This article mainly describes a fermentation and purification method for expressing recombinant collagen protein in Escherichia coli. The method comprises constructing engineered bacteria expressing human type III collagen and adopting a strategy of feeding in batches for high-density fermentation. The rapid proliferation of bacterial cells is promoted at 37°C, and then the culture is inoculated into a fermentation tank with different carbon sources for growth. When glycerol is used as the main carbon source, the yield of recombinant collagen protein can reach 0.25 to 0.40 g/L. The method allows exploration of the differences in recombinant collagen production with different carbon sources in order to identify the most suitable fermentation medium component. The human type III collagen produced by the method has the typical structure of collagen, with high cell adhesion and the stability of tissue structure. Therefore, it can be used as the raw material for various collagen products, especially facial fillers, dressings, freeze-dried fibers, and gels. © 2025 Wiley Periodicals LLC. Basic Protocol: Fermentation and purification of recombinant human type III collagen expressed in Escherichia coli.
Cancer drug resistance, whether intrinsic or acquired, underlies most relapses and treatment failures. Reliable preclinical models are crucial to define resistance mechanisms and test counterstrategies. This overview article concisely compares three model classes: (1) clinical, patient‑derived xenografts that retain tumor heterogeneity and can mirror patient resistance; (2) induced‑resistance models produced by prolonged drug selection in vivo or in vitro that recapitulate tumor evolution under therapy; and (3) engineered isogenic cell lines that isolate specific resistance drivers. We summarize some key resistance mechanisms revealed and potential therapeutic approaches informed by these models, including rational combinations, mutation‑targeted inhibitors/degraders, efflux/epigenetic modulators, and immune-related combinations. Each model has trade‑offs, but integrating them accelerates mechanistic insight and translational drug development. This overview guides selection of preclinical models and design of strategies to overcome cancer drug resistance. © 2025 Wiley Periodicals LLC.
Tumor-treating fields (TTFields) represent an innovative approach to cancer treatment that leverages low-intensity (1-3 V/cm) alternating electric fields operating at intermediate frequencies (100-300 kHz). These electric fields are specifically designed to disrupt the mitotic process, thereby inhibiting the proliferation of malignant cells. Recent research has shown that TTFields not only induce direct cytotoxic effects but also modulate the immune system by triggering immunogenic cancer cell death, thereby enhancing immune cell infiltration into tumor sites. This dual mechanism opens up new possibilities for synergistic integration with immunotherapy, which has already revolutionized oncology through the advent of immune-checkpoint inhibitors, adoptive cell therapies, and cancer vaccines. Given the immune-modulatory properties of TTFields, there is growing interest in exploring their potential synergistic effect to enhance immunotherapeutic efficacy. Recognizing the complementary role of TTFields in cancer treatment paves the way for innovative combinatorial strategies that may further improve patient outcomes by enhancing the effectiveness of both TTFields and immunotherapy. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.
Unattended endotoxin (ETX) contamination in biological samples constitute a major challenge for in vitro and in vivo applications. Besides being potentially life-threatening, ETX contamination is especially relevant in global transcriptome analyses, where competing ETX stimulation can significantly skew the final gene expression profile. Our studies in mice and cultured skin epithelial cells (epidermal keratinocytes) aiming to characterize the effect of antibodies such as AK23 immunoglobulins (IgG directed against the cell-cell adhesion molecule desmoglein [DSG] 3) in the autoimmune disease pemphigus vulgaris (PV) revealed that laboratory-produced and even commercial control antibodies can exhibit non-negligible ETX contaminations. Moreover, these contaminants are extremely difficult to remove. To overcome these challenges, we have devised a simple yet nontoxic and scalable two-step protocol to efficiently reduce ETX levels during or after the IgG purification process. It consists firstly of 0.5 M NaOH pre-treatment of all devices, including the protein A resin, used during IgG sanitization and purification, in parallel with meticulous in-process monitoring of ETX levels. Secondly, before IgG elution from protein A, ETX is stripped from IgG by ion-exchange with the common amino acid arginine. This two-step approach successfully reduces ETX by >95% from hybridoma-derived, laboratory-produced AK23 IgG, as well as patient PV and control IgG, resulting in an 85% IgG recovery rate and ETX levels compatible with U.S. Pharmacopeia guidelines. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sanitization of devices and protein A resin with 0.5 M NaOH Basic Protocol 2: On-column stripping of ETX from AK23 IgG Basic Protocol 3: Quality control of sanitized AK23 IgG.
