Vala Sciences, Inc. | Date: 2013-12-16
Video recordings from two or more optical channels are produced, processed, and analyzed simultaneously in order to provide quantitative analysis of action potentials, calcium transients and ionic flux in excitable cells loaded with voltage or ion sensitive dyes with distinct excitation and emission wavelengths, The specific wavelengths of fluorescent light emitted from each dye are separated and recorded. The recordings are mutually registered and cytometric analysis is performed to provide a quantitative analysis of the action potentials calcium transient, and/or ionic flux on a cell-by-cell and well-by-well basis in microtiter plates. The cells are then fixed, labeled for other biomarkers, and scanned again. The resulting fixed cell images are registered with the live cell recordings and analyzed; missing cells that were washed off are detected relative to the live recordings, and cytometry data from live and fixed cell scans is collated cell-by-cell.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 761.59K | Year: 2016
DESCRIPTION provided by applicant Defects in heart formation during embryonic and fetal development likely contribute to miscarriages which terminate up to of all pregnancies and Congential Heart Defects CHDs which are among the most common birth defects Environmental factors may influence miscarriages and up to of all CHDs but little is known about how such chemicals affect formation of the heart during early embryonic differentiation The goal of this SBIR project is to develop a Cardiopoiesis Assay heart formation assay to enable testing of compounds to determine if they influence the emergence of cardiac myocytes from multipotent mesodermal progenitors a critical early decision point in heart formation During the previous Phase I portion of the project a version of the assay was developed using mouse embryonic stem cells and this assay has proven useful in identifying chemicals siRNAs and miRs that modify cardiac differentiation For Phase II we propose to develop the assay using human induced pluripotent stem cells hiPSCs which will be derived from multiple donors to increase the genetic diversity represented by the assay The Cardiopoiesis Assay will be useful in predicting whether chemical compounds affect heart formation in the human embryo which is of critical importance to human health The assay will be of interest to government agencies in the US such as the Environmental Protection Agency and worldwide concerned with environmental toxicology PUBLIC HEALTH RELEVANCE Defects of the heart are the most common birth defects and may be caused in part by toxic compounds from the environment that interfere with heart formation prior to birth We propose to develop a test system to screen chemicals for dangerous effects on heart formation We propose to use human stem cells which we will derive from skin samples obtained from volunteer donors the skin samples are obtained by a painless procedure that is risk free Such stem cells can be cultured in a manner that they form beating heart cells in laboratory dishes We will develop a procedure to test if chemicals prevent formation of heart cells from stem cells as such chemicals are likely to cause heart birth defects
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.84K | Year: 2013
The technical objectives from the original solicitation are presented below, along with a brief discussion relevant to the Neural Crest Cell Migration assay. 1) Develop a working assay in a 384 microwell format (the Phase II objective will be to adapt the assay to the 1536 well format). Cells will be cultured to confluence in a 384 well dish; the cell monolayer will then be scratched to create a cell-free region within the center of each well. Migration of cells into the central region will be quantified in the presence of test compounds by imaging the cells for nuclei using fluorescence microscopy. Mitochondrial health will also be assessed utilizing a fluorescent dye (MitoTrackerOrange, or related dye) which will be imaged in a separate channel. 2) Characterize the sensitivity, specificity, variability, reproducibility, signal: background, dynamic range, and accuracy of the assay, utilizing standard positive and negative controls, Z' values gt0.5. The goal is to achieve Z' values of 0.50 or greaterfor the effect of standard compounds to inhibit cell migration or mitochondrial function. Standard compounds to be considered are cytochalasin D for cell migration, and FCCP for mitochondrial health. 3) Demonstrate the utility of the assay by characterizing its ability to detect the effects of compounds known to affect the pathway/cellular phenotype, with a throughput of at least 10,000 samples/day with workstation automation. The goal is to achieve a throughput of at least 10,000 samples/day for dataacquisition which we plan to achieve utilizing Vala's proprietary IC200 microscopy workstation, CyteSeer'i# cell image analysis program, and methods to be further developed in the proposed funding period. 4) Develop an SOP to be submitted to NCA Ts forevaluation . A detailed SOP will be developed for the assay for submission to NCAT. PUBLIC HEALTH RELEVANCE
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase II | Award Amount: 1.23M | Year: 2013
DESCRIPTION (provided by applicant): Monitoring physiology of individual cardiomyocytes in high throughput has not been reported. The inability to perform high throughput physiological measurements limits many basic and applied studies, including the use of stem cell derived cardiomycoytes in cardiotoxicity testing. Current automated cardiotoxicity tests have poor predictive value because they use tumor cell lines engineered with single channels (e.g. hERG), and physiologically relevant tests are reserved for few candidates during the relatively late stages of development. The poor biological relevance of these models contributes to the high failure rate of drug candidates before FDA approval and even after commercialization. We have automated recording frommyocytes for Calcium Transients, but are still limited by use of electrode devices for pacing that prevents miniaturization beyond 96- well format. Furthermore, Action Potential measurement, the most relevant physiological parameter in excitable cells, isstill reserved to low throughput analysis. We propose