Constructing Smartphone-Controlled Optogenetic Switches in Mammalian Cells.

With the increasing indispensable role of smartphones in our daily lives, the mobile health care system coupled with embedded physical sensors and modern communication technologies make it an attractive technology for enabling the remote monitoring of an individual's health. Using a multidisciplinary design principle coupled with smart electronics, software, and optogenetics, the investigators constructed smartphone-controlled optogenetic switches to enable the ultraremote-control transgene expression. A custom-designed SmartController system was programmed to process wireless signals from smartphones, enabling the regulation of therapeutic outputs production by optically engineered cells via a far-red light (FRL)-responsive optogenetic interface. In the present study, the investigators describe the details of the protocols for constructing smartphone-controlled optogenetic switches, including the rational design of an FRL-triggered transgene expression circuit, the procedure for cell culture and transfection, the implementation of the smartphone-controlled far-red light-emitting diode (LED) module, and the reporter detection assay.

Straightforward neuron micropatterning and neuronal network construction on cell-repellent polydimethylsiloxane using microfluidics-guided functionalized Pluronic modification.

Neuronal cell microengineering involving micropatterning and polydimethylsiloxane (PDMS) microfluidics enables promising advances in microscale neuron control. However, a facile methodology for the precise and effective manipulation of neurons on a cell-repellent PDMS substrate remains challenging. Herein, a simple and straightforward strategy for neuronal cell patterning and neuronal network construction on PDMS based on microfluidics-assisted modification of functionalized Pluronic is described. The cell patterning process simply involves a one-step microfluidic modification and routine in vitro culture. It is demonstrated that multiple types of neuronal cell arrangements with various spatial profiles can be conveniently produced using this patterning tool. The precise control of neuronal cells with high patterning fidelity up to single cell resolution, as well as high adhesion and differentiation, is achieved too. Furthermore, neuronal network construction using the respective cell population and single cell patterning prove to be applicable. This achievement provides a convenient and feasible methodology for engineering neuronal cells on PDMS substrates, which will be useful for applications in many neuron-related microscale analytical research fields, including cell engineering, neurobiology, neuropharmacology, and neuronal sensing.

Ca2+-Mediated Surface Polydopamine Engineering to Program Dendritic Cell Maturation.

Engineering of cell surfaces holds promise in manipulating cellular activities in a physicochemical route as a complement to the biological approach. Mediated by Ca2+, a quick and convenient yet cytocompatible method is used to achieve surface engineering, by which polydopamine nanostructures can be in situ grown onto dendritic cell (DC) surfaces within 10 min. Ca2+, as the physical bridge between the negative cell surface and polydopamine, avoids the direct chemical polymerization of polydopamine onto the cell surface, critically important to maintain the cell viability. As a proof of concept in potential applications, this cell surface engineering shows a good control toward DC maturation. Upon surface polydopamine engineering, bone-marrow-derived DC exhibits a unique bidirectional control of maturation. The polydopamine structure enables effective suppression of DC activation by acting as an efficient scavenger of reactive oxygen species, a key signal during maturation. Conversely, an 808 nm laser irradiation can remotely relieve the suppressed state and effectively activate DC maturation by the photoheat effect of polydopamine (39 °C). The work provides an easily implemented, straightforward approach to achieve cell surface engineering, through which the DC maturation can be controlled.

In Vitro Differentiation of T Cell: From CAR-Modified T-iPSC.

T cells engineered to express chimeric antigen receptor (CAR) against the B cell antigen CD19 are achieving remarkable clinical effects on hematological malignancies. Allogeneic transplantation approach is promising for broaden application of CART therapy. iPSCs are one of the ideal cell sources for this approach. CAR-engineered iPSCs are demonstrated to give rise to CAR-engineered T cell and exert their effector function. In this section, we describe the method to generate CAR-engineered iPSCs and differentiate them into T cells.

A miniaturized optical tomography platform for volumetric imaging of engineered living systems.

