Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Three-dimensional refractive index estimation based on deep-inverse non-interferometric optical diffraction tomography (ODT-Deep).

Optical diffraction tomography (ODT) solves an inverse scattering problem to obtain label-free, 3D refractive index (RI) estimation of biological specimens. This work demonstrates 3D RI retrieval methods suitable for partially-coherent ODT systems supported by intensity-only measurements consisting of axial and angular illumination scanning. This framework allows for access to 3D quantitative RI contrast using a simplified non-interferometric technique. We consider a traditional iterative tomographic solver based on a multiple in-plane representation of the optical scattering process and gradient descent optimization adapted for focus-scanning systems, as well as an approach that relies solely on 3D convolutional neural networks (CNNs) to invert the scattering process. The approaches are validated using simulations of the 3D scattering potential for weak phase 3D biological samples.

Stabilization of uric acid mixed crystals by melamine.

Melamine stabilizes heterogeneous nucleation of calcium crystals by increasing the retention time and decreasing the rate of dissolution. Stabilization of such mixed crystals limit the efficacy of non-invasive treatment options for kidney stones. Crystalline forms of uric acid (UA) are also involved in urolithiasis or UA kidney stones; however, its interactions with contaminating melamine and the resulting effects on the retention of kidney stones remain unknown. Since melamine augments calcium crystal formation, it provides an avenue for us to understand the stability of UA-calcium phosphate (CaP) crystals. We show here that melamine facilitates UA+CaP crystal formation, resulting in greater aggregates. Moreover, melamine induced mixed crystal retention through a time-dependent manner in presence and/or absence of hydroxycitrate (crystal inhibitor), indicating its abridged effectiveness as conventional remedy. CaP was also shown to modify optical properties of UA+CaP mixed crystals. Differential staining of individual crystals revealed enhanced co-aggregation of UA and CaP. The dissolution rate of UA in presence of melamine was faster than its heterogeneous crystallization form with CaP, although the size was comparatively much smaller, suggesting disparity in regulation between UA and CaP crystallization. While melamine stabilized UA, CaP and mixed crystals in relatively physiological conditions (artificial urine), the retentions of those crystals were further augmented by melamine, even in presence of hydroxycitrate, thus reducing treatment efficacy.

Computational multi-wavelength phase synthesis using convolutional neural networks [Invited].

Multi-wavelength digital holographic microscopy (MWDHM) provides indirect measurements of the refractive index for non-dispersive samples. Successive-shot MWDHM is not appropriate for dynamic samples and single-shot MWDHM significantly increases the complexity of the optical setup due to the need for multiple lasers or a wavelength tunable source. Here we consider deep learning convolutional neural networks for computational phase synthesis to obtain high-speed simultaneous phase estimates on different wavelengths and thus single-shot estimates of the integral refractive index without increased experimental complexity. This novel, to the best of our knowledge, computational concept is validated using cell phantoms consisting of internal refractive index variations representing cytoplasm and membrane-bound organelles, respectively, and a simulation of a realistic holographic recording process. Specifically, in this work we employed data-driven computational techniques to perform accurate dual-wavelength hologram synthesis (hologram-to-hologram prediction), dual-wavelength phase synthesis (unwrapped phase-to-phase prediction), direct phase-to-index prediction using a single wavelength, hologram-to-phase prediction, and 2D phase unwrapping with sharp discontinuities (wrapped-to-unwrapped phase prediction).

An Oral-mucosa-on-a-chip sensitively evaluates cell responses to dental monomers.

Knowledge of human gingival cell responses to dental monomers is critical for the development of new dental materials. Testing standards have been developed to provide guidelines to evaluate biological functionality of dental materials and devices. However, one shortcoming of the traditional testing platforms is that they do not recapitulate the multi-layered configuration of gingiva, and thus cannot evaluate the layer-specific cellular responses. An oral mucosa-chip with two cell layers was previously developed as an alternative platform to assess the oral mucosa responses to dental biomaterials. The mucosa-chip consists of an apical keratinocyte layer attached to a fibroblast-embedded collagen hydrogel through interconnecting pores in a three-microchannel network. Here, cell responses in the mucosa-chip were evaluated against 2-hydroxyethyl methacrylate (HEMA), a common monomer used in restorative and aesthetic dentistry. The response of mucosal cell viability was evaluated by exposing the chip to HEMA of concentrations ranging from 1.56 to 25 mM and compared to cells in conventional well-plate monoculture. The co-cultured cells were then stained and imaged with epifluorescence and confocal microscopy to determine the layer-specific responses to the treatment. Mucosa-chips were demonstrated to be more sensitive to assess HEMA-altered cell viability than well-plate cultures, especially at lower doses (1.56 and 6.25 mM). The findings suggest that the mucosa-chip is a promising alternative to traditional platforms or assays to test a variety of biomaterials by offering a multi-layered tissue geometry, accessible layer-specific information, and higher sensitivity in detecting cellular responses.

