RESUMO

Tumor-on-chips (ToCs) are useful platforms for studying the physiology of tumors and evaluating the efficacy and toxicity of anti-cancer drugs. However, the design and fabrication of a ToC system is not a trivial venture. We introduce a user-friendly, flexible, 3D-printed microfluidic device that can be used to culture cancer cells or cancer-derived spheroids embedded in hydrogels under well-controlled environments. The system consists of two lateral flow compartments (left and right sides), each with two inlets and two outlets to deliver cell culture media as continuous liquid streams. The central compartment was designed to host a hydrogel in which cells and microtissues can be confined and cultured. We performed tracer experiments with colored inks and 40 kDa fluorescein isothiocyanate dextran to characterize the transport/mixing performances of the system. We also cultured homotypic (MCF7) and heterotypic (MCF7-BJ) spheroids embedded in gelatin methacryloyl hydrogels to illustrate the use of this microfluidic device in sustaining long-term micro-tissue culture experiments. We further demonstrated the use of this platform in anticancer drug testing by continuous perfusion of doxorubicin, a commonly used anti-cancer drug for breast cancer. In these experiments, we evaluated drug transport, viability, glucose consumption, cell death (apoptosis), and cytotoxicity. In summary, we introduce a robust and friendly ToC system capable of recapitulating relevant aspects of the tumor microenvironment for the study of cancer physiology, anti-cancer drug transport, efficacy, and safety. We anticipate that this flexible 3D-printed microfluidic device may facilitate cancer research and the development and screening of strategies for personalized medicine.

Assuntos

Antineoplásicos , Neoplasias da Mama , Impressão Tridimensional , Esferoides Celulares , Humanos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Feminino , Células MCF-7 , Hidrogéis/química , Dispositivos Lab-On-A-Chip , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Dextranos/química , Gelatina/química , Doxorrubicina/farmacologia , Doxorrubicina/química , Sobrevivência Celular/efeitos dos fármacos , Metacrilatos

RESUMO

COVID-19 made explicit the need for rethinking the way in which we conduct testing for epidemic emergencies. During the COVID-19 pandemic, the dependence on centralized lab facilities and resource-intensive methodologies (e.g., RT-qPCR methods) greatly limited the deployment of widespread testing efforts in many developed and underdeveloped countries. Here, we illustrate the development of a simple and portable diagnostic kit that enables self-diagnosis of COVID-19 at home from saliva samples. We describe the development of a do-it-yourself (DIY) incubator for Eppendorf tubes that can be used to conduct SARS-CoV-2 detection with competitive sensitivity and selectivity from saliva at home. In a proof-of-concept experiment, we assembled Eppendorf-tube incubators at our home shop, prepared a single-tube mix of reagents and LAMP primers in our lab, and deployed these COVID-19 detection kits using urban delivery systems (i.e., Rappifavor or Uber) to more than 15 different locations in Monterrey, México. This straightforward strategy enabled rapid and cost-effective at-home molecular diagnostics of SARS-CoV-2 from real saliva samples with a high sensitivity (100%) and high selectivity (87%).

RESUMO

Biopolymer microgels present many opportunities in biomedicine and tissue engineering. To understand their in vivo behavior in therapeutic interventions, long-term monitoring is critical, which is usually achieved by incorporating fluorescent materials within the hydrogel matrix. Current research is limited due to issues concerning the biocompatibility and instability of the conventional fluorescent species, which also tend to adversely affect the bio-functionality of the hydrogels. Here, we introduce a microfluidic-based approach to generate nitrogen-functionalized graphene quantum dot (NGQD) incorporated gelatin methacryloyl (GelMA) hydrogel microspheres, capable of long-term monitoring while preserving or enhancing the other favorable features of 3D cell encapsulation. A multilayer droplet-based microfluidic device was designed and fabricated to make monodisperse NGQD-loaded GelMA hydrogel microspheres encapsulating skeletal muscle cells (C2C12). Control over the sizes of microspheres could be achieved by tuning the flow rates in the microfluidic device. Skeletal muscle cells encapsulated in these microgels exhibited high cell viability from day 1 (82.9 ± 6.50%) to day 10 (92.1 ± 3.90%). The NGQD-loaded GelMA microgels encapsulating the cells demonstrated higher metabolic activity compared to the GelMA microgels. Presence of sarcomeric α-actin was verified by immunofluorescence staining on day 10. A fluorescence signal was observed from the NGQD-loaded microgels during the entire period of the study. The investigation reveals the advantages of integrating NGQDs in microgels for non-invasive imaging and monitoring of cell-laden microspheres and presents new opportunities for future therapeutic applications.

Assuntos

Grafite , Microgéis , Pontos Quânticos , Engenharia Tecidual , Hidrogéis , Gelatina , Metacrilatos

RESUMO

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

RESUMO

Chronic wounds have become one of the most important issues for healthcare systems and are a leading cause of death worldwide. Wound dressings are necessary to facilitate wound treatment. Engineering wound dressings may substantially reduce healing time, reduce the risk of recurrent infections, and reduce the disability and costs associated. In the path of engineering of an ideal wound dressing, hydrogels have played a leading role. Hydrogels are 3D hydrophilic polymeric structures that can provide a protective barrier, mimic the native extracellular matrix (ECM), and provide a humid environment. Due to their advantages, hydrogels (with different architectural, physical, mechanical, and biological properties) have been extensively explored as wound dressing platforms. Here we describe recent studies on hydrogels for wound healing applications with a strong focus on the interplay between the fabrication method used and the architectural, mechanical, and biological performance achieved. Moreover, we review different categories of additives which can enhance wound regeneration using 3D hydrogel dressings. Hydrogel engineering for wound healing applications promises the generation of smart solutions to solve this pressing problem, enabling key functionalities such as bacterial growth inhibition, enhanced re-epithelialization, vascularization, improved recovery of the tissue functionality, and overall, accelerated and effective wound healing.