Volumetric optical microscopy approaches that enable acquisition of three-dimensional (3D) information from a biological sample are attractive for numerous non-invasive imaging applications. The unprecedented structural details that these techniques provide have helped in our understanding of different aspects of architecture of cells, tissues, and organ systems as they occur in their natural states. Nonetheless, the instrumentation for most of these techniques is sophisticated, bulky, and costly, and is less affordable to most laboratory settings. Several miniature imagers based on webcams or low-cost sensors featuring easy assembly have been reported, for in situ imaging of biological structures at low costs. However, they have not been able to achieve the ability of 3D imaging throughout the entire volumes for spatiotemporal analyses of the structural changes in these specimens. Here we present a miniaturized optical tomography (mini-Opto) platform for low-cost, volumetric characterization of engineered living systems through hardware optimizations as well as applications of an optimized algebraic algorithm for image reconstruction.

Generation of CAR-T Cells for Cancer Immunotherapy.

T cells engineered with chimeric antigen receptors (CARs) are emerging as powerful cancer immunotherapies. Remarkable efficacies have been demonstrated in treating B-cell malignancies with CAR-T cells, leading to the FDA's first approval of gene therapy. Currently, numerous clinical trials for hematological malignancies and solid tumors are underway worldwide. Production of CAR-T cells with proper qualities is essential for CAR-T success in vivo. Here we detail optimized protocols for the generation of CAR-T cells for preclinical studies using lentiviral gene transfer, expansion of CAR-T cells in culture, detection of CAR expression, and evaluation of CAR-T cellular cytotoxicity in vitro.

Engineering cell heterogeneity into organs-on-a-chip.

Organ-on-a-chip development is an application that will benefit from advances in cell heterogeneity characterization because these culture models are intended to mimic in vivo microenvironments, which are complex and dynamic. Due in no small part to advances in microfluidic single cell analysis methods, cell-to-cell variability is an increasingly understood feature of physiological tissues, with cell types from as common as 1 out of every 2 cells to as rare as 1 out of every 100 000 cells having important roles in the biochemical and biological makeup of tissues and organs. Variability between neighboring cells can be transient or maintained, and ordered or stochastic. This review covers three areas of well-studied cell heterogeneity that are informative for organ-on-a-chip development efforts: tumors, the lung, and the intestine. Then we look at how recent single cell analysis strategies have enabled better understanding of heterogeneity within in vitro and in vivo tissues. Finally, we provide a few work-arounds for adapting current on-chip culture methods to better mimic physiological cell heterogeneity including accounting for crucial rare cell types and events.

Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples.

Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells.

With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena in vitro - disentangled from the complex environment of a cell. Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature's own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.

Bioprocessing automation in cell therapy manufacturing: Outcomes of special interest group automation workshop.

Phacilitate held a Special Interest Group workshop event in Edinburgh, UK, in May 2017. The event brought together leading stakeholders in the cell therapy bioprocessing field to identify present and future challenges and propose potential solutions to automation in cell therapy bioprocessing. Here, we review and summarize discussions from the event. Deep biological understanding of a product, its mechanism of action and indication pathogenesis underpin many factors relating to bioprocessing and automation. To fully exploit the opportunities of bioprocess automation, therapeutics developers must closely consider whether an automation strategy is applicable, how to design an 'automatable' bioprocess and how to implement process modifications with minimal disruption. Major decisions around bioprocess automation strategy should involve all relevant stakeholders; communication between technical and business strategy decision-makers is of particular importance. Developers should leverage automation to implement in-process testing, in turn applicable to process optimization, quality assurance (QA)/ quality control (QC), batch failure control, adaptive manufacturing and regulatory demands, but a lack of precedent and technical opportunities can complicate such efforts. Sparse standardization across product characterization, hardware components and software platforms is perceived to complicate efforts to implement automation. The use of advanced algorithmic approaches such as machine learning may have application to bioprocess and supply chain optimization. Automation can substantially de-risk the wider supply chain, including tracking and traceability, cryopreservation and thawing and logistics. The regulatory implications of automation are currently unclear because few hardware options exist and novel solutions require case-by-case validation, but automation can present attractive regulatory incentives.

Human Neurospheroid Arrays for In Vitro Studies of Alzheimer's Disease.

