A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01-20 µg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Retrospective analysis of trends in surgery volumes between 2016 and 2019 and impact of the insurance deductible: Cross-sectional study.

BACKGROUND: Understanding trends in surgical volumes can help Ambulatory Surgery Centers (ASCs) prevent clinician burnout and provide adequate staffing while maintaining the quality of patient care throughout the year. Health insurance deductibles reset in January each year and may contribute to an annual rhythm where the levee of year-end deductibles is breached in the last few months of every year, resulting in a flood of cases and several accompanying challenges. This study aims to identify and analyze monthly and yearly surgical volume patterns in ASCs and explore a relationship with the deductible reset. METHODS: De-identified, aggregate visit data for 2016-2019 were obtained retrospectively from 14 ambulatory surgery centers within the same benchmarking consortium in the Southeast. The ASCs subspecialty types consisted of orthopedics, urology, otolaryngology, and multispecialty. Kaiser Family Foundation survey data from 2016 to 2019 was used to inform deductible trends. Augmented Dickey-Fuller tests, linear regressions, and two-sample T-tests were conducted to explore and establish patterns in surgical volume between 2016 and 2019. RESULTS: Overall, average orthopedic surgical volume increased 38.04% from January to December in 2016-2019 with an average difference of 64 cases (95% CI: 47-80), while that of all ASCs combined increased 19.24% within the same timeframe with an average difference of 37 cases (95% CI: 21-52). Average health insurance deductibles rose 12% from $1476 to $1655 within the same timeframe. Regression analysis showed a stronger association between year and volume for orthopedic ASCs (R (Claxton et al., 2019) [2] = 0.796) than for all ASCs combined (R (Claxton et al., 2019) [2] = 0.645). Regression analysis also showed a stronger association between month and volume for orthopedic ASCs (R (Claxton et al., 2019) [2] = 0.488-0.805) than for all ASCs combined (R (Claxton et al., 2019) [2] = 0.115-0.493). CONCLUSION: This study is first to identify regular and predictable yearly and monthly increases in orthopedic ASCs surgical volume. The study also identifies yearly increases in surgical volume for all ASCs. The combination of increasing yearly demand for orthopedic surgery and growing association between month and volume leads to an unnecessary year-end rush. The study aims to inform future policy decisions as well as help ASCs better manage resources throughout the year.

Pumpless, unidirectional microphysiological system for testing metabolism-dependent chemotherapeutic toxicity.

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments. HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing "colon tumor", "liver," and "bone marrow" tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusion-limited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on dose-limiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.

A Tissue Engineering Approach to Metastatic Colon Cancer.

Colon cancer remains the third most common cause of cancer in the US, and the third most common cause of cancer death. Worldwide, colon cancer is the second most common cause of cancer and cancer deaths. At least 25% of patients still present with metastatic disease, and at least 25-30% will develop metastatic colon cancer in the course of their disease. While chemotherapy and surgery remain the mainstay of treatment, understanding the fundamental cellular niche and mechanical properties that result in metastases would facilitate both prevention and cure. Advances in biomaterials, novel 3D primary human cells, modelling using microfluidics and the ability to alter the physical environment, now offers a unique opportunity to develop and test impactful treatment.

Differential Monocyte Actuation in a Three-Organ Functional Innate Immune System-on-a-Chip.

A functional, human, multiorgan, pumpless, immune system-on-a-chip featuring recirculating THP-1 immune cells with cardiomyocytes, skeletal muscle, and liver in separate compartments in a serum-free medium is developed. This in vitro platform can emulate both a targeted immune response to tissue-specific damage, and holistic proinflammatory immune response to proinflammatory compound exposure. The targeted response features fluorescently labeled THP-1 monocytes selectively infiltrating into an amiodarone-damaged cardiac module and changes in contractile force measurements without immune-activated damage to the other organ modules. In contrast to the targeted immune response, general proinflammatory treatment of immune human-on-a-chip systems with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) causes nonselective damage to cells in all three-organ compartments. Biomarker analysis indicates upregulation of the proinflammation cytokines TNF-α, IL-6, IL-10, MIP-1, MCP-1, and RANTES in response to LPS + IFN-γ treatment indicative of the M1 macrophage phenotype, whereas amiodarone treatment only leads to an increase in the restorative cytokine IL-6 which is a marker for the M2 phenotype. This system can be used as an alternative to humanized animal models to determine direct immunological effects of biological therapeutics including monoclonal antibodies, vaccines, and gene therapies, and the indirect effects caused by cytokine release from target tissues in response to a drug's pharmacokinetics (PK)/pharmacodynamics (PD) profile.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Suture Button Suspensionplasty in the Treatment of Carpometacarpal Arthritis: A Retrospective Analysis of One Surgeon's Experience Over 9 Years.

