Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

Benzene Derivatives Analysis Using Aluminum Nitride Waveguide Raman Sensors.

Raman spectroscopy using aluminum nitride (AlN) optical waveguides was demonstrated for organic compound analysis. The AlN waveguide device was prepared by reactive sputtering deposition and complementary-metal-oxide semiconductor (CMOS) processes. A fundamental waveguide mode was observed over a broad visible spectrum and the waveguide evanescent wave was used to excite the Raman signals of the test analytes. The performance of the waveguide sensor was characterized by measuring the Raman spectra of the benzene derivative mixtures consisting of benzene, anisole, and toluene. The compositions and concentrations were resolved by correlating the obtained Raman spectrum with the characteristic Raman peaks associated with C-C, C-H, and C-O functional groups. With the advantages of real-time detection and enhanced Raman signal intensity, the AlN waveguides provided a sensor platform for nondestructive and online chemical compound monitoring.

Light scattering by pulmonary alveoli and airway surface liquid using a concentric sphere model.

We employ a concentric sphere Mie scattering model to describe light scattering by pulmonary alveoli and airway surface liquid (ASL). Using this layered sphere model, we compare alveolar scattering at different points along the respiratory cycle and observe the effect of ASL thickness on light scattering in the lung. We have also extrapolated the model to investigate alveolar scattering in various animal models of pulmonary disease. This model of pulmonary light scattering can estimate in vivo optical properties for normal and pathological states, potentially aiding the design of optical systems for diagnosis and investigation of pulmonary pathologies.

Volumetric structured illumination microscopy enabled by a tunable-focus lens.

We present a mechanical-scan-free method for volumetric imaging of biological tissue. The optical sectioning is provided by structured illumination, and the depth of the imaging plane is varied using an electrically tunable-focus lens. We characterize and evaluate the ability of this axial-scanning mechanism in structured illumination microscopy and demonstrate its ability to perform subcellular resolution imaging in oral mucosa ex vivo. The proposed mechanism can potentially convert any wide-field microscope to a 3D-imaging platform without the need for mechanical scanning of imaging optics and/or sample.

Estimation of the number of fluorescent end-members for quantitative analysis of multispectral FLIM data.

Multispectral fluorescence lifetime imaging (m-FLIM) can potentially allow identifying the endogenous fluorophores present in biological tissue. Quantitative description of such data requires estimating the number of components in the sample, their characteristic fluorescent decays, and their relative contributions or abundances. Unfortunately, this inverse problem usually requires prior knowledge about the data, which is seldom available in biomedical applications. This work presents a new methodology to estimate the number of potential endogenous fluorophores present in biological tissue samples from time-domain m-FLIM data. Furthermore, a completely blind linear unmixing algorithm is proposed. The method was validated using both synthetic and experimental m-FLIM data. The experimental m-FLIM data include in-vivo measurements from healthy and cancerous hamster cheek-pouch epithelial tissue, and ex-vivo measurements from human coronary atherosclerotic plaques. The analysis of m-FLIM data from in-vivo hamster oral mucosa identified healthy from precancerous lesions, based on the relative concentration of their characteristic fluorophores. The algorithm also provided a better description of atherosclerotic plaques in term of their endogenous fluorophores. These results demonstrate the potential of this methodology to provide quantitative description of tissue biochemical composition.

Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells.

Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.

Flexible endoscope for continuous in vivo multispectral fluorescence lifetime imaging.

Fluorescence lifetime imaging (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. There has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed endoscopy systems. Unfortunately, the predominant multispectral FLIM approaches suffer from limitations that impede the development of endoscopy systems that are suitable for in vivo tissue imaging. We present a compact wide-field time-gated FLIM flexible endoscope capable of continuous lifetime imaging of up to three fluorescence emission bands simultaneously. This endoscope design will facilitate the evaluation of FLIM for in vivo applications.

Exploring the Clinical Utility of Raman Spectroscopy for Point-of-Care Cardiovascular Disease Biomarker Detection.

A variety of innovative point-of-care (POC) solutions using Raman systems have been explored. However, the vast effort is in assay development, while studies of the characteristics required for Raman spectrometers to function in POC applications are lacking. In this study, we tested and compared the performance of eight commercial Raman spectrometers ranging in size from benchtop Raman microscopes to portable and handheld Raman spectrometers using paper fluidic cartridges, including their ability to detect cardiac troponin I and heart fatty acid binding protein, both of which are well-established biomarkers for evaluating cardiovascular health. Each spectrometer was evaluated in terms of excitation wavelength, laser characteristics, and ease of use to investigate POC utility. We found that the Raman spectrometers equipped with 780 and 785 nm laser sources exhibited a reduced background signal and provided higher sensitivity compared to those with 633 and 638 nm laser sources. Furthermore, the spectrometer equipped with the single acquisition line readout functionality showed improved performance when compared to the point scan spectrometers and allowed measurements to be made faster and easier. The portable and handheld spectrometers also showed similar detection sensitivity to the gold standard instrument. Lastly, we reduced the laser power for the spectrometer with single acquisition line readout capability to explore the system performance at a laser power that change the classification from a Class 3B laser device to a Class 3R device and found that it showed comparable performance. Overall, these findings show that portable Raman spectrometers have the potential to be used in POC settings with accuracy comparable to laboratory-grade instruments, are relatively low-cost, provide fast signal readout, are easy to use, and can facilitate access for underserved communities.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth.