Neurospheroids are commonly used for in vitro disease modeling and drug screening. However, the heterogeneity in size of the neurospheroids mixtures available through current methods limits their utility when employed for basic mechanistic studies of neurodegenerative diseases or screening for new interventions. Here, we generate neurospheroids from immortalized neural progenitor cells and human induced pluripotent stem cells that are uniform in size, into large-scale arrays. In proof of concept experiments, we validate the neurospheroids array as a sensitive and robust tool for screening compounds over extended time. We show that when suspended in three-dimensional extracellular matrix up to several weeks, the stem cell-derived neurospheroids display extensive neurite outgrowth and extend thick bundles of dendrites outward. We also cultivate genetically-engineered stem cell-derived neurospheroids with familial Alzheimer's disease mutations for eight weeks in our microarray system. Interestingly, we observed robust accumulation of amyloid-ß and phosphorylated tau, key hallmarks of Alzheimer's disease. Overall, our in vitro model for engineering neurospheroid arrays is a valuable tool for studying complex neurodegenerative diseases and accelerating drug discovery.

Pneumatically actuated cell-stretching array platform for engineering cell patterns in vitro.

Cellular response to mechanical stimuli is a well-known phenomenon known as mechanotransduction. It is widely accepted that mechanotransduction plays an important role in cell alignment which is critical for cell homeostasis. Although many approaches have been developed in recent years to study the effect of external mechanical stimuli on cell behaviour, most of them have not explored the ability of mechanical stimuli to engineer cell alignment to obtain patterned cell cultures. This paper introduces a simple, yet effective pneumatically actuated 4 × 2 cell stretching array for concurrently inducing a range of cyclic normal strains onto cell cultures to achieve predefined cell alignment. We utilised a ring-shaped normal strain pattern to demonstrate the growth of in vitro patterned cell cultures with predefined circumferential cellular alignment. Furthermore, to ensure the compatibility of the developed cell stretching platform with general tools and existing protocols, the dimensions of the developed cell-stretching platform follow the standard F-bottom 96-well plate. In this study, we report the principle design, simulation and characterisation of the cell-stretching platform with preliminary observations using fibroblast cells. Our experimental results of cytoskeleton reorganisation such as perpendicular cellular alignment of the cells to the direction of normal strain are consistent with those reported in the literature. After two hours of stretching, the circumferential alignment of fibroblast cells confirms the capability of the developed system to achieve patterned cell culture. The cell-stretching platform reported is potentially a useful tool for drug screening in 2D mechanobiology experiments, tissue engineering and regenerative medicine.

A Microfluidic Biodisplay.

Synthetically engineered cells are powerful and potentially useful biosensors, but it remains problematic to deploy such systems due to practical difficulties and biosafety concerns. To overcome these hurdles, we developed a microfluidic device that serves as an interface between an engineered cellular system, environment, and user. We created a biodisplay consisting of 768 individually programmable biopixels and demonstrated that it can perform multiplexed, continuous sampling. The biodisplay detected 10 µg/L sodium-arsenite in tap water using a research grade fluorescent microscope, and reported arsenic contamination down to 20 µg/L with an easy to interpret "skull and crossbones" symbol detectable with a low-cost USB microscope or by eye. The biodisplay was designed to prevent release of chemical or biological material to avoid environmental contamination. The microfluidic biodisplay thus provides a practical solution for the deployment and application of engineered cellular systems.

Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development.

