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Periodic Reporting for period 1 - Als-on-a-chip (A tissue-on-a-chip platform for systems-level studies of ALS pathology and drug screening)

Reporting period: 2015-09-30 to 2017-09-29

Amyotrophic Lateral Sclerosis (ALS) is a complex disease, characterized by diverse pathology that causes progressive motor neuron death. Unfortunately, most ALS patients die within 5 years. The single FDA-approved ALS drug (riluzole) has modest effects, so there is urgent need for new treatments to save patient lives. Currently, the main tool for preclinical ALS studies is mice overexpressing human SOD1G93A. However, despite promising results in this mouse model, more than 30 candidate ALS drugs failed to show efficacy in clinical trials over the past 20 years. These failures have been partly attributed to poor knowledge of ALS origins and pathology, key differences in the physiology of human and mice nerve cells, and the complexity of ALS pathology that cannot be blocked by “magic bullets”.
The objective of the ALS-on-a-chip project was to develop a novel tissue-on-a-chip platform for systems-level studies of ALS pathology and drug screening. The proposed platform combines a novel cell-scaffold in vitro system with high-throughput quantification in order to quantify the effects of candidate ALS drugs on appropriate mouse and human cellular models of ALS. The proposed technology enables quantification of large arrays of 3D tissue analogs by modern quantification methods, providing novel ways describe ALS pathology and quantify drug effects based on advanced 3D models of the central nervous system (CNS).
The project provided novel in vitro tools than could accelerate the development of new ALS treatments by improving the preclinical evaluation of candidate drugs in appropriate in vitro models. The proposed platform could be easily expanded/adapted in order to develop various kinds of physiologically-relevant models for central nervous system (CNS) physiology and pathology.
The project consisted of 3 parts: 1) design the in vitro device (“ALS chip”) for conducting experiments on ALS pathology. 2) developing assays for quantifying ALS pathology in ALS-chip devices. 3) Quantifying the effects of candidate drugs in cells of mice and human origin and identify similarities and differences between human and mice pathology..

The project faced four major engineering and scientific challenges: 1) develop a novel facility for fabricating porous scaffolds and devices 2) develop appropriate cellular models of ALS pathology of mouse or human origin inside ALS-chips, 3) develop protocols and computational tools for quantifying cells in ALS-chips, 4) demonstrate the ability of ALS-chips to provide information that can enhance pre-clinical discovery for ALS.

BIOMATERIALS: ALS-on-chip devices utilized porous scaffolds, similar to scaffolds utilized in regenerative medicine. All required equipment for fabricating scaffolds was setup from scratch. New protocols for fabricating scaffolds of various chemical composition were developed. The resulting scaffolds were characterized by SEM, mechanical testing, and optical spectroscopy.

DEVICE DESIGN: ALS-on-a-chip device design focused on ease of use and easy quantification by high-content imaging and proteomics. Novel fabrication methods were developed in order to prepare devices of repeatable properties. Various PDMS superstructures were prepared using 3D printed molds. A robotic liquid handler was programmed in order to precisely control the chemical composition of device scaffolds and provide experimental versality.

CELL PLAYERS. Motor neurons (MN) are the key cell of interest in ALS studies. The mouse MN cell line NSC34 was kindly provided by Prof. P. Shaw (University of Sheffield) and utilized to optimize the platform. NSC34 grew long neurites in standard 2D culture but attached poorly to porous scaffolds. Co-culture of NSC34 with C2C12 myoblasts enhanced NSC34 attachment and neurite outgrowth. Several attempts to differentiate primary mouse E13.5 cortical neural stem cells (NSC) to MN failed. Instead, mouse MN were derived by differentiating E14 mouse embryonic stem cells. Experimental protocols were adapted in order to differentiate and seed MN as single cells mode inside ALS-chips.

QUANTIFICATION AND COMPUTATION: Confocal microscopy and multiphoton microscopy were found to provide high-resolution 3D images of cells throughout the thickness of ALS-chip scaffolds. Device scaffolds were fabricated in the footprint of a 96-well plate so that they could be imaged by standard high-content microscopes. A specific adaptor was developed in order to quanitfy ALS-chips in multiple pieces of laboratory equipment, including HCS and platereaders. Computational tools were developed to process acquired images and obtain single-cell quantification. Proteomics were utilized to quantify intracellular responses induced by external stimuli and drugs in ALS-chips. Results demonstrated that both stimuli-free and stimuli-induced phosphorylation response depended on the physicohemical properties of the scaffolds.
ORGAN ON CHIP DESIGN: Several organ-on-chip platforms have been developed over the past years to provide advanced in vitro modeling of tissues and organs. Most of them utilize 3D cultures of cells inside hydrogels and microfluidic systems. Despite their engineering elegance, most designs suffer from issues that limit their application in drug discovery (complex use, cost, require extensive training, low sample throughput). The ALS-on-chip project succeeded in developing a novel device design that utilized miniature porous scaffolds and emphasized ease of use, customization, and quantification by high-throughput methods. Compared to existing organ-on-chip designs, ALS-chip device design offers great versality and enables conducting large scale experiments that match preclinical drug discovery needs (sometime there is need to screen thousands of compounds,sometimes there is need to understand the mode of action of a drug before proceeding to costly clinical trials .

ALS DRUG DISCOVERY: Preliminary experiments were conducted to test the neuroprotective ability of microneurotrophins (MNTs; small molecule analogs of neurotrophins; a novel class of neuroptotective compounds). While previous studies on MNTs focused on compounds that mimic NGF, research in ALS-on-chip (still under way) focuses towards less-studied microneurotrophins that mimic BDNF, the key neurotrophin for MN. Collaborations were setup with two startup companies in Greece and a non-profit that focuses on ALS drug discovery in order to design experiments that can explain differences in drug effects in human versus mouse models.

NEW RESEACH DIRECTIONS: Based on the technology developed, several novel collaborations were established. Examples include, but are not limited to: Neuroprotection by MNTs, drug delivery via biomaterials, laser microfabrication, systems neuroscience, scaffold vascularization, stem cell differentiation, cell printing, in vitro models of Alzheimer’s disease, mechanical testing of porous scaffolds.

TECHNOLOGY COMMERCIALIZATION: Based on the technology developed, a team of scientists and engineers has been formed in order to set up a spinoff company that will provide novel in vitro models to address challenges in modern drug discovery. The team aspires to make real-world impact for patients that suffer from ALS (or other neurodegenerative diseases) by enhancing preclinical drug discovery, identify better candidate drugs and provide guidance to future clinical trials.
3d culture of primary corical neurons inside an als-chip device
3D network of stem cell-derived motor neurons that formed inside an als-chip device
Prototype of the als-on-chip device highlighting the porous nature of its scaffolds
Co-culture of NSC34 motor neuron cell line with C2C12 cells in an als-on-chip device