Purpose: Trapeziectomy with suture button suspensionplasty (SBS) to treat thumb carpometacarpal (CMC) arthritis has been proposed as an alternative to ligament reconstruction tendon interposition. There have been limited large-scale or long-term reports regarding SBS outcomes. Single-surgeon intermediate follow-up is reported. Methods: We conducted a retrospective review of patients undergoing SBS procedures by a single surgeon. Implant manufacturer and postoperative immobilization protocol were recorded. Surgical outcomes, complications, and revision procedures were identified. Postoperative Disabilities of the Arm, Shoulder, and Hand scores were collected. Results: A total of 242 SBS surgeries were included, involving 215 patients, average age 64.82 years (range, 42-86 years). Average follow-up was 35 ± 25 months. In all, 183 Arthrex and 59 Stryker systems were used, 42 of which were immobilized for 6 weeks after surgery and 200 of which were mobilized at 2 weeks afterward. Postoperative Disabilities of the Arm, Shoulder, and Hand surveys were completed by 122 patients (57%), with an average score of 12. No scaphometacarpal abutment was reported. Thirteen complications were reported (5%), 7 of which were implant-associated (3%) and 6 of which were not (2%). Implant-associated complications consisted of 3 suture button pull-outs, 2 thumb-index metacarpal abutments, one suture tail irritation, and one index metacarpal fracture. Operative revision was required in 4 of 7 implant-associated cases and 5 of 6 non-implant associated cases. No suture button pull-outs required revision surgery. Conclusions: Results for a large series of SBS for CMC arthroplasty with intermediate follow-up revealed excellent clinical outcomes and low complication rates. Clinical relevance: Suture button suspensionplasty as an alternative to ligament reconstruction tendon interposition may be a viable option for treating thumb CMC arthritis. In addition, a technique to manage thumb-index metacarpal abutment is described.

Effectiveness of Amniotic Fluid Injection in the Treatment of Trigger Finger: A Pilot Study.

Purpose: To assess the efficacy and safety of amniotic fluid therapy injections in patients with mild to moderate trigger finger. Methods: All participants received 1 mL of amniotic fluid injected into the tendon sheath of the affected tendon. Pretreatment and posttreatment data were collected for triggering frequency, Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire scores, and numerical pain rating scale scores. Results: Of 111 digits from 96 patients, 51% experienced clinically notable improvement and did not receive an alternative treatment. Average length of follow-up was 11 months. From baseline to end of follow-up, average pain score (0-10) decreased from 5.19 to 1.19 (P < .001), median triggering per day decreased from 5 to 0 (P < .001), and median DASH score (1-100) decreased from 20 to 6.03 (P < .001). There was a 50% success rate in patients with diabetes and a 52.6% success rate in digits diagnosed with concomitant Dupuytren contracture in the same hand. Conclusions: Amniotic fluid therapy injections may offer a biologic alternative for conservative treatment of trigger finger, particularly for patients with diabetes. Decreased pain, decreased triggering, and improved DASH scores offer preliminary evidence supporting the use of amniotic injections for stenosing tenosynovitis. Type of study/level of evidence: Therapeutic IV.

Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development.

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system ("Body-on-a-Chip") designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is "pumpless" and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a "breathable" component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the "lung" using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a "breathable" lung module that maintains an air-liquid interface.

Strategies for using mathematical modeling approaches to design and interpret multi-organ microphysiological systems (MPS).

Recent advances in organ-on-a-chip technology have resulted in numerous examples of microscale systems that faithfully mimic the physiology and pathology of human organs and diseases. The next step in this field, which has already been partially demonstrated at a proof-of-concept level, would be integration of organ modules to construct multiorgan microphysiological systems (MPSs). In particular, there is interest in "body-on-a-chip" models, which recapitulate complex and dynamic interactions between different organs. Integration of multiple organ modules, while faithfully reflecting human physiology in a quantitative sense, will require careful consideration of factors such as relative organ sizes, blood flow rates, cell numbers, and ratios of cell types. The use of a mathematical modeling platform will be an essential element in designing multiorgan MPSs and interpretation of experimental results. Also, extrapolation to in vivo will require robust mathematical modeling techniques. So far, several scaling methods and pharmacokinetic and physiologically based pharmacokinetic models have been applied to multiorgan MPSs, with each method being suitable to a subset of different objectives. Here, we summarize current mathematical methodologies used for the design and interpretation of multiorgan MPSs and suggest important considerations and approaches to allow multiorgan MPSs to recapitulate human physiology and disease progression better, as well as help in vitro to in vivo translation of studies on response to drugs or chemicals.

Correction: UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems.

Correction for 'UniChip enables long-term recirculating unidirectional perfusion with gravity-driven flow for microphysiological systems' by Ying I. Wang and Michael L. Shuler, Lab Chip, 2018, 18, 2563-2574.

On the potential of in vitro organ-chip models to define temporal pharmacokinetic-pharmacodynamic relationships.

Functional human-on-a-chip systems hold great promise to enable quantitative translation to in vivo outcomes. Here, we explored this concept using a pumpless heart only and heart:liver system to evaluate the temporal pharmacokinetic/pharmacodynamic (PKPD) relationship for terfenadine. There was a time dependent drug-induced increase in field potential duration in the cardiac compartment in response to terfenadine and that response was modulated using a metabolically competent liver module that converted terfenadine to fexofenadine. Using this data, a mathematical model was developed to predict the effect of terfenadine in preclinical species. Developing confidence that microphysiological models could have a transformative effect on drug discovery, we also tested a previously discovered proprietary AstraZeneca small molecule and correctly determined the cardiotoxic response to its metabolite in the heart:liver system. Overall our findings serve as a guiding principle to future investigations of temporal concentration response relationships in these innovative in vitro models, especially, if validated across multiple time frames, with additional pharmacological mechanisms and molecules representing a broad chemical diversity.

Author Correction: A recellularized human colon model identifies cancer driver genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multi-organ system for the evaluation of efficacy and off-target toxicity of anticancer therapeutics.

A pumpless, reconfigurable, multi-organ-on-a-chip system containing recirculating serum-free medium can be used to predict preclinical on-target efficacy, metabolic conversion, and measurement of off-target toxicity of drugs using functional biological microelectromechanical systems. In the first configuration of the system, primary human hepatocytes were cultured with two cancer-derived human bone marrow cell lines for antileukemia drug analysis in which diclofenac and imatinib demonstrated a cytostatic effect on bone marrow cancer proliferation. Liver viability was not affected by imatinib; however, diclofenac reduced liver viability by 30%. The second configuration housed a multidrug-resistant vulva cancer line, a non-multidrug-resistant breast cancer line, primary hepatocytes, and induced pluripotent stem cell-derived cardiomyocytes. Tamoxifen reduced viability of the breast cancer cells only after metabolite generation but did not affect the vulva cancer cells except when coadministered with verapamil, a permeability glycoprotein inhibitor. Both tamoxifen alone and coadministration with verapamil produced off-target cardiac effects as indicated by a reduction of contractile force, beat frequency, and conduction velocity but did not affect viability. These systems demonstrate the utility of a human cell-based in vitro culture system to evaluate both on-target efficacy and off-target toxicity for parent drugs and their metabolites; these systems can augment and reduce the use of animals and increase the efficiency of drug evaluations in preclinical studies.

Piezoelectric BioMEMS Cantilever for Measurement of Muscle Contraction and for Actuation of Mechanosensitive Cells.

A piezoelectric biomedical microelectromechanical system (bioMEMS) cantilever device was designed and fabricated to act as either a sensing element for muscle tissue contraction or as an actuator to apply mechanical force to cells. The sensing ability of the piezoelectric cantilevers was shown by monitoring the electrical signal generated from the piezoelectric aluminum nitride in response to the contraction of iPSC-derived cardiomyocytes cultured on the piezoelectric cantilevers. Actuation was demonstrated by applying electrical pulses to the piezoelectric cantilever and observing bending via an optical detection method. This piezoelectric cantilever device was designed to be incorporated into body-on-a-chip systems.

Engineering a Bioartificial Human Colon Model Through Decellularization and Recellularization.

The tissue engineering method of decellularization and recellularization has been successfully used in a variety of regenerative medicine applications. The protocols used to de/recellularize various organs and tissues are largely different. Here we describe a method to effectively engineer a bioartificial colon by completely removing original cells from human intestinal tissues followed by repopulating the acellular tissue matrix with cell cultures. This method provides a novel approach for human intestinal regeneration and can be used to identify potential cancer driver genes.