Simulation of digital holographic recording and reconstruction using a generalized matrix method.

In recent years, research efforts in the field of digital holography have expanded significantly, due to the ability to obtain high-resolution intensity and phase images. The information contained in these images have become of great interest to the machine learning community, with applications spanning a wide portfolio of research areas, including bioengineering. In this work, we seek to demonstrate a high-fidelity simulation of holographic recording. By accurately and numerically simulating the propagation of a coherent light source through a series of optical elements and the object itself, we accurately predict the optical interference of the object and reference wave at the recording plane, including diffraction effects, aberrations, and speckle. We show that the optical transformation that predicts the complex field at the recording plane can be generalized for arbitrary holographic recording configurations using a matrix method. In addition, we provide a detailed description of digital phase reconstruction and aberration compensation for a variety of off-axis holographic configurations. Reconstruction errors are presented for the various holographic recording geometries and complex field objects. While the primary objective of this work is not to evaluate phase reconstruction approaches, the reconstruction of simulated holograms provides validation of the generalized simulation method. The long-term goal of this work is that the generalized holographic simulation motivates the use of phase reconstruction of the simulated holograms to populate databases for training machine-learning algorithms aimed at classifying relevant objects recorded through a variety of holographic setups.

Morphology, Motility, and Cytoskeletal Architecture of Breast Cancer Cells Depend on Keratin 19 and Substrate.

Cancer cells gain motility through events that accompany modulation of cell shape and include altered expression of keratins. However, the role of keratins in change of cancer cell architecture is not well understood. Therefore, we ablated the expression of keratin 19 (K19) in breast cancer cells of the MDA-MB-231 cell line and found that cells lacking K19 become more elongated in culture, with morphological reversion toward the parental phenotype upon transduction of KRT19. Also, the number of actin stress fibers and focal adhesions were significantly reduced in KRT19 knockout (KO) cells. The altered morphology of KRT19 KO cells was then characterized quantitatively using digital holographic microscopy (DHM), which not only confirmed the phenotypic change of KRT19 KO cells but also identified that the K19-dependent morphological change is dependent on the substrate type. A new quantitative method of single cell analysis from DHM, via average phase difference maps, facilitated evaluation of K19-substrate interactive effects on cell morphology. When plated on collagen substrate, KRT19 KO cells were less elongated and resembled parental cells. Assessing single cell motility further showed that while KRT19 KO cells moved faster than parental cells on a rigid surface, this increase in motility became abrogated when cells were plated on collagen. Overall, our study suggests that K19 inhibits cell motility by regulating cell shape in a substrate-dependent manner. Thus, this study provides a potential basis for the altered expression of keratins associated with change in cell shape and motility of cancer cells. © 2020 International Society for Advancement of Cytometry.

Microstructural densification and alignment by aspiration-ejection influence cancer cell interactions with three-dimensional collagen networks.

Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.

Microfluidic fabrication of stable collagen microgels with aligned microstructure using flow-driven co-deposition and ionic gelation.

The controlled biofabrication of stable, aligned collagen hydrogels within microfluidic devices is critically important to the design of more physiologically accurate, longer-cultured on-chip models of tissue and organs. To address this goal, collagen-alginate microgels were formed in a microfluidic channel by calcium crosslinking of a flowing collagen-alginate solution through a cross-channel chitosan membrane spanning a pore allowing ion diffusion but not convection. The gels formed within seconds as isolated islands in a single channel, and their growth was self-limiting. Total gel thickness was controlled by altering the concentration of calcium and collagen-alginate flow rate to reach an equilibrium of calcium diffusion and solution convection at the gel boundary, for a desired thickness of 30-200 µm. Additionally, less calcium and higher flow produced greater compression of the gel, with regions farther from the pore compressing more. An aligned, stable collagen network was demonstrated by collagen birefringence, circumferential texture orientation, and little change in gel dimensions with de-chelation of calcium from alginate by prolonged flow of EDTA in the channel. Resultant gels were most stable and only slightly asymmetric when formed from solutions containing 8 mg ml-1 collagen. Diffusion of 4 kDa and 70 kDa fluorescently-labeled dextran indicated size-dependent diffusion across the gel, and accessibility of the construct to appropriately-sized bioactive molecules. This work demonstrates the physicochemical parameter control of collagen gel formation in microfluidic devices, with utility toward on-chip models of dense extracellular matrix invasion, cancer growth and drug delivery to cells within dense extracellular matrix bodies.