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Portable, multi-modal Raman and fluorescence spectroscopic platform for point-of-care applications.

Significance: Point-of-care (POC) platforms utilizing optical biosensing strategies can achieve on-site detection of biomarkers to improve the quality of care for patients in low-resource settings. Aim: We aimed to develop a portable, multi-modal spectroscopic platform capable of performing Raman and fluorescence measurements from a single sample site. Approach: We designed the spectroscopic platform in OpticStudio using commercial optical components and built the system on a portable optical breadboard. Two excitation and collection arms were utilized to detect the two optical signals. The multi-modal functionality was validated using ratiometric Raman/fluorescence samples, and the potential utility was demonstrated using a model bioassay for cardiac troponin I. Results: The designed spectroscopic platform achieved a spectral resolution of 0.67 ± 0.2 nm across the Raman detection range (660 to 770 nm). The ratiometric Raman/fluorescence samples demonstrated no crosstalk between the two detector arms across a gradient of high molar concentrations. Testing of the model bioassay response showed that the integrated approach improved the linearity of the calibration curve from (R2 = 0.977) for the Raman only and (R2 = 0.972) for the fluorescence only to (R2 = 0.988) for the multi-modal approach. Conclusion: These findings demonstrate the potential impact of a multi-modal POC spectroscopic platform to improve the sensitivity and robustness necessary for biomarker detection.

In vivo microscopy as an adjunctive tool to guide detection, diagnosis, and treatment.

SIGNIFICANCE: There have been numerous academic and commercial efforts to develop high-resolution in vivo microscopes for a variety of clinical use cases, including early disease detection and surgical guidance. While many high-profile studies, commercialized products, and publications have resulted from these efforts, mainstream clinical adoption has been relatively slow other than for a few clinical applications (e.g., dermatology). AIM: Here, our goals are threefold: (1) to introduce and motivate the need for in vivo microscopy (IVM) as an adjunctive tool for clinical detection, diagnosis, and treatment, (2) to discuss the key translational challenges facing the field, and (3) to propose best practices and recommendations to facilitate clinical adoption. APPROACH: We will provide concrete examples from various clinical domains, such as dermatology, oral/gastrointestinal oncology, and neurosurgery, to reinforce our observations and recommendations. RESULTS: While the incremental improvement and optimization of IVM technologies should and will continue to occur, future translational efforts would benefit from the following: (1) integrating clinical and industry partners upfront to define and maintain a compelling value proposition, (2) identifying multimodal/multiscale imaging workflows, which are necessary for success in most clinical scenarios, and (3) developing effective artificial intelligence tools for clinical decision support, tempered by a realization that complete adoption of such tools will be slow. CONCLUSIONS: The convergence of imaging modalities, academic-industry-clinician partnerships, and new computational capabilities has the potential to catalyze rapid progress and adoption of IVM in the next few decades.

Quantifiable Intravital Light Sheet Microscopy.

Live imaging of zebrafish embryos that maintains normal development can be difficult to achieve due to a combination of sample mounting, immobilization, and phototoxicity issues that, once overcome, often still results in image quality sufficiently poor that computer-aided analysis or even manual analysis is not possible. Here, we describe our mounting strategy for imaging the zebrafish midbrain-hindbrain boundary (MHB) with light sheet fluorescence microscopy (LSFM) and pilot experiments to create a study-specific set of parameters for semiautomatically tracking cellular movements in the embryonic midbrain primordium during zebrafish segmentation.

Automated mesenchymal stem cell segmentation and machine learning-based phenotype classification using morphometric and textural analysis.

Purpose: Mesenchymal stem cells (MSCs) have demonstrated clinically relevant therapeutic effects for treatment of trauma and chronic diseases. The proliferative potential, immunomodulatory characteristics, and multipotentiality of MSCs in monolayer culture is reflected by their morphological phenotype. Standard techniques to evaluate culture viability are subjective, destructive, or time-consuming. We present an image analysis approach to objectively determine morphological phenotype of MSCs for prediction of culture efficacy. Approach: The algorithm was trained using phase-contrast micrographs acquired during the early and mid-logarithmic stages of MSC expansion. Cell regions are localized using edge detection, thresholding, and morphological operations, followed by cell marker identification using H-minima transform within each region to differentiate individual cells from cell clusters. Clusters are segmented using marker-controlled watershed to obtain single cells. Morphometric and textural features are extracted to classify cells based on phenotype using machine learning. Results: Algorithm performance was validated using an independent test dataset of 186 MSCs in 36 culture images. Results show 88% sensitivity and 86% precision for overall cell detection and a mean Sorensen-Dice coefficient of 0.849 ± 0.106 for segmentation per image. The algorithm exhibited an area under the curve of 0.816 ( CI 95 = 0.769 to 0.886) and 0.787 ( CI 95 = 0.716 to 0.851) for classifying MSCs according to their phenotype at early and mid-logarithmic expansion, respectively. Conclusions: The proposed method shows potential to segment and classify low and moderately dense MSCs based on phenotype with high accuracy and robustness. It enables quantifiable and consistent morphology-based quality assessment for various culture protocols to facilitate cytotherapy development.

