A multi-laboratory preclinical trial in rodents to assess treatment candidates for acute ischemic stroke.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Evaluation of Activated Carbon and Platinum Black as High-Capacitance Materials for Platinum Electrodes.

The application of direct current (DC) produces a rapid and reversible nerve conduction block. However, prolonged injection of charge through a smooth platinum electrode has been found to cause damage to nervous tissue. This damage can be mitigated by incorporating high-capacitance materials (HCM) (e.g., activated carbon or platinum black) into electrode designs. HCMs increase the storage charge capacity (i.e., "Q value") of capacitive devices. However, consecutive use of these HCM electrodes degrades their surface. This paper evaluates activated carbon and platinum black (PtB) electrode designs in vitro to determine the design parameters which improve surface stability of the HCMs. Electrode designs with activated carbon and PtB concentrations were stressed using soak, bend and vibration testing to simulate destructive in vivo environments. A Q value decrease represented the decreased stability of the electrode-HCM interface. Soak test results supported the long-term Q value stabilization (mean = 44.3 days) of HCM electrodes, and both HCMs displayed unique Q value changes in response to soaking. HCM material choices, Carbon Ink volume, and application of Nafion™ affected an electrode's ability to resist Q value degradation. These results will contribute to future developments of HCM electrodes designed for extended DC application for in vivo nerve conduction block.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

User-Centered Design Process of an mHealth App for Health Professionals: Case Study.

BACKGROUND: User-centered design processes are infrequently employed and not fully explored for building mobile health (mHealth) apps that are particularly targeted to health professionals as end users. The authors have used a user-centered design-based approach to build an mHealth app for health professionals, tasked to deliver medical laboratory-related information on a daily basis. OBJECTIVE: Our objective is to generate a simple and functional user-centered design process for mHealth apps for health professionals. This paper presents the key learnings from design activities. METHODS: A stratified random sample of doctors and nurses was recruited for the study. The design activities were planned in the following sequence: focus group discussion for situation analysis and information architecture, design activity 1 for wireframe designing, design activity 2 for wireframe testing, and user testing sessions 1 and 2. RESULTS: The final design and functions of the app, information architecture, and interactive elements were largely influenced by the participatory design-based user-centered design activities. As a result of the design process, we could identify the mental models of processing requests for information and personal preferences based on the experience. These findings were directly or indirectly incorporated into the app design. Furthermore, finding alternative ways of working within time constraints and cultural barriers and the methods employed to manage the challenges of interdisciplinary discourse stood out among the lessons learned. CONCLUSIONS: We recommend a user-centered design process based on a participatory design approach in mHealth app design, enriched with focus group discussions where possible.

Analysis of a Cancer Stem Cell-Derived Single Colony Raised in a Microwell Array.

Cancer stem cells (CSCs) were reported to play important roles in cancer initialization, progression, and metastasis. In order to study the variation between CSCs and non-CSCs, single-cell analysis is conducted but technically complicated. In the current work, a microwell array made by an agarose hydrogel was developed for the study of a CSC-derived single colony. This approach is simple, convenient, and compatible with the setting and skill set of existing biological laboratories. Single cells, double cells, and multiple cells were distributed in the microwells. Isolation of CSCs could be achieved after a 5 day starvation culture course. Then, a CSC-derived single colony was formed by culturing the CSCs in a nutritious culture medium for another 5 days. The results revealed that a single CSC presented a high colony formation rate. Multiple cells containing CSCs and non-CSCs could raise a larger single colony than the multiple cells with CSCs only. Although CSCs possess an aggressive characteristic, development of a solid tumor requires the proactive involvement of non-CSCs. This work showed a practical demonstration of using a microwell array for the investigation of a CSC-derived single colony.

Visualization and Quantification of 3D Tumor Cell Migration under Extracellular Stimulation.

To investigate tumor cell migration capability, the scratch/wound healing assay and the Transwell assay are the most commonly used assays in the current biomedical research laboratory. However, both assays have their limitations and may mislead the interpretation of the results. In the current study, visualization and quantification of tumor cell migration process was realized in a three-dimensional (3D) environment. The tumor cells horizontally migrated along a Matrigel-filled microchannel under extracellular stimulation. The cell migration process was visualized under a microscope, and the migration speed could be calculated based on the traveling distance of the cells and the time required. Here, three demonstrations were conducted, respectively, including cells attracted by nutrient gradient, stimulated by cytokine, and coculturing with fibroblasts. The results revealed that the cell migration capability could be visually and quantitatively correlated to the extracellular stimulation. The current protocol is compatible to the existing laboratory setup and provides a persuasive result for the study of the 3D cell migration process. Understanding of the molecular and intercellular mechanism of cancer metastasis can potentially develop effective therapeutic strategy.

Investigation of uniform sized multicellular spheroids raised by microwell arrays after the combined treatment of electric field and anti-cancer drug.

Nowadays, cancer disease is continuously identified as the leading cause of mortality worldwide. Cancer chemotherapeutic agents have been continuously developing to achieve high curative effectiveness and low side effects. However, solid tumors present the properties of low drug penetration and resistance of quiescent cells. Radiation therapy is concurrently given in some cases; but it induces different levels of adverse effects. In the current work, uniform sized multicellular spheroids were raised by microwell arrays to mimic the architecture of solid tumors. Investigation of the response of the spheroids was conducted after the treatment of alternating electric field. The result showed that the electric field could induce early apoptosis by disturbing cell membrane. Moreover, combined treatment of electric field and anti-cancer drug was applied to the spheroids. The electric field synergistically enhanced the treatment efficacy because the anti-cancer drug could permeate through the disrupted cell membrane. Significant improvement of late apoptosis was shown by the combined treatment. Because the electric field treatment induces limited side effect to the patient, lower dosage of anti-cancer drug may be applied to the patients for achieving curative effectiveness.

Paper/polymer composited microfluidic platform for screening cell viability and protein expression under a chemical gradient environment.

Culturing cells in three-dimensional (3D) environment can obtain a better clinical prediction for evaluating chemotherapy protocols and become a standard culture practice in cancer research. However, it involves tedious and time consuming operation. In the current work, a paper/polymer composited microfluidic platform was developed for screening cell viability and protein expression under chemical gradient environment. Cells were cultured in a paper sheet and expressed cell properties in 3D environment. The paper sheet was encapsulated in the microfluidic platform generating chemical gradient. After the culture course, investigations of cell viability and protein expression were respectively achieved by directly adding reagent and conducting on-paper immunoassay. Activation of respective signaling pathway could be identified and responded to different stimulations including nutrient gradient, IL-6 cytokine gradient, and anti-cancer drug gradient. On-paper analysis of protein expression could be completed within 1.5 h. The present technique integrates tedious operations on a single paper substrate. It provides a first-tier screening tool for cellular response under chemical gradient.

Proliferation arrest, selectivity, and chemosensitivity enhancement of cancer cells treated by a low-intensity alternating electric field.

Elimination of serious side effects is a desired feature of cancer therapy. Alternating electric field treatment is one approach to the non-invasive treatment of cancer. The efficacy and safety of this novel therapy are confirmed for the treatment of glioblastoma multiforme. In the current study, we co-cultured cancer cells and normal cells to investigate the selectivity and chemosensitivity enhancement of an electric field treatment. Cancer cells (cell line: HeLa and Huh7) and fibroblasts (cell line: HEL299) were cultured in an in-house-developed cell culture device embedded with stimulating electrodes. A low-intensity alternating electric field was applied to the culture. The field significantly induced proliferation arrest of the cancer cells, while had limited influence on the fibroblasts. Moreover, in combination with the anti-cancer drug, damage to the cancer cells was enhanced by the electric field. Thus, a lower dosage of the drug could be applied to achieve the same treatment effectiveness. This study provides evidence that low-intensity electric field treatment selectively induced proliferation arrest and enhanced the chemosensitivity of the cancer cells. This electro-chemotherapy could be developed and applied as a regional cancer therapy with minimal side effects.