Machine Learning with Optical Phase Signatures for Phenotypic Profiling of Cell Lines.

Robust and reproducible profiling of cell lines is essential for phenotypic screening assays. The goals of this study were to determine robust and reproducible optical phase signatures of cell lines for classification with machine learning and to correlate optical phase parameters to motile behavior. Digital holographic microscopy (DHM) reconstructed phase maps of cells from two pairs of cancer and non-cancer cell lines. Seventeen image parameters were extracted from each cell's phase map, used for linear support vector machine learning, and correlated to scratch wound closure and Boyden chamber chemotaxis. The classification accuracy was between 90% and 100% for the six pairwise cell line comparisons. Several phase parameters correlated with wound closure rate and chemotaxis across the four cell lines. The level of cell confluence in culture affected phase parameters in all cell lines tested. Results indicate that optical phase features of cell lines are a robust set of quantitative data of potential utility for phenotypic screening and prediction of motile behavior. © 2019 International Society for Advancement of Cytometry.

Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning.

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility. © 2017 International Society for Advancement of Cytometry.

Electrical Programming of Soft Matter: Using Temporally Varying Electrical Inputs To Spatially Control Self Assembly.

The growing importance of hydrogels in translational medicine has stimulated the development of top-down fabrication methods, yet often these methods lack the capabilities to generate the complex matrix architectures observed in biology. Here we show that temporally varying electrical signals can cue a self-assembling polysaccharide to controllably form a hydrogel with complex internal patterns. Evidence from theory and experiment indicate that internal structure emerges through a subtle interplay between the electrical current that triggers self-assembly and the electrical potential (or electric field) that recruits and appears to orient the polysaccharide chains at the growing gel front. These studies demonstrate that short sequences (minutes) of low-power (∼1 V) electrical inputs can provide the program to guide self-assembly that yields hydrogels with stable, complex, and spatially varying structure and properties.

Automatic phase aberration compensation for digital holographic microscopy based on deep learning background detection.

We propose a fully automatic technique to obtain aberration free quantitative phase imaging in digital holographic microscopy (DHM) based on deep learning. The traditional DHM solves the phase aberration compensation problem by manually detecting the background for quantitative measurement. This would be a drawback in real time implementation and for dynamic processes such as cell migration phenomena. A recent automatic aberration compensation approach using principle component analysis (PCA) in DHM avoids human intervention regardless of the cells' motion. However, it corrects spherical/elliptical aberration only and disregards the higher order aberrations. Traditional image segmentation techniques can be employed to spatially detect cell locations. Ideally, automatic image segmentation techniques make real time measurement possible. However, existing automatic unsupervised segmentation techniques have poor performance when applied to DHM phase images because of aberrations and speckle noise. In this paper, we propose a novel method that combines a supervised deep learning technique with convolutional neural network (CNN) and Zernike polynomial fitting (ZPF). The deep learning CNN is implemented to perform automatic background region detection that allows for ZPF to compute the self-conjugated phase to compensate for most aberrations.

HistoMosaic Detecting KRAS G12V Mutation Across Colorectal Cancer Tissue Slices through in Situ PCR.

We report on HistoMosaic, a novel technique for genetic analysis of formalin-fixed, paraffin-embedded tissue slices. It combines microfluidic compartmentalization, in situ allele-specific PCR, and fluorescence microscopy. The experimental proof of principle was achieved by in situ detection of KRAS G12V mutation in colorectal cancer tissues and is presented herein. HistoMosaic offers the ability to detect mutations over the entire tissue slide simultaneously, rapidly, economically, and without selection bias, while coregistering the genetic information with the preserved morphological information. Thus, HistoMosaic has wide applicability in basic science as a tool to map genetic heterogeneity. It is also a platform to build companion diagnostics for targeted therapies in oncology, to help ensure that the right drug is given to the right patient, thereby saving healthcare resources and improving patient outcomes.

Accurate quantitative phase digital holographic microscopy with single- and multiple-wavelength telecentric and nontelecentric configurations.