RESUMO

Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.

RESUMO

The development of novel cancer therapeutic strategies has garnered increasing interest in cancer research. Among the therapeutic choices, chemosensitizers have shown exciting prospects. Peptides are an attractive alternative among the molecules that may be used as chemosensitizers. We rationally designed a new-to-nature peptide, nurP28, derived from the 22-kDa α-zein protein sequence (entry Q00919_MAIZE). The resultant sequence of the nurP28 peptide after the addition of arginine residues was LALLALLRLRRRATTAFIIP, and we added acetyl and amide groups at the N- and C-terminus, respectively, for capping. We evaluated the cytotoxicity of the nurP28 peptide alone and in combination with docetaxel in fibroblast monolayers and breast cancer monolayers and spheroids. Our results indicated that nurP28 is not cytotoxic to human fibroblasts or cancer cells. Nevertheless, when combined with 1 µM docetaxel, 3 ng/mL nurP28 induced equivalent (in MCF7 monolayers) and higher (in MCF7 spheroids) cytotoxic effects than 10-fold higher doses of docetaxel alone. These findings suggest that nurP28 may act as a chemosensitizer in breast cancer treatment. This study describes the enhancing "anti-cancer" effects of nurP28 in breast cancer 2D and 3D cultures treated with docetaxel. Further studies should explore the mechanisms underlying these effects and assess the clinical potential of our findings using animal models.

Assuntos

Antineoplásicos , Neoplasias da Mama , Zeína , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Docetaxel/farmacologia , Feminino , Humanos , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Esferoides Celulares

RESUMO

Light-based bioprinter manufacturing technology is still prohibitively expensive for organizations that rely on accessing three-dimensional biological constructs for research and tissue engineering endeavors. Currently, most of the bioprinting systems are based on commercial-grade-based systems or modified DIY (do it yourself) extrusion apparatuses. However, to date, few examples of the adoption of low-cost equipment have been found for light-based bioprinters. The requirement of large volumes of bioinks, their associated cost, and the lack of information regarding the parameter selection have undermined the adoption of this technology. This paper showcases the retrofitting and assessing of a low-cost Light-Based 3D printing system for tissue engineering. To evaluate the potential of a proposed design, a manufacturability test for different features, machine parameters, and Gelatin Methacryloyl (GelMA) concentrations for 7.5% and 10% was performed. Furthermore, a case study of a previously seeded hydrogel with C2C12 cells was successfully implemented as a proof of concept. On the manufacturability test, deviational errors were found between 0.7% to 13.3% for layer exposure times of 15 and 20 s. Live/Dead and Actin-Dapi fluorescence assays after 5 days of culture showed promising results in the cell viability, elongation, and alignment of 3D bioprinted structures. The retrofitting of low-cost equipment has the potential to enable researchers to create high-resolution structures and three-dimensional in vitro models.

RESUMO

Loop-mediated isothermal amplification (LAMP) has been recently studied as an alternative method for cost-effective diagnostics in the context of the current COVID-19 pandemic. Recent reports document that LAMP-based diagnostic methods have a comparable sensitivity and specificity to that of RT-qPCR. We report the use of a portable Arduino-based LAMP-based amplification system assisted by pH microelectrodes for the accurate and reliable diagnosis of SARS-CoV-2 during the first 3 min of the amplification reaction. We show that this simple system enables a straightforward discrimination between samples containing or not containing artificial SARS-CoV-2 genetic material in the range of 10 to 10,000 copies per 50 µL of reaction mix. We also spiked saliva samples with SARS-CoV-2 synthetic material and corroborated that the LAMP reaction can be successfully monitored in real time using microelectrodes in saliva samples as well. These results may have profound implications for the design of real-time and portable quantitative systems for the reliable detection of viral pathogens including SARS-CoV-2.

Assuntos

COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Microeletrodos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Fosfoproteínas/genética , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/metabolismo , Tempo de Reação , SARS-CoV-2/isolamento & purificação , Saliva/virologia

RESUMO

Multi-material and multilayered micro- and nanostructures are prominently featured in nature and engineering and are recognized by their remarkable properties. Unfortunately, the fabrication of micro- and nanostructured materials through conventional processes is challenging and costly. Herein, we introduce a high-throughput, continuous, and versatile strategy for the fabrication of polymer fibers with complex multilayered nanostructures. Chaotic electrospinning (ChE) is based on the coupling of continuous chaotic printing (CCP) and electrospinning, which produces fibers with an internal multi-material microstructure. When a CCP printhead is used as an electrospinning nozzle, the diameter of the fibers is further scaled down by 3 orders of magnitude while preserving their internal structure. ChE enables the use of various polymer inks for the creation of nanofibers with a customizable number of internal nanolayers. Our results showcase the versatility and tunability of ChE to fabricate multilayered structures at the nanoscale at high throughput. We apply ChE to the synthesis of unique carbon textile electrodes composed of nanofibers with striations carved into their surface at regular intervals. These striated carbon electrodes with high surface areas exhibit 3- to 4-fold increases in specific capacitance compared to regular carbon nanofibers; ChE holds great promise for the cost-effective fabrication of electrodes for supercapacitors and other applications.