Large-scale genomics efforts led to the identification of an increasing number of mutations in various cancers. However, the functional role of a vast majority of these mutations in disease pathogenesis remains unknown. For enzymes whose activity can be blocked by approved drugs, knowledge regarding the effect of novel or uncommon mutations on inhibitor sensitivity helps in opting for effective treatment strategies. However, it is impossible to experimentally evaluate pathogenic effect and drug sensitivity for all mutations that are being identified in multiple diseases. Therefore, computational predictions of pathogenicity and drug sensitivity can potentially help in the design of an individualized treatment approach. This article includes computational methods to: (a) predict the pathogenicity of mutations based on primary and tertiary structures of the target enzyme, (b) study the effect of mutations on protein conformation, and (c) predict the binding affinity of mutant structures towards targeted therapeutics. All the methods utilize freely available computational tools and have considerable translational value in improving patient outcomes with targeted therapy. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Pathogenicity prediction of mutations based on primary and tertiary structures Basic Protocol 2: Homology modeling of mutant protein structures Basic Protocol 3: Understanding the effect of mutations on protein conformation Basic Protocol 4: Predicting the binding affinities of mutant proteins towards specific inhibitors.
Acinetobacter baumannii is a high-risk pathogen associated with increased patient morbidity and mortality. Host-pathogen interactions amplify its virulence, in part by promoting biofilm formation-a crucial factor in antimicrobial resistance and persistence. Given the bacterium's strong propensity for acquiring resistance, antimicrobial susceptibility testing (AST) is essential for guiding effective therapeutic interventions. However, discrepancies have been observed between in vitro AST results and therapeutic outcomes, with some antimicrobials being deemed to show in vivo efficacy despite appearing ineffective in vitro. This discordance may stem from traditional AST protocols, which rely on bacteriological media such as Mueller Hinton broth (MHB) optimized for bacterial growth but not for mimicking the host environment. Moreover, conventional AST does not account for virulence traits such as biofilm formation, which further contribute to treatment failure. Incorporating physiologically relevant culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, alongside assessment of biofilm formation may improve the predictive value of AST. This work outlines two complementary protocols for improving AST interpretation in A. baumannii infections. Basic Protocol 1 compares minimum inhibitory concentration (MIC) values generated using MHB and RPMI. Basic Protocol 2 evaluates biofilm formation in MHB, tryptic soy broth (TSB; control), and RPMI, with and without antimicrobial exposure. Together, these approaches aim to inform alternative AST strategies that better reflect in vivo conditions and optimize therapeutic decision-making. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Comparing A. baumannii minimum inhibitory concentration (MIC) results in bacteriological (MHB) versus physiological (RPMI) media Basic Protocol 2: Comparing A. baumannii isolate(s) biofilm formation following assays completed in bacteriological culture media (MHB and control TSB) and physiological medium (RPMI).
Histone deacetylases (HDACs) are crucial epigenetic regulators involved in the modulation of gene expression and are promising therapeutic targets for treating various diseases, including central nervous system disorders and cancer. HDAC inhibitors exhibit neuroprotective, antiepileptogenic, and antidepressant properties in animal models, underscoring their clinical relevance. Quantifying HDAC activity is essential for identifying inhibitors and evaluating their effects under physiological or pathological conditions. This article outlines a comprehensive, sensitive, and robust assay to quantify HDAC activity in tissue lysates, with specific application to brain tissue. The assay is based on the catalytic removal of an acetyl group from the Boc-Lys(Ac)-AMC substrate by HDAC enzymes. Following nuclear protein extraction, tissue HDAC activity can be quantitatively assessed using a fluorometric Boc-Lys(Ac) HDAC activity kit. Adding a developer containing trypsin then converts the deacetylated product into a measurable fluorophore. This enables precise quantification of HDAC activity levels across different tissues, making the method suitable for screening putative HDAC inhibitors and assessing their effects on epigenetically modulated phenotypes. We validated these protocols using brain tissue samples from mice subjected to traumatic brain injury, a condition known to elevate HDAC activity levels. This assay provides an efficient, scalable tool for exploring HDAC function, evaluating therapeutic interventions, and advancing the understanding of HDAC-mediated mechanisms in animal models. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of nuclear protein from brain and other tissues Support Protocol 1: Harvesting and microdissection of brain and other tissues Support Protocol 2: Estimation of extracted protein using the Pierce bicinchoninic acid (BCA) assay Basic Protocol 2: HDAC activity fluorometric assay in the brain and other tissues.