several conceptual advances to solve these problems by developing a miniaturized, cell-based optogenetic pacing device for high throughput analysis of human Induced Pluripotent Stem Cell (hIPSC)-derived cardiomyocytes in an automated platform for cell-by-cell cytometric analysis of cardiomyocyte physiology. We will also develop automatic segmentation/analysis of Action Potentials (AP) through fluorescent voltage probes and post-recording tracking to identify the same cells after fixation and immunostaining analysis. Calcium Transient (CT) analysis, already developed in a previous SBIR contract, will converge with AP and post-recording tracking to generate single cell multiparametric measurement of all these endpoints conducted in High Throughput. Extensive evaluation will be conducted with drugs that alter AP through different mechanisms to validate the platform. Preliminary data show that stable cell lines expressing the light-triggered protein Channelrhodopsin-2 (ChR2) will electrically couple to cardiomyocytes, allowing optically controlled stimulation of AP without disruption of normal cardiomyocyte physiology. Membrane AP can be recorded in cardiomyocytes through voltage probes and are suitable to image segmentation analysis. Automatic CT measurement and hIPSC-derived cardiomyocytes are an effective model to test cardiotoxic effects of reference drugs. The Aims will advance the use of fluorescent probes to measure action potential, calcium flux and cell characteristics in response to the stimulation. Cardiomyocyte physiology will be quantified by image analysis software that records and analyzes single-cell AP and CT in relation to cardiac subtype or specific protein expression. The software will segment the images into single cell recordings, thus all measurements and data analysis will be on a cell-by- cell basis. The format will be evaluated for 384- and 1536-well to conduct screening on hundreds of cells per individual data point (e.g. compound tested), allowing throughput of tens to hundreds of thousands of datapoints in a single screen by the end of the funding period. Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing. PUBLIC HEALTH RELEVANCE Large-scale studies of heart safety early during the drug development process are not currently possible or have low value because physiological testing is too arduous to perform on more than a few cells in a single experiment. Here we propose a conceptual advance of a previous design, where we have automated some of the steps enabling moderate throughput but that still rely on traditional methods to stimulate cardiomyocytes and analyze only one parameter relevant to cardiac physiology. We propose to develop a cell-based miniaturized pacing device that is stimulated by light and then activates the cardiomyocytes. We will also automate the process of Action Potential measurement and will link to Calcium Transient and cardiomyocytes subtypes to generate a comprehensive platform that analyze several aspect of cardiomyocyte physiology. The device will enable many applications, including large physiological screening for new cardioactive drugs and early testing of drug candidates for adverse cardiac toxicity, which is a major reason for drug failure during development, costing 2.5 billion annually.
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 300.00K | Year: 2013
DESCRIPTION (provided by applicant): The proposed project is a Phase I STTR collaboration between Vala Sciences Inc, a small business that specializes in the development of cell-based high content/high throughput assays, and the Sanford Burnham Medical Research Institute, a worldwide leader in biomedical research. The project will develop an assay (Kinetic Image Cytometer-Neuron, or KIC-Neuron) to quantify the effect of test compounds on neuronal transmission and the development of neuronal cell circuits utilizing optogenetic methods. For KIC-Neuron, primary rat hippocampal neurons will be cultured in a high throughput format (96 or 384 well), then transduced with lenti-viruses encoding light-activated channel rhodopsin, and loaded with fluorescent indicators of DNA (to visualize the nuclei) and intracellular calcium. This will enable depolarization of the cells via use of a flash of light, which leads to neurotransmission across the neuronal cultures and elevation of intracellular calcium in post-synaptic neurons, which is quantified via automated microscopy and image analysis techniques. Following the activity assay, the cells are also fixed, labeled, and imaged for neuronal biomarkers, which can be correlated on a cell-by-cell basis to neuroactivity. The assay system will be used to quantify the effects of short -term exposure to compounds (such as the presence of ion channel blockers or antagonists of neural transmitter receptors during the activity assay), and also to quantify potential long-term effectsof compounds on the neural circuits. The latter phenomenon is relevant to the Chemo-brain condition, which is a loss cognitive ability that develops in cancer patients following chemotherapy, and may be related to other forms of memory loss and dementia,as well, including Alzheimer's. The assay will be of interest to government agencies including the US Environmental Protection Agency, and NIH institutes (NCI and NHCLBI) as well as to both pharmaceutical and academic researchers. KIC-NEURON will be commercialized for Vala's contract research screening as well as developed into a product line featuring kits, software, and instrumentation. PUBLIC HEALTH RELEVANCE In human patients treated with anti-cancer drugs, or suffering from inherited disorders, brainneuron function is lost, leading to impaired speech, understanding, and decision making. It is unclear why this occurs, no therapies have yet been found to improve brain function. In this project, we will develop methods to produce brain in a dish neuronal cultures that are very small (each dish features 96 individual cultures) along with a specialized microscope that will record the activity of the neurons in an automated manner. The methods developed in this project will enable neurological researchersat universities, pharmaceutical companies, and government agencies to test hundreds to thousands of compounds for potential toxic or beneficial effects on brain function.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.55K | Year: 2013
