cell printing experiment designs, an outline is given for experimental designs for tuning single particulate dispensing probability to any value desired between 0 and 1. The focus of Chapter 5 relates to reactive inkjet printing of ultrathin films on surfaces. For systems with moderately good surface wetting, such as polar solvents on glass or metal oxides, inkjet printed droplets result in features ranging approximately from 20 to 300 µm in diameter per droplet. By first printing a thiol-functionalized heterochelic linker and covalently bonding it to the print surface, the surface will accommodate subsequent thiol-ene click reactions only with original monolayer, and only where the first and second deposition features overlap. This combination of spatial selectivity as well as chemoselectivity allows for the preparation of a wide range of monolayers on a printed surface, in a format well-suited to automated surface characterization techniques, as was illustrated using XPS. In Chapters 6, two different categories of irreversible polymerization reactions are described, where print features are reacted in a specific pattern that is process unique. Printable ionogels are developed, which impart conductivity to printed patterns, and consequently, functionality to only those locations where the material has been deposited. Also in Chapter 6, the first example of a moisture-sensitive reactive printing is outlined, where a diisocyanate is combined with different polyols within seconds to create highly crosslinked, ultra-stiff surfaces, which can be built up into three dimensions by successive layering. The topics outlined in this thesis are intended to illustrate the breadth of how inkjet technology can be utilized to support a diverse field of materials science applications — particularly when coupled with modular, off-the-shelf synthetic transformations. The incorporation of synthetic chemistry into inkjet extends the application of inkjet from dispensing static materials merely from a cartridge onto a target, into a dynamic tool for transforming these materials into something new. At the same time, inkjet printing and other allied microfluidics tools enable chemistry experiments (and by extension, life science experiments) on a scale that would otherwise be challenging to realize by other means. The two driving forces of high-throughput experiment design, miniaturization and automation, are both embodied in this dispensing technique, and consequently inkjet printing is a rapidly evolving discipline; it is the intent of this work and the examples given to underscore the diversity offered by this technology

Characterising fitness landscapes of protein evolution by next-generation sequencing

A protein’s amino acid sequence determines its structural, chemical and physical properties, yet how sequence variation influences protein function is still incompletely understood. Protein fitness landscapes powerfully describe the sequence-function relationship by dividing sequence space into functional hills and valleys. This representation is often invoked yet lacks experimental evidence; the immense vastness of possible sequence space makes comprehensive high-quality datasets difficult to obtain. Laboratory directed evolution has focused on optimal utilisation of substitution libraries, however examination of functional innovation in Nature shows that short insertions and deletions (InDels) also play a key role. Beyond rare targeted studies of specific InDels, high-throughput data on fitness landscape for mutations other than substitutions are lacking entirely. In my PhD, I worked towards experimentally describing the fitness landscapes of InDels and substitutions in three systems: GFP, phosphotriesterase (PTE) and the kinase MKK1 docking domain. Towards this goal, I established two experimental assays (GFP, PTE) for deep mutational scanning and a new software toolkit, InDelScanner, for interpreting resulting data that contain InDels. With GFP, I sorted the deletions and substitution libraries into three activity fractions using FACS, then deep sequenced them with Illumina MiSeq to obtain a pilot dataset. The comparison of deletion effects between different lengths of deletions (-3, -6 and -9 bp) indicates that deletions are partially tolerated in eGFP, with tolerance improved for short deletions and in the stabilised starting point GFP8. Further interpretation of data was complicated by limited resolution in the sequencing dataset stemming from poor FACS separation, so I optimised the conditions for better sorting resolution using the mKate2 fluorescent protein as an expression reporter. In the second iteration of the activity sorting I additionally included UMIs in the plasmid design to improve the utilisation of NGS capacity. In the case of PTE, I performed proof-of-concept experiments for microfluidic droplet sorting in an integrated device with an in-line incubation line and a fluorescent sorting design. In parallel, testing of solubility and activity of random InDel variants showed that functional InDels do not necessarily suffer from a stability handicap, making InDel mutagenesis a viable strategy for gene randomisation in directed evolution. One challenge of InDel library data analysis is that InDels are not compatible with existing, substitution-focused software. Using the GFP deletions dataset, I developed the InDelScanner scripts which accurately detect, aggregate and filter insertions, deletions and substitutions. Using the scripts for composition analysis of TRIAD libraries in PTE showed these libraries are well balanced and highly diverse. Finally, I used the InDelScanner scripts to interpret a deep mutational scanning dataset that recorded the sequence preferences in the MKK1 docking domain, acting to activate ERK2. This experiment showed that the fitness landscape in this kinase pair is shaped by the activating effect of hydrophobic residues in the docking groove, as well as widespread positive epistasis. Together, the projects in this thesis demonstrate that deep mutational scanning experiments are a powerful method for exploring the sequence-function relationship in proteins, which can extend into comparison of different types of mutations as well as probing their (epistatic) interactions.BBSRC BB/M011194/

Supported Engineered Extracellular Matrices for 3D Cell Culture

In the current shift away from 2D tissue culture polystyrene and towards 3D cell culture models, several important design criteria have yet to be considered: (1) provision of large open areas where cells can create their own niche (2) fabrication of a scaffold that is chemically and mechanically tunable and (3) presentation of proteins that mimic native extracellular matrix (ECM). Polymer scaffolds fabricated by 3D jet writing provide extensive void space for maximum cell-cell and cell-ECM interactions. This work expands on such electrospinning technologies to establish a micromanufacturing process that modulates the flow of various polymer solutions through a manifold. The resulting scaffolds contain spatially distinct domains that can be customized to exhibit specific bulk or surface properties. Such tunability is not limited to the synthetic design space. We have discovered that hydrodynamically induced fibrillogenesis can yield remarkably stable networks of protein fibrils suspended across a support or scaffold that recapitulate important structural and functional hallmarks of cell-secreted ECM. These engineered networks of fibronectin serve as a breast cancer microenvironment, making it possible to culture an unfractionated patient sample (n=14), where less than 5% are cancer cells, into a self-selected composition of differentiated cancer cells, stem-like cancer cells, and various stromal cells. An average of 40% increase in the tumor-initiating population and at least a 7-fold increase in the cancer cell population was observed after six days (n=3). This user-defined 3D cell culture platform will enable investigation into the bidirectional relationship between cells and the ECM, not just for breast cancer but a variety of diseased or healthy tissue types.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140892/1/stacyram_1.pd