Gout is a chronic disease caused by the deposition of monosodium urate monohydrate (MSU) crystals within the body, particularly in one or more joints, which can lead to sudden severe attacks of pain, swelling, redness, and tenderness, known as gout flares. Historically termed the "disease of kings," gout is one of the oldest joint diseases and remains the most common form of inflammatory arthritis haunting humans in the 21st century. It is associated with cardiovascular, metabolic, and renal comorbidities and can lead to reduced mobility and impaired physical function and contributing to work absenteeism. Given its increasing global incidence, novel therapies for gouty arthritis disease are urgently needed. Experimental gout models are indispensable tools for deciphering disease pathogenesis and evaluating the efficacy and side effect of newly developed therapeutics at preclinical stage. Herein, we described a series of highly reproducible acute gout flare and air pouch models in rodents and rabbits that can be used to address various scientific questions relevant to pathological changes and immune responses during and after a gout attack. Animal gout flare models, elicited by MSU crystals, mimic the main histopathological features of human gouty arthritis, including damage to cartilage and joint swelling. Meanwhile, air pouch models serve as a tool to evaluate robust inflammatory cytokine secretion and neutrophil infiltration. This article provides a detailed description of procedures and troubleshooting tips required to reproducibly induce gout flare and air pouch models in animals and critically evaluate the pathogenesis of the disease. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Preparation of monosodium urate crystalline Basic Protocol 2: Development of MSU-induced gout flare model in mice Support Protocol 1: Histological assessment of mouse ankle tissues Basic Protocol 3: Development of MSU-induced gout flare model in rats Basic Protocol 4: Development of MSU-induced gout flare model in rabbits Basic Protocol 5: Development and validation of reference articles in MSU-induced air pouch model in rats Basic Protocol 6: Development and validation of reference articles in MSU-induced air pouch model in mice Support Protocol 2: Flow cytometry of mouse neutrophils in air pouch lavage samples.
To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.
Cancer‐associated cachexia is defined by loss of weight and muscle mass, and by the potential loss of adipose tissue accompanied by insulin resistance and increased resting energy expenditure. Cachexia is most prevalent in pancreatic cancer, the third leading cause of cancer‐related deaths. While various factors interact to induce cachexia, the precise mechanisms underlying this clinical condition are not fully understood. Clinically relevant animal models of cachexia are needed given the lack of standard diagnostic methods or treatments for this condition. Described in this article are in vitro and in vivo models used to study the role of macrophages in the induction of cachexia in pancreatic cancer. Included are procedures for isolating and culturing bone marrow−derived macrophages, harvesting tumor‐ and macrophage‐derived conditioned medium, and studying the effect of conditioned medium on C2C12 myotubes. Also described are procedures involving the use of an orthotopic model of pancreatic cancer, including a method for examining skeletal muscle atrophy in this model. © 2020 Wiley Periodicals LLC.
In vitro assessment of topical (dermal) pharmacokinetics is a critical aspect of the drug development process for semi‐solid products (e.g., solutions, foams, sprays, creams, gels, lotions, ointments), allowing for informed selection of new chemical entities, optimization of prototype formulations during the nonclinical stage, and determination of bioequivalence of generics. It can also serve as a tool to further understand the impact of different excipients on drug delivery, product quality, and formulation microstructure when used in parallel with other techniques, such as analyses of rheology, viscosity, microscopic characteristics, release rate, particle size, and oil droplet size distribution. The in vitro permeation test (IVPT), also known as in vitro skin penetration/permeation test, typically uses ex vivo human skin in conjunction with diffusion cells, such as Franz (or vertical) or Bronaugh (or flow‐through) diffusion cells, and is the technique of choice for dermal pharmacokinetics assessment. Successful execution of the IVPT also involves the development and use of fit‐for‐purpose bioanalytical methods and procedures. The protocols described herein provide detailed steps for execution of the IVPT utilizing flow‐through diffusion cells and for key aspects of the development of a liquid chromatography–tandem mass spectrometry method intended for analysis of the generated samples (epidermis, dermis, and receptor solution). © 2020 Wiley Periodicals LLC.
