Microfabricated Physiological Models for In Vitro Drug Screening Applications

Microfluidics and microfabrication have recently been established as promising tools for developing a new generation of in vitro cell culture microdevices. The reduced amounts of reagents employed within cell culture microdevices make them particularly appealing to drug screening processes. In addition, latest advancements in recreating physiologically relevant cell culture conditions within microfabricated devices encourage the idea of using such advanced biological models in improving the screening of drug candidates prior to in vivo testing. In this review, we discuss microfluidics-based models employed for chemical/drug screening and the strategies to mimic various physiological conditions: fine control of 3D extra-cellular matrix environment, physical and chemical cues provided to cells and organization of co-cultures. We also envision future directions for achieving multi-organ microfluidic devices

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Supplement: "Localization and broadband follow-up of the gravitational-wave transient GW150914" (2016, ApJL, 826, L13)

This Supplement provides supporting material for Abbott et al. (2016a). We briefly summarize past electromagnetic (EM) follow-up efforts as well as the organization and policy of the current EM follow-up program. We compare the four probability sky maps produced for the gravitational-wave transient GW150914, and provide additional details of the EM follow-up observations that were performed in the different bands

Modelling of 3D spatially controlled compartmentalized tissues in microfluidics

Organs-on-chip have been widely addressed as potential tools for recreating tissue structure and functions within microdevices. In perspective, the possibility of engineering cellular threedimensional constructs with behaviour similar to physiological tissues or organs is a paramount aim for improving basic research studies and drug screening processes [1]. Biological functional structures are typically characterized by a compartmental architecture where multiple cellular units made up of different cell types and/or extracellular matrix are spatially organized to interact and contribute to biological homeostasis and function. Nevertheless, it is currently challenging to neatly interface multi-compartmental 3D biological constructs within microfluidic systems and there is a need for techniques that allow fine spatial control of 3D cell-laden matrices [2]. We here present a novel microfluidic technique for engineering complex micro-tissue structures made of controlled multi-compartmental three-dimensional cellular constructs. By employing molding PDMS layers, we show how to form pure composites of two stacked or flanking tissue constructs (made of human bone marrow-derived mesenchymal stem cells, Panel A and B) within existing microfluidic systems commonly used for controlled presentation of soluble factors (differentiation factors, drug compounds, etc.) or application of medium perfusion. We then applied this technique to form endothelialized constructs with vessel-like structures within microtissues. We demonstrate cell viability, continuity of composite constructs and endothelial barrier formation. As no confining structures (pillars or phaseguides) are present at the generated interfaces, this technique holds promise for advanced modelling of complex multi-compartmental tissues/organs and ongoing work is aimed at generating micro-tissues of relevant physiological structures (blood-brain barrier and osteochondral interface)

Generating Multicompartmental 3D Biological Constructs Interfaced through Sequential Injections in Microfluidic Devices

A novel technique is presented for molding and culturing composite 3D cellular constructs within microfluidic channels. The method is based on the use of removable molding polydimethylsiloxane (PDMS) inserts, which allow to selectively and incrementally generate composite 3D constructs featuring different cell types and/or biomaterials, with a high spatial control. The authors generate constructs made of either stacked hydrogels, with uniform horizontal interfaces, or flanked hydrogels with vertical interfaces. The authors also show how this technique can be employed to create custom-shaped endothelial barriers and monolayers directly interfaced with 3D cellular constructs. This method dramatically improves the significance of in vitro 3D biological models, enhancing mimicry and enabling for controlled studies of complex biological districts

Generation of functional cardiac microtissues in a beating heart-on-a-chip

With the increasing attention on cardiovascular disorders and the current inability of pre-clinical models to accurately predict human physiology, the need for advanced and reliable heart in vitro models is paramount. Microfabrication technologies provide potential solutions in the organs-on-chip systems: microengineered devices where cell cultures can be hosted and cultured to develop three-dimensional models or microtissues with high similarity to human physiology. We here described the fabrication and operation procedures for a beating heart-on-a-chip. The device features a culture region for a 3D cardiac microtissue and a system for applying tuned mechanical stimulation during culture to improve cardiac development. We additionally describe procedures for characterizing tissue maturation via immunofluorescence and functional evaluations of microtissue contractility

Towards the joint on a chip: double layered directly interfaced tissues to mimic the Osteoarthritic cartilage-subchondral interface

Osteoarthritis (OA) is the most diffused musculoskeletal disease and a worldwide cause of pain and disability. Although OA’s most evidently affected tissue is cartilage (AC), pathological changes regard the whole joint. An altered biomechanic at the interface between AC and bone, namely widening of a layer of mineralized hypertrophic cartilage (HC), has been suggested among pathological causes. Most OA in vitro models, however, do not account for joints’ biomechanics nor for the multiplicity of tissues affected by OA. In this work we present an OoC model of OA joint enabling to culture two directly interfaced 3D-tissues differentiated in AC and HC and coupled with a mechanism to provide both vertically aligned 3D constructs with mechanical compression. Theory and Experimental procedure Starting from a recently developed OA cartilage on chip model, we first investigated culture conditions to properly differentiate 3D micro-constructs into AC and HC within the miniaturized model. Articular chondrocytes (CH) were used as cell source for AC, mesenchymal stromal cells (MSCSs) for HC. Tissues were cultured either singularly or in coculture where a direct interface between 3D micro-construct was achieved through removable PDMS molds. A new device was then designed to provide vertically interfaced micro-constructs, with mechanical stimulation. ECM deposition and gene expression were analyzed both in single and coculture. Results A tailored culture medium allowed to reach differentiation of both AC and HC. Aggrecan and Collagen type-II(Col2a1) characterized AC matrix, while HC matrix, still Col2a1 positive, was also rich in collagen type-X and calcium deposits, hallmarks of the hypertrophic tissue. MSCSs and ACs maintained different expression levels of distinctive markers (e.g. ACAN, COL10A1 and OPN) in both cultures. Moreover, a proof-of-concept of the possibility to stack the two tissues in a knee joint-like vertical configuration was achieved by means of a new device. Notably it was possible to couple it with an actuation layer for OA induction through mechanical overload. Conclusions and discussion Successful differentiation of both tissues and achievement of a direct interface between AC and HC opens the path for a more representative Joint-like model. Mechanical stimulation, furthermore, allows for the study of biomechanics role in OA onset. Indeed, these results could allow an in vitro representation of the tidemark and for investigation on the causes (e.g. mechanical injuries) of the transition of AC to HC which distinguish OA’s onset. Acknowledgments Fondazione Cariplo (#2018-0551) MSCA IF (#841975). SNF (310030_175660). PoliFab CleanRoo