Congenital cardiac defects (CCDs) are among the most common birth defects and fetal cardiac defects likely contribute to miscarriages, which terminate up to 25% of all pregnancies. Environmental factors may influence up to 80% of all CCDs, but little is known about how chemicals from the environment may affect formation of the heart. We propose to develop a cardiopoiesis (formation of the heart) assay to screen chemicals using stem cells, which recapitulate early event in heart formation, in which cardiac myocytes, vascular endothelial cells, and vascular smooth muscle cells differentiate from mesodermal multipotent cardiac precursors. The assay is performed in 384 well plates and uses murine embryonic stem cells (mESCs) bioengineered with reporter genes toreport the proportion of cardiac myocytes, vascular endothelial cells, and vascular smooth that emerge from the cultures. In preliminary experiments, 550 known chemical pathway modulators were screened with the cardiopoiesis assay, and inhibitors of the Wnt pathway were found to strongly upregulate production of cardiac myocytes. This is consistent with the known influence of the Wnt pathway on cardiac development and the suspected role of dysregulated Wnt activity in producing CCDs. Phase I goals will be to further improve the assay by identifying compounds or genomic constructs that can serve as reliable positive or negative effectors in the system, to develop methods to maximize the information that can be garnered from each screening run, and to developmethods to improve the consistency and further miniaturize the assay. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Defects of the heart are among the most common birth defects and might be caused, in part, by toxic compounds from the environment interfering with heart development before the baby is born. However, there are few methods to test chemicals for such effects. We propose to develop a test to screen chemicals for dangerous effects on heart formation, by using approved stem cells, which can becultured in a manner so that heart cells form within the cultures. In fact, heart cells derived from stem cells, beat, rhythmically, jst like a human heart. If a chemical interferes with the development of heart cells from stem cells, it will likely have harmful effects on heart development in human embryos, as well.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.93K | Year: 2015
DESCRIPTION provided by applicant Parkinsonandapos s disease PD is the nd most common neurodegenerative affliction affecting approx of the human population The symptoms of PD include tremors in motor function of limbs due to progressive death of dopaminergic neurons in the brain While certain forms of PD are inherited it is clear that environmental toxins including pesticides and likely industrial solvents contribute to PD but the pathways via which this occurs are incompletely understood Synuclein Syn a protein of unknown function participates in PD pathology likely due to mis folding aggregation and toxicity to mitochondrial function Further environmental toxins linked to PD also inhibit mitochondrial function A mutation in Syn A T has been identified which causes an early onset of PD Induced pluripotent stem cells iPSCs have been prepared from a subject harboring the Syn A T mutation and dopaminergic neurons derived from these iPSCs display increased PD related responses to pesticides compared to isogenic control iPSC cells The goal of this Phase I SBIR project will be to develop a high throughput screen to test environmental toxins for their ability to increase the risk of PD using iPSC derived dopaminergic neurons harboring the A T mutation To identify the biomarkers that will provide the best indication of PD related pathologies we will test pesticides on these neurons and quantify effects on synaptic structures mitochondria and the generation of reactive oxygen and nitrogen species Customers for the assay include government agencies such as the US EPA Vala Sciences Inc specializes in developing cell based assays relevant to environmental toxicity and is an assay provider for the EPA ToxCast program The Phase II goals will be to further refine and expand the capability of the assays that can be done with these neurons PUBLIC HEALTH RELEVANCE Parkinsonandapos s disease PD is the nd most common neurodegenerative affliction affecting approx of the human population The symptoms of PD include tremors in motor function of limbs due to progressive death of certain neurons in the brain While certain forms of PD are inherited it is clear that environmental toxins including pesticides and likely industrial solvents contribute to PD Recently a patient with an early onset form of PD was identified and found to have a mutation in a certain protein and researchers were able to prepare stem cells from a sample of the patientandapos s skin that can be used to form neurons in a dish Furthermore these neurons have proven to be sensitive to environmental toxins We propose to develop methods so that the stem cell derived neurons from the PD patient can be used to screen chemicals that might escape into the environment for their potential to cause PD using the stem cell derived neurons The methods will involve the use of automated robotic microscopes
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 223.05K | Year: 2014
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.07M | Year: 2014
Vala Sciences, Inc. | Date: 2013-04-05
An analytical reference for digital histochemistry is constituted of an on-slide standard used to calibrate biomarker expression in biopsy material and tissue sections. The standard consists of one or more calibration standards, which are derived from cell lines that express biomarkers, and are arranged into a calibration array. Expression of the desired biomarkers is validated by Western blotting, flow cytometry, or other methods. The calibration array is applied to a microscopy slide in such a manner that test samples from biopsies or tissue sections, or other cellular material, can also be mounted on the same slide, adjacent to the calibration array. Data obtained from the calibration array can be used to quantify expression of biomarkers in the biopsy material, tissue sections, tissue microarrays, and cellular material, via digital histochemistry.