Stephanie Knowlton

Bioprinting for cancer research

Bioprinting offers the ability to create highly complex 3D architectures with living cells. This cutting-edge technique has significantly gained popularity and applicability in several fields. Bioprinting methods have been developed to effectively and rapidly pattern living cells, biological macromolecules, and biomaterials. These technologies hold great potential for applications in cancer research. Bioprinted cancer models represent a significant improvement over previous 2D models by mimicking 3D complexity and facilitating physiologically relevant cell-cell and cell-matrix interactions. Here we review bioprinting methods based on inkjet, microextrusion, and laser technologies and compare 3D cancer models with 2D cancer models. We discuss bioprinted models that mimic the tumor microenvironment, providing a platform for deeper understanding of cancer pathology, anticancer drug screening, and cancer treatment development.

3D-printed microfluidic devices

Microfluidics is a flourishing field, enabling a wide range of biochemical and clinical applications such as cancer screening, micro-physiological system engineering, high-throughput drug testing, and point-of-care diagnostics. However, fabrication of microfluidic devices is often complicated, time consuming, and requires expensive equipment and sophisticated cleanroom facilities. Three-dimensional (3D) printing presents a promising alternative to traditional techniques such as lithography and PDMS-glass bonding, not only by enabling rapid design iterations in the development stage, but also by reducing the costs associated with institutional infrastructure, equipment installation, maintenance, and physical space. With the recent advancements in 3D printing technologies, highly complex microfluidic devices can be fabricated via single-step, rapid, and cost-effective protocols, making microfluidics more accessible to users. In this review, we discuss a broad range of approaches for the application of 3D printing technology to fabrication of micro-scale lab-on-a-chip devices.

Sickle cell detection using a smartphone.

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening “silent” strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement.

3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs

Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering.

Towards Single-Step Biofabrication of Organs on a Chip via 3D Printing

Organ-on-a-chip engineering employs microfabrication of living tissues within microscale fluid channels to create constructs that closely mimic human organs. With the advent of 3D printing, we predict that single-step fabrication of these devices will enable rapid design and cost-effective iterations in the development stage, facilitating rapid innovation in this field.

Smart-Phone Based Magnetic Levitation for Measuring Densities

Magnetic levitation, which uses a magnetic field to suspend objects in a fluid, is a powerful and versatile technology. We develop a compact magnetic levitation platform compatible with a smart-phone to separate micro-objects and estimate the density of the sample based on its levitation height. A 3D printed attachment is mechanically installed over the existing camera unit of a smart-phone. Micro-objects, which may be either spherical or irregular in shape, are suspended in a paramagnetic medium and loaded in a microcapillary tube which is then inserted between two permanent magnets. The micro-objects are levitated and confined in the microcapillary at an equilibrium height dependent on their volumetric mass densities (causing a buoyancy force toward the edge of the microcapillary) and magnetic susceptibilities (causing a magnetic force toward the center of the microcapillary) relative to the suspending medium. The smart-phone camera captures magnified images of the levitating micro-objects through an additional lens positioned between the sample and the camera lens cover. A custom-developed Android application then analyzes these images to determine the levitation height and estimate the density. Using this platform, we were able to separate microspheres with varying densities and calibrate their levitation heights to known densities to develop a technique for precise and accurate density estimation. We have also characterized the magnetic field, the optical imaging capabilities, and the thermal state over time of this platform.

A Bioprinted Liver-on-a-Chip for Drug Screening Applications

The need for a liver-on-a-chip tissue model for drug screening is particularly important in tissue engineering because of the high frequency of drug-induced liver injury. Recently, a liver tissue model conducive to hepatotoxicity testing was developed by bioprinting hepatic spheroids encapsulated in a hydrogel scaffold into a microfluidic device.

Advancing cancer research using bioprinting for tumor-on-a-chip platforms

There is an urgent for a novel approach to cancer research with 1.7 million new cases of cancer occurring every year in the United States of America. Tumor models offer promise as a useful platform for cancer research without the need for animal models, but there remains a challenge to fabricate a relevant model which mimics the structure, function and drug response of human tumors. Bioprinting can address this need by fabricating three-dimensional constructs that mimic tumor heterogeneity, vasculature and spheroid structures. Furthermore, bioprinting can be used to fabricate tissue constructs within microfluidic platforms, forming tumor-on-a-chip devices which are ideal for high-throughput testing in a biomimetic microenvironment. Applications of tumors-on-a-chip include facilitating basic research to better understand tumor development, structure and function as well as drug screening to improve the efficiency of cancer drug discovery.

Utilizing stem cells for three-dimensional neural tissue engineering

Three-dimensional neural tissue engineering has made great strides in developing neural disease models and replacement tissues for patients. However, the need for biomimetic tissue models and effective patient therapies remains unmet. The recent push to expand 2D neural tissue engineering into the third dimension shows great potential to advance the field. Another area which has much to offer to neural tissue engineering is stem cell research. Stem cells are well known for their self-renewal and differentiation potential and have been shown to give rise to tissues with structural and functional properties mimicking natural organs. Application of these capabilities to 3D neural tissue engineering may be highly useful for basic research on neural tissue structure and function, engineering disease models, designing tissues for drug development, and generating replacement tissues with a patients genetic makeup. Here, we discuss the vast potential, as well as the current challenges, unique to integration of 3D fabrication strategies and stem cells into neural tissue engineering. We also present some of the most significant recent achievements, including nerve guidance conduits to facilitate better healing of nerve injuries, functional 3D biomimetic neural tissue models, physiologically relevant disease models for research purposes, and rapid and effective screening of potential drugs.

Continuous-Ink, Multiplexed Pen-Plotter Approach for Low-Cost, High-Throughput Fabrication of Paper-Based Microfluidics

There is an unmet need for high-throughput fabrication techniques for paper-based microanalytical devices, especially in limited resource areas. Fabrication of these devices requires precise and repeatable deposition of hydrophobic materials in a defined pattern to delineate the hydrophilic reaction zones. In this study, we demonstrated a cost- and time-effective method for high-throughput, easily accessible fabrication of paper-based microfluidics using a desktop pen plotter integrated with a custom-designed multipen holder. This approach enabled simultaneous printing with multiple printing heads and, thus, multiplexed fabrication. Moreover, we proposed an ink supply system connected to commercial technical pens to allow continuous flow of the ink, thereby increasing the printing capacity of the system. We tested the use of either hot- or cold-laminating layers to improve (i) the durability, stability, and mechanical strength of the paper-based devices and (ii) the seal on the back face of the chromatography paper to prevent wetting of the sample beyond the hydrophilic testing region. To demonstrate a potential application of the paper-based microfluidic devices fabricated by the proposed method, colorimetric urine assays were implemented and tested: nitrite, urobilinogen, protein, blood, and pH.