The expanding number of research studies utilizing the imiquimod-induced psoriasiform dermatitis model attests to the usefulness of this procedure. Advantages of this model include rapid development of the skin response and cost-effectiveness. A major limitation is that application of imiquimod cream over large areas of skin, as well as licking and ingestion of the cream, may lead to severe systemic inflammation, which can cause a general decline in health, splenomegaly, and death. In this protocol, Finn chambers are used to localize the imiquimod cream to a small area of the skin. This results in production of severe and reproducible psoriatic skin reactions with significantly less imiquimod, greatly reducing the possibility of untoward systemic effects. Moreover, having psoriasiform and control skin areas on the same mice decreases inter-animal differences. The protocol can be readily adapted for other skin disease models involving topical application of test agents. This article also details functional measurements performed during assays, including skin thickness, blood perfusion, semiquantitative histopathological evaluation, determination of scaling score to monitor psoriatic symptoms, and collection of spleen and body weight data to identify systemic effects. © 2020 The Authors. Basic Protocol: Use of Finn chambers to induce psoriasiform skin reactions with imiquimod Support Protocol 1: Measurement of double-fold dorsal skin thickness Support Protocol 2: Measurement of blood perfusion Support Protocol 3: Determination of scaling score Support Protocol 4: Semiquantitative histopathological scoring Support Protocol 5: Assessment of systemic side effects in response to imiquimod application.
With the rapid approval of immune checkpoint inhibitors for lung, melanoma, breast, genitourinary, and hematological malignancies, the hematopoietic cells in the tumor microenvironment (TME) are now considered an important, if not essential, consideration for cancer scientists. In many instances, syngeneic murine models have not been highly predictive for responsiveness in clinical trials. Our limited understanding of the human TME have, therefore, precluded a rational translation of immunotherapeutic combinations. This has led to the adoption of hematopoietic humanized murine models for the study of human tumor immunology in vivo. However, concerns about chimerism rates, HLA mismatching, and incomplete reconstitution of the innate immune system have driven a quest for improvements in these allogeneic humanized murine systems. Presented in this article is a completely autologous xenotransplantation method for reconstituting the human tumor immune microenvironment in vivo without the use of a patient's peripheral blood which is known to be associated with low engraftment rates. With this new approach, the current limitations of allogeneic humanized models are avoided by using matched bone marrow cells (BMCs) and derived tumor xenoplants (PDXs) from solid tumors in cancer patients. This autologous system provides a platform for studying endogenous lymphocytic and myeloid cell infiltration into the human tumor in vivo. © 2020 Wiley Periodicals LLC. Basic Protocol : Autologous reconstitution of human tumors Support Protocol 1 : Transduction of BMCs and/or tumor cells prior to autologous reconstitution Support Protocol 2 : Modeling immunotherapeutic agents in an autologously humanized model
The clinical success of immune checkpoint modulators and the development of next‐generation immune‐oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug) and triple‐transgenic NOG‐EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV‐IL3, CSF2)], which expresses human IL‐3 and GM‐CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post‐transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T‐cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC‐HIS model can be limited due to onset of xenogeneic graft‐versus‐host disease (xGVHD) within 3 to 5 weeks post‐implantation. Host deficiency in MHC class I, as occurs in beta‐2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood–derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC‐HIS and PBMC‐HIS mouse models are provided. In addition, the co‐engraftment and growth kinetics of patient‐derived and cell line–derived xenograft tumors in humanized mice and recovery of tumor‐infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors.
This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate Vhalf , the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online Vhalf measurement to obtain a concentration response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol. In our hands, the sample reference demonstrated good state- and use-dependent inhibition of Nav1.7. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Comparing Vhalf measurements using offline calculation and a new adaptive online estimation Basic Protocol 2: Concentration response experiments using a state-dependent protocol Basic Protocol 3: Concentration response experiments using a use-dependent protocol Support Protocol: Cell culture protocol.
Ligand-gated ion channels (LGICs) are a group of diverse ion channels that are gated by ligands and play important roles in normal physiological and pathological conditions. Many of them are drug targets that have been pursued, are being pursued, and will likely be pursued in the future by pharmaceutical companies and academic groups for a variety of diseases. One of those LGICs is the GABAA receptor, a heterooligomeric chloride channel that can be blocked and modulated at various sites. In order to study the receptor's functional response to compounds, the manual patch-clamp method provides a detailed but low-throughput electrophysiological characterization. QPatch II, a next-generation automated patch clamp machine that was recently developed by Sophion Bioscience, provides an automated electrophysiological study of ion channels. In this article, we use the GABAA receptor as an example for studying LGICs and describe two detailed protocols for using QPatch II to carry out pharmacological studies on the receptor. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Ligand concentration-response experiment (GABAA receptor) on QPatch II Alternate Protocol: Non-cumulative ligand concentration-response experiment (GABAA receptor) on QPatch II Support Protocol 1: Cell culture of HEK293-hGABAA (α5β3γ2) Support Protocol 2: Data analysis for Basic Protocol 1 Support Protocol 3: Data analysis for Alternate Protocol Basic Protocol 2: Antagonist dose-response experiment (GABAA receptor) on QPatch II Support Protocol 3: Data analysis for Basic Protocol 2.
