Traditional Cell Culture Methods Research Articles

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

The study on 4D culture system of squamous cell carcinoma of tongue

Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinβ3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.

Tape-assisted fabrication method for constructing PDMS membrane-containing culture devices with cyclic radial stretching stimulation.

Advanced in vitro culture systems have emerged as alternatives to animal testing and traditional cell culture methods in biomedical research. Polydimethylsiloxane (PDMS) is frequently used in creating sophisticated culture devices owing to its elastomeric properties, which allow mechanical stretching to simulate physiological movements in cell experiments. We introduce a straightforward method that uses three types of commercial tape-generic, magic and masking-to fabricate PDMS membranes with microscale thicknesses (47.2 µm for generic, 58.1 µm for magic and 89.37 µm for masking) in these devices. These membranes are shaped as the bases of culture wells and can perform cyclic radial movements controlled via a vacuum system. In experiments with A549 cells under three mechanical stimulation conditions, we analysed transcriptional regulators responsive to external mechanical stimuli. Results indicated increased nuclear yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) activity in both confluent and densely packed cells under cyclically mechanical strains (Pearson's coefficient (PC) of 0.59 in confluent and 0.24 in dense cells) compared with static (PC = 0.47 in confluent and 0.13 in dense) and stretched conditions (PC = 0.55 in confluent and 0.20 in dense). This technique offers laboratories without microfabrication capabilities a viable option for exploring cellular behaviour under dynamic mechanical stimulation using PDMS membrane-equipped devices.

Integration of secreted signaling molecule sensing on cell monitoring platforms: a critical review.

Monitoring cell secretion in complex microenvironments is crucial for understanding cellular behavior and advancing physiological and pathological research. While traditional cell culture methods, including organoids and spheroids, provide valuable models, real-time monitoring of cell secretion of signaling molecules remains challenging. Integrating advanced monitoring technologies into these systems often disrupts the delicate balance of the microenvironment, making it difficult to achieve sensitivity and specificity. This review explored recent strategies for integrating the monitoring of cell secretion of signaling molecules, crucial for understanding and replicating cell microenvironments, within cell culture platforms, addressing challenges such as non-adherent cell models and the focus on single-cell methodologies. We highlight advancements in biosensors, microfluidics, and three-dimensional culture methods, and discuss their potential to enhance real-time, multiplexed cell monitoring. By examining the advantages, limitations, and future prospects of these technologies, we aim to contribute to the development of integrated systems that facilitate comprehensive cell monitoring, ultimately advancing biological research and pharmaceutical development.

Enhancing Dengue Virus Production and Immunogenicity with Celcradle™ Bioreactor: A Comparative Study with Traditional Cell Culture Methods.

The dengue virus, the primary cause of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, is the most widespread mosquito-borne virus worldwide. In recent decades, the prevalence of dengue fever has increased markedly, presenting substantial public health challenges. Consequently, the development of an efficacious vaccine against dengue remains a critical goal for mitigating its spread. Our research utilized Celcradle™, an innovative tidal bioreactor optimized for high-density cell cultures, to grow Vero cells for dengue virus production. By maintaining optimal pH levels (7.0 to 7.4) and glucose concentrations (1.5 g/L to 3.5 g/L) during the proliferation of cells and viruses, we achieved a peak Vero cell count of approximately 2.46 × 109, nearly ten times the initial count. The use of Celcradle™ substantially decreased the time required for cell yield and virus production compared to conventional Petri dish methods. Moreover, our evaluation of the immunogenicity of the Celcradle™-produced inactivated DENV4 through immunization of mice revealed that sera from these mice demonstrated cross-reactivity with DENV4 cultured in Petri dishes and showed elevated antibody titers compared to those from mice immunized with virus from Petri dishes. These results indicate that the dengue virus cultivated using the Celcradle™ system exhibited enhanced immunogenicity relative to that produced in traditional methods. In conclusion, our study highlights the potential of the Celcradle™ bioreactor for large-scale production of inactivated dengue virus vaccines, offering significant promise for reducing the global impact of dengue virus infections and accelerating the development of effective vaccination strategies.

Abstract 512: Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles

Abstract Background: Traditional cell culture methods involve growing cells on flat surfaces, which do not fully replicate the human physiological conditions. In contrast, 3D cell cultures accurately reproduce aspects of the in-vitro malignant and non-malignant cell behavior. However, differences in tumor sensitivity platforms between 2D vs. 3D models might be relevant for some, but not for other drugs. Methods: The colorectal cancer cell line HCT-116 was cultured and plated in flat bottom well plates for the 2D condition and in Ultra Low Attachment round-bottom plates for the 3D condition. After plating, cells were treated with chemotherapies and targeted therapies for four days; cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50s were quantified to understand the differences in responses between 2D and 3D cellular environments. Results: HCT-116 exhibited similar responses to platinum drugs, taxanes, and several other tested drugs in both platforms, with the exception of gemcitabine (2D IC50 0.04 mcM; 3D IC50 12.8 mcM), lapatinib (2D IC50 23; 3D IC50 418), tucatinib (2D IC50 23.3; 3D IC50 79.3), and palbociclib (2D IC50 7.0; 3D IC50 24.0). Interestingly, these drugs share a common mechanism of action, which is the inhibition of cell cycle progression from G1 to S phase. Hence, we hypothesize that this shared mechanism may account for the observed differences in drug efficacy between the two environments. Conclusions: Similar dose responses have been observed with various drugs on both platforms, yet drugs that inhibit cell cycle progression from G1 to S phase, revealed higher sensitivity in 2D and more resistance in the 3D platform. Confirmatory cell-cycle results analyses are in progress. Further research is needed to understand if this applies to patient samples. Citation Format: Jahanvi Kumar, Chiara Maestri, Rajeshwar Nitiyanandan, Ivan Trus, Ricardo J. Parker, Brigitte Apfel, Christian Apfel. Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 512.

Gnotobiotic pig models: Illuminating the enigma of human norovirus infection and immunity

Gnotobiotic pig models: Illuminating the enigma of human norovirus infection and immunity Dr Lijuan Yuan and her team have studied human noroviruses (HuNoV) in gnotobiotic pigs for over 15 years. Here, she explains how such research is advancing our understanding of HuNoV pathogenesis, infectivity, and immunity. The intricate interplay between pathogens and their hosts has been a focal point of infectious disease research, especially for elusive viruses like human noroviruses (HuNoV). With their capacity to cause acute gastroenteritis and notorious ability to evade traditional cell culture methods, the study of HuNoV has been particularly challenging. However, gnotobiotic pig models have emerged as a potent tool in unveiling the complex dynamics of HuNoV pathogenesis, infectivity, and immunity. This review delves into the application of gnotobiotic pig models in deciphering the enigmatic world of HuNoV, shedding light on their elusive interactions with the host.

Rapid and Sensitive Pathogen Detection in Serum for Early Sepsis Diagnostics Using an Electrical-Double-Layer Gated Field-Effect Transistor-Based Sensor Array

Sepsis is a major medical inflammatory response to bloodstream infection caused bypathogens. Over 30 million infections occur due to sepsis in a year throughout the globe.With over 6 million deaths, sepsis treatment demands timely diagnosis and medicalinterventions to prevent large outspread of the infection. Rapid and sensitive detection ofinfectious bacteria plays an