In non-alcoholic steatohepatitis (NASH), hepatic stellate cells (HSC) differentiate into myofibroblast-like cells that cause fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. Thus, modeling interactions between activated HSCs and hepatocytes in vitro can aid in the development of anti-NASH/fibrosis therapeutics and lead to a better understanding of disease progression. Species-specific differences in drug metabolism and disease pathways now necessitate the supplementation of animal studies with data acquired using human liver models; however, current models do not adequately model the negative effects of primary human activated HSCs on the phenotype of otherwise well-differentiated primary human hepatocytes (PHHs) as in vivo. Therefore, here we first determined the long-term effects of primary human activated HSCs on PHH phenotype in a micropatterned co-culture (MPCC) platform while using 3T3-J2 murine embryonic fibroblasts as the control cell type since it has been shown previously to stabilize PHH functions for 4-6 weeks. We found that HSCs were not able to stabilize the PHH phenotype to the same magnitude and longevity as the fibroblasts, which subsequently inspired the development of a micropatterned tri-culture (MPTC) platform in which (a) micropatterned PHHs were functionally stabilized using fibroblasts, and (b) the PHH phenotype was modulated by culturing HSCs within the fibroblast monolayer at physiologically-relevant ratios with PHHs. Transwell inserts containing HSCs were placed atop MPCCs containing fibroblasts to confirm the effects of paracrine signaling between PHHs and HSCs. We found that while albumin and urea secretions were relatively similar in MPTCs and MPCCs (suggesting well-differentiated PHHs), increasing HSC numbers within MPTCs downregulated hepatic cytochrome-P450 (2A6, 3A4) and transporter activities, and caused steatosis over 2 weeks. Furthermore, MPTCs secreted higher levels of pro-inflammatory interleukin-6 (IL-6) cytokine and C-reactive protein (CRP) than MPCCs. Treatment of MPCCs with HSC-conditioned culture medium confirmed that HSC secretions mediate the altered phenotype of PHHs observed in MPTCs, partly via IL-6 signaling. Lastly, we found that NADPH oxidase (NOX) inhibition and farnesoid X receptor (FXR) activation using clinically relevant drugs alleviated hepatic dysfunctions in MPTCs. In conclusion, MPTCs recapitulate symptoms of NASH- and early fibrosis-like dysfunctions in PHHs and have utility for drug discovery in this space.

Manufacturing Cell Therapies Using Engineered Biomaterials.

Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tunable properties offer an intriguing tool to provide or deliver cues to retain stemness, direct differentiation, promote reprogramming, manipulate the genome, or select functional phenotypes. This review discusses the use of engineered biomaterials to control human cell manufacturing. Future work exploiting engineered biomaterials has the potential to generate manufacturing processes that produce standardized cells with well-defined critical quality attributes appropriate for clinical testing.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Cell Processing Engineering for Regenerative Medicine : Noninvasive Cell Quality Estimation and Automatic Cell Processing.

The cell processing engineering including automatic cell processing and noninvasive cell quality estimation of adherent mammalian cells for regenerative medicine was reviewed. Automatic cell processing necessary for the industrialization of regenerative medicine was introduced. The cell quality such as cell heterogeneity should be noninvasively estimated before transplantation to patient, because cultured cells are usually not homogeneous but heterogeneous and most protocols of regenerative medicine are autologous system. The differentiation level could be estimated by two-dimensional cell morphology analysis using a conventional phase-contrast microscope. The phase-shifting laser microscope (PLM) could determine laser phase shift at all pixel in a view, which is caused by the transmitted laser through cell, and might be more noninvasive and more useful than the atomic force microscope and digital holographic microscope. The noninvasive determination of the laser phase shift of a cell using a PLM was carried out to determine the three-dimensional cell morphology and estimate the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. The noninvasive discrimination of cancer cells from normal cells by measuring the phase shift was performed based on the difference in cytoskeleton density. Chemical analysis of the culture supernatant was also useful to estimate the differentiation level of a cell population. A probe beam, an infrared beam, and Raman spectroscopy are useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.

Directing the spatial patterning of motor neuron differentiation in engineered microenvironments.

Embryonic development of the spinal cord proceeds through a carefully orchestrated temporal and spatial sequence of chemical cues to provide precise patterning of adult cell types. Recreating this complex microenvironment in a standard cell culture dish is difficult, if not impossible. In this paper, a microfluidic device is used to recapitulate, in vitro, the graded patterning events which occur during early spinal cord development. The microdevice design is developed using COMSOL modeling, with which the spatiotemporal profiles of multiple, diffusible morphogens are simulated. Four independently addressed source/sinks are employed to generate two overlapping orthogonal gradients within a cell culture chamber, mimicking the dorsoventral and anteroposterior axes of the developing embryo. Mouse embryonic stem cells are directed therein to differentiate into motor neurons in a spatially organized manner, reminiscent of a neural tube.