In this work, we investigate, both theoretically and experimentally, single-wavelength and multiwavelength digital holographic microscopy (DHM) using telecentric and nontelecentric configurations in transmission and reflection modes. A single-wavelength telecentric imaging system in DHM was originally proposed to circumvent the residual parabolic phase distortion due to the microscope objective (MO) in standard nontelecentric DHM configurations. However, telecentric configurations cannot compensate for higher order phase aberrations. As an extension to the telecentric and nontelecentric arrangements in single-wavelength DHM (SW-DHM), we propose multiple-wavelength telecentric DHM (MW-TDHM) in reflection and transmission modes. The advantages of MW-TDHM configurations are to extend the vertical measurement range without phase ambiguity and optically remove the parabolic phase distortion caused by the MO in traditional MW-DHM. These configurations eliminate the need for a second reference hologram to subtract the two-phase maps and make digital automatic aberration compensation easier to apply compared to nontelecentric configurations. We also discuss a reconstruction algorithm that eliminates the zero-order and virtual images using spatial filtering and another algorithm that minimizes the intensity of fluctuations using apodization. In addition, we employ two polynomial models using 2D surface fitting to compensate digitally for chromatic aberration (in the multiwavelength case) and for higher order phase aberrations. A custom-developed user-friendly graphical user interface is employed to automate the reconstruction processes for all configurations. Finally, TDHM is used to visualize cells from the highly invasive MDA-MB-231 cultured breast cancer cells.

Recombinant Human Keratinocyte Growth Factor Ameliorates Cancer Treatment-Induced Oral Mucositis on a Chip.

Oral mucositis (OM) is a severe complication of cancer therapies caused by off-target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM-OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane-lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM-OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32-36h. Presence of KGF in OM-OC protected tissues from damage caused by cancer treatments in a dose-dependent manner, being more effective at 10 ng/mL. As verified by F-actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM-OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL-8 and TNF-a expression at Day 12 and 18, and TGF-ß1 at Day 18 in OM-OC. Taken together, these findings support the utility of OM-OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.

Programmable Physical Properties of Freestanding Chitosan Membranes Electrofabricated in Microfluidics.

Microfluidic-integrated freestanding membranes with suitable biocompatibility and tunable physicochemical properties are in high demand for a wide range of life science and biological studies. However, there is a lack of facile and rapid methods to integrate such versatile membranes into microfluidics. A recently invented interfacial electrofabrication of chitosan membranes offers an in-situ membrane integration strategy that is flexible, controllable, simple, and biologically friendly. In this follow-up study, we explored the ability to program the physical properties of these chitosan membranes by varying the electrofabrication conditions (e.g., applied voltage and pH of alginate). We found a strong association between membrane growth rate, properties, and fabrication parameters: high electrical stimuli and pH of alginate resulted in high optical retardance and low permeability, and vice versa. This suggests that the molecular alignment and density of electrofabricated chitosan membranes could be actively tailored according to application needs. Lastly, we demonstrated that this interfacial electrofabrication could easily be expanded to produce chitosan membrane arrays with higher uniformity than the previously well-established flow assembly method. This study demonstrates the tunability of the electrofabricated membranes' properties and functionality, thus expanding the utility of such membranes for broader applications in the future.

Digital Holographic Microscopy to Assess Cell Behavior.

Digital holographic microscopy is an imaging technique particularly well suited to the study of living cells in culture, as no labeling is required and computed phase maps produce high contrast, quantitative pixel information. A full experiment involves instrument calibration, cell culture quality checks, selection and setup of imaging chambers, a sampling plan, image acquisition, phase and amplitude map reconstruction, and parameter map post-processing to extract information about cell morphology and/or motility. Each step is described below, focusing on results from imaging four human cell lines. Several post-processing approaches are detailed, with an aim of tracking individual cells and dynamics of cell populations.

Oral mucositis on a chip: modeling induction by chemo- and radiation treatments and recovery.

Oral mucositis (OM) is a debilitating complication affecting roughly 70% of head and neck cancer patients receiving chemotherapy and/or radiation treatment. No broadly effective preventative treatment for OM exists. Therefore, anin vitromodel of cancer treatment-induced OM would aid studies into possible origins of the pathology and future drug targets to ameliorate it. In this study, we present a microfluidic oral mucosa triculture tissue construct consisting of a keratinocyte layer attached to a subepithelial fibroblast and endothelial cell-embedded collagen gel. To address the typically low stability of mucosal constructs in microfluidics, ruthenium-catalyzed photocrosslinking was implemented to strengthen the collagen gel and prevent the invasion of keratinocytes, thus maintaining tissue construct geometry and oral mucosa barrier function for over 18 d of culture. Next, the OM chip was exposed to cisplatin (day 10) and damaging radiation (day 11, ± cisplatin at day 10), mimicking damage from cancer therapy. Damage to and then recovery of the tissue layers and function were observed over days 11-18. Therefore, several important features of OM induction and resolution were modeled in microfluidic culture. The OM model on a chip allows for more sophisticated studies into mechanisms of OM and potential treatments.