Anatoly Kachurin

Tissue Engineering by Self-Assembly of Cells Printed into Topologically Defined Structures

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

ABSTRACT In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO4 or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.

Direct-Write Construction of Tissue-Engineered Scaffolds

A computer-controlled xyz dispensing system called the Biological Architecture Tool (BAT) has been extensively tested in the creation of multilayered and three-dimensional biological objects: tissue scaffolds and plain and patterned cellular-array slides. The BAT dispensing system has proven its versatility and reliability in tissue engineering and biological experiments. The potential employments of modified versions of the xyz dispensers for in vivo minimally invasive surgery and other in vitro aspects of biological and medical research are discussed.

Rapid prototyping of electronics via direct writing and laser processing

High-quality electronic circuit elements are being written directly onto substrates with relatively low temperature tolerances by means of a pen-dispensing process. Conductor, resistor, and dielectric paste formulations are deposited onto the substrate, then thermally treated with a laser to reach final form. The laser-treatment process is facilitated by using optical pyrometer temperature measurements as real-time feedback for laser power control. The paste deposition and laser processing technologies have been incorporated into the development of a machine tool for conducting rapid prototyping of electronic circuits deposited onto conformal substrates.High-quality electronic circuit elements are being written directly onto substrates with relatively low temperature tolerances by means of a pen-dispensing process. Conductor, resistor, and dielectric paste formulations are deposited onto the substrate, then thermally treated with a laser to reach final form. The laser-treatment process is facilitated by using optical pyrometer temperature measurements as real-time feedback for laser power control. The paste deposition and laser processing technologies have been incorporated into the development of a machine tool for conducting rapid prototyping of electronic circuits deposited onto conformal substrates.

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders.

Laser-machined microfluidic bioreactors with printed scaffolds and integrated optical waveguides

Laser micromachining combined with digital printing allows rapid prototyping of complex bioreactors with reduced fabrication times compared to multi-mask photolithography. Microfluidic bioreactors with integrated optical waveguides for diagnostics have been fabricated via ultrashort pulse laser micromachining and digital printing. The microfluidic channels are directly laser machined into poly(dimethylsiloxane) (PDMS) silicone elastomer. Multimode optical waveguides are formed by coating the PDMS with alternating refractive index polymer layers and laser machining to define the waveguide geometry. Tapered alignment grooves are also laser machined to aid in coupling optical fibers to the waveguides. Three-dimensional (3-D) bio-scaffold matrices comprising liquid solutions that can be selectively and rapidly gelled are digitally printed inside the bioreactors and filled with nutrient rich media and cells. This paper will describe the maskless fabrication of complex 3-D bioreactors and discuss their performance characteristics.

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue.

CAD/CAM for MEMS and BioMEMS

Novel devices can be relatively simple in theory and modeling, but difficult and many times unfeasible to fabricate in a traditional cleanroom environment. We have developed a CAD/CAM tool capable of integrating multiple materials in the electronic, photonic, and biological regimes for applications in both MEMS and BioMEMS devices. Some materials are known and more fully characterized, such as thick film resistors or conductors, while other materials such as biodegradable scaffolding are new but showing promise to realize heterogenous tissue engineered constructs and drug delivery devices. The tool does not discriminate, but rather places these materials in specified locations with precision volumetric control, gently, conformally, and in 3-D. This paper will describe the enabling aspect of true 3-D maskless fabrication as well as describe multiple device structures and demonstrations.