Automated patch-clamp (APC) systems have become indispensable tools of drug-discovery programs by allowing high-throughput electrophysiology-based screening of ion channel compounds. The recent development and introduction of microfluidics-based APC systems have made it possible to study the interactions of ligand-gated ion channels with pharmacological reagents, such as agonists, antagonists, or positive allosteric modulators (PAMs), with reliable pharmacological results comparable to those of the gold-standard manual patch-clamp technique while maintaining high-throughput capacity. Many ligand-gated ion channels exhibit rapid desensitization upon repetitive introduction of ligands; this loss of channel activity in the absence of pharmacological interaction poses a challenge for developing accurate, precise, and robust assays with high success rate, low run-down, and reliable pharmacological results. Here we present procedures to study nicotinic acetylcholine receptors (nAChRs) with the IonFlux™, an automated patch-clamp system with continuous flow and precise fluidic exchange; these procedures can also be generalized to the study of other ligand-gated ion channels. We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand-gated ion channels, generating data that closely mimic those from manual patch-clamp conditions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring agonist concentration-dependent response Basic Protocol 2: Measuring antagonist concentration-dependent response Basic Protocol 3: Measuring positive allosteric modulator (PAM) concentration-dependent response Support Protocol 1: Basic IonFlux system operation Support Protocol 2: Plate care and filling Support Protocol 3: Plate preparation for water rinsing Support Protocol 4: Water rinsing of plates Support Protocol 5: Plate priming Support Protocol 6: General assay Support Protocol 7: Editing the compound addition sequence (compound list) Support Protocol 8: Creating compound list for agonist concentration-dependent response Support Protocol 9: Creating compound list for antagonist or PAM concentration-dependent response Support Protocol 10: Defining the different compounds used or compound list Support Protocol 11: Maintenance Support Protocol 12: Data analysis Support Protocol 13: Cell culture.
Western blotting with fluorescence detection offers the possibility of detecting multiple targets simultaneously on a single blot. Primary antibodies are increasingly available from multiple hosts, and there are now a wide variety of dye labels to exploit multiple imaging channels. If primary and secondary antibodies are selected so that individual targets can be discriminated, multiple antigens can be detected and quantified in a single experiment. Current fluorescence imaging instrumentation offers multiple detection channels and gives sensitivity comparable to other methods. The method described in this article allows multiple targets to be quantified simultaneously and reduces the need for stripping and re-probing. It also allows loading controls to be detected alongside the targets of interest. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Five-plex western blot detection, including tubulin detection for loading control.
Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high-throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC-CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1-2.3), which conduct the cardiac inward rectifier potassium current (IK1 ). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC-CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1 ) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC-CMs. In this article, we describe the method of using hiPSC-CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add-on (Dynamite8 ). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology at physiologic temperatures.
The only drugs currently approved for the treatment of Alzheimer's Disease (AD) are four acetylcholinesterase inhibitors and the NMDA antagonist memantine. Apart from these drugs, which have minimal to no clinical benefit, the 40-year search for effective therapeutics to treat AD has resulted in a clinical failure rate of 100% not only for compounds that prevent brain amyloid deposition or remove existing amyloid plaques but also those acting by a variety of other putative disease-associated mechanisms. This indicates that the preclinical data generated from current AD targets to support the selection, optimization, and translation of new chemical entities (NCEs) and biologics to clinical trials is seriously compromised. While many of these failures reflect flawed hypotheses or a lack of adequate characterization of the preclinical pharmacodynamic and pharmacokinetic (PD/PK) properties of lead NCEs—including their bioavailability and toxicity—the conceptualization, validation, and interrogation of the current animal models of AD represent key limitations. The overwhelming majority of these AD models are transgenic, based on aspects of the amyloid hypothesis and the genetics of the familial form of the disease. As a result, these generally lack construct and predictive validity for the sporadic form of the human disease. The 170 or so transgenic models, perhaps the largest number ever focused on a single disease, use rodents, mainly mice, and in addition to amyloid also address aspects of tau causality with more complex multigene models including other presumed causative factors together with amyloid. This overview discusses the current animal models of AD in the context of both the controversies surrounding the causative role of amyloid in the disease and the need to develop validated models of cognitive function/dysfunction that more appropriately reflect the phenotype(s) of human aged-related dementias. © 2019 by John Wiley & Sons, Inc.
