Culture of cancer spheroids and evaluation of anti-cancer drugs in 3D-printed miniaturized continuous stirred tank reactors (mCSTRs).

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliablein vitrocancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e. derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 ml mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 d in ultra-low-attachment plates to allow formation of discoid cell aggregates (∼600µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5-6 d of culture to reach a steady diameter between 600 and 800µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7-derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1andABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

Fabrication of Multilayered Composite Nanofibers Using Continuous Chaotic Printing and Electrospinning: Chaotic Electrospinning.

Multi-material and multilayered micro- and nanostructures are prominently featured in nature and engineering and are recognized by their remarkable properties. Unfortunately, the fabrication of micro- and nanostructured materials through conventional processes is challenging and costly. Herein, we introduce a high-throughput, continuous, and versatile strategy for the fabrication of polymer fibers with complex multilayered nanostructures. Chaotic electrospinning (ChE) is based on the coupling of continuous chaotic printing (CCP) and electrospinning, which produces fibers with an internal multi-material microstructure. When a CCP printhead is used as an electrospinning nozzle, the diameter of the fibers is further scaled down by 3 orders of magnitude while preserving their internal structure. ChE enables the use of various polymer inks for the creation of nanofibers with a customizable number of internal nanolayers. Our results showcase the versatility and tunability of ChE to fabricate multilayered structures at the nanoscale at high throughput. We apply ChE to the synthesis of unique carbon textile electrodes composed of nanofibers with striations carved into their surface at regular intervals. These striated carbon electrodes with high surface areas exhibit 3- to 4-fold increases in specific capacitance compared to regular carbon nanofibers; ChE holds great promise for the cost-effective fabrication of electrodes for supercapacitors and other applications.

Characterization of Soft Tooling Photopolymers and Processes for Micromixing Devices with Variable Cross-Section.

In this paper, we characterized an assortment of photopolymers and stereolithography processes to produce 3D-printed molds and polydimethylsiloxane (PDMS) castings of micromixing devices. Once materials and processes were screened, the validation of the soft tooling approach in microfluidic devices was carried out through a case study. An asymmetric split-and-recombine device with different cross-sections was manufactured and tested under different regime conditions (10 < Re < 70). Mixing performances between 3% and 96% were obtained depending on the flow regime and the pitch-to-depth ratio. The study shows that 3D-printed soft tooling can provide other benefits such as multiple cross-sections and other potential layouts on a single mold.

Influence of Controlled Cooling on Crystallinity of Poly (L-Lactic Acid) Scaffolds after Hydrolytic Degradation.

The use of hybrid manufacturing to produce bimodal scaffolds has represented a great advancement in tissue engineering. These scaffolds provide a favorable environment in which cells can adhere and produce new tissue. However, there are several areas of opportunity to manufacture structures that provide enough strength and rigidity, while also improving chemical integrity. As an advancement in the manufacturing process of scaffolds, a cooling system was introduced in a fused deposition modeling (FDM) machine to vary the temperature on the printing bed. Two groups of polylactic acid (PLA) scaffolds were then printed at two different bed temperatures. The rate of degradation was evaluated during eight weeks in Hank's Balanced Salt Solution (HBSS) in a controlled environment (37 °C-120 rpm) to assess crystallinity. Results showed the influence of the cooling system on the degradation rate of printed scaffolds after the immersion period. This phenomenon was attributable to the mechanism associated with alkaline hydrolysis, where a higher degree of crystallinity obtained in one group induced greater rates of mass loss. The overall crystallinity was observed, through differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA), and Fourier transformed infrared spectroscopy (FTIR) analysis, to increase with time because of the erosion of some amorphous parts after immersion.

Using chaotic advection for facile high-throughput fabrication of ordered multilayer micro- and nanostructures: continuous chaotic printing.

This paper introduces the concept of continuous chaotic printing, i.e. the use of chaotic flows for deterministic and continuous extrusion of fibers with internal multilayered micro- or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (∼102 cm2 cm-3) at high resolution (∼10 µm). This creates structures with extremely high surface area to volume ratio (SAV). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output. In an exciting new development, we demonstrate a method for scaling down these microstructures by 3 orders of magnitude, to the nanoscale level (∼150 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including-but not limited to-bioprinting of multi-scale layered biological structures such as bacterial communities, living tissues composed of organized multiple mammalian cell types, and fabrication of smart multi-material and multilayered constructs for biomedical applications.

Influence of Controlled Cooling in Bimodal Scaffold Fabrication Using Polymers with Different Melting Temperatures.

The combination of different materials and capabilities to manufacture at several scales open new possibilities in scaffold design for bone regeneration. This work is focused on bimodal scaffolds that combine polylactic acid (PLA) melt extruded strands with polycaprolactone (PCL) electrospun fibers. This type of bimodal scaffold offers better mechanical properties, compared to the use of PCL for the extruded strands, and provides potential a means for controlled drug and/or growth factor delivery through the electrospun fibers. The technologies of fused deposition modeling (FDM) and electrospinning were combined to create 3D bimodal constructs. The system uses a controlled cooling system allowing the combination of polymers with different melting temperatures to generate integrated scaffold architecture. The thermoplastic polymers used in the FDM process enhance the mechanical properties of the bimodal scaffold and control the pore structure. Integrated layers of electrospun microfibers induce an increase of the surface area for cell culture purposes, as well as potential in situ controlled drug and/or growth factor delivery. The proposed bimodal scaffolds (PLA extruded strands and PCL electrospun fibers) show appropriate morphology and better mechanical properties when compared to the use of PCL extruded strands. On average, bimodal scaffolds with overall dimensions of 30 × 30 × 2.4 mm³ (strand diameter of 0.5 mm, strand stepover of 2.5 mm, pore size of 2 mm, and layer height of 0.3 mm) showed scaffold stiffness of 23.73 MPa and compression strength of 3.85 MPa. A cytotoxicity assay based human fibroblasts showed viability of the scaffold materials.

Characterization of interdigitated electrode structures for water contaminant detection using a hybrid voltage divider and a vector network analyzer.

Interdigitated capacitive electrode structures have been used to monitor or actuate over organic and electrochemical media in efforts to characterize biochemical properties. This article describes a method to perform a pre-characterization of interdigitated electrode structures using two methods: a hybrid voltage divider (HVD) and a vector network analyzer (VNA). Both methodologies develop some tests under two different conditions: free air and bi-distilled water media. Also, the HVD methodology is used for other two conditions: phosphate buffer with laccase (polyphenoloxidase; EC 1.10.3.2) and contaminated media composed by a mix of phosphate buffer and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The purpose of this study is to develop and validate a characterization methodology using both, a hybrid voltage divider and VNA T-# network impedance models of the interdigitated capacitive electrode structure that will provide a shunt RC network of particular interest in detecting the amount of contamination existing in the water solution for the media conditions. This methodology should provide us with the best possible sensitivity in monitoring water contaminant media characteristics. The results show that both methods, the hybrid voltage divider and the VNA methodology, are feasible in determining impedance modeling parameters. These parameters can be used to develop electric interrogation procedures and devices such as dielectric characteristics to identify contaminant substances in water solutions.