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy.

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis.

From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.

A scalable system for generation of mesenchymal stem cells derived from induced pluripotent cells employing bioreactors and degradable microcarriers.

Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3-fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5-minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost-effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.

Clinical label-free biochemical and metabolic fluorescence lifetime endoscopic imaging of precancerous and cancerous oral lesions.

INTRODUCTION: Incomplete head and neck cancer resection occurs in up to 85% of cases, leading to increased odds of local recurrence and regional metastases; thus, image-guided surgical tools for accurate, in situ and fast detection of positive margins during head and neck cancer resection surgery are urgently needed. Oral epithelial dysplasia and cancer development is accompanied by morphological, biochemical, and metabolic tissue and cellular alterations that can modulate the autofluorescence properties of the oral epithelial tissue. OBJECTIVE: This study aimed to test the hypothesis that autofluorescence biomarkers of oral precancer and cancer can be clinically imaged and quantified by means of multispectral fluorescence lifetime imaging (FLIM) endoscopy. METHODS: Multispectral autofluorescence lifetime images of precancerous and cancerous lesions from 39 patients were imaged in vivo using a novel multispectral FLIM endoscope and processed to generate widefield maps of biochemical and metabolic autofluorescence biomarkers of oral precancer and cancer. RESULTS: Statistical analyses applied to the quantified multispectral FLIM endoscopy based autofluorescence biomarkers indicated their potential to provide contrast between precancerous/cancerous vs. healthy oral epithelial tissue. CONCLUSION: To the best of our knowledge, this study represents the first demonstration of label-free biochemical and metabolic clinical imaging of precancerous and cancerous oral lesions by means of widefield multispectral autofluorescence lifetime endoscopy. Future studies will focus on demonstrating the capabilities of endogenous multispectral FLIM endoscopy as an image-guided surgical tool for positive margin detection during head and neck cancer resection surgery.

Fluorescence modeling of in vivo optical detection of Mycobacterium tuberculosis.

Tuberculosis is one of the deadliest infectious diseases worldwide. New tools to study pathogenesis and monitor subjects in pre-clinical studies to develop treatment regimens are critical for progress. We developed an improved optical system for detecting bacteria in lungs of mice using internal illumination. We present a computational optical model of the full mouse torso to characterize the optical system. Simulated theoretical limits for the lowest detectable bacterial load support the experimental improvements with an internal illumination source, and suggest that protocol improvements could further lower the detection threshold.

An ultraviolet-curable, core-shell vaccine formed via phase separation.

One of the central challenges in the field of vaccine delivery is to develop a delivery method that maintains antigen stability while also enabling control over the system's release kinetics. Addressing these challenges would not only allow for expanded access to vaccines worldwide but would also help significantly reduce mortality rates in developing countries. In this article, we report the development of single-injection vaccine depots for achieving novel delayed burst release. Synthesized poly(ε-caprolactone) and poly(ε-caprolactone) triacrylate were used to form stationary bubbles within an aqueous solution of 10% carboxymethylcellulose. These polymeric bubbles (referred to as "polybubbles") can then be injected with an aqueous solution of cargo, resulting in the formation of a polymeric shell. The puncture resulting from cargo injection self-heals prior to ultraviolet (UV) curing. UV curing and lyophilization were shown to enhance the stability of the polybubbles. BSA- CF 488 and HIV1 gp120/41 were used as the antigen in the study as a proof-of-concept. Further endeavors to automate the production of polybubbles are underway.

Fabrication and characterization of cylindrical light diffusers comprised of shape memory polymer.

We developed a technique for constructing light diffusing devices comprised of a flexible shape memory polymer (SMP) cylindrical diffuser attached to the tip of an optical fiber. The devices are fabricated by casting an SMP rod over the cleaved tip of an optical fiber and media blasting the SMP rod to create a light diffusing surface. The axial and polar emission profiles and circumferential (azimuthal) uniformity are characterized for various blasting pressures, nozzle-to-sample distances, and nozzle translation speeds. The diffusers are generally strongly forward-directed and consistently withstand over 8 W of incident IR laser light without suffering damage when immersed in water. These devices are suitable for various endoluminal and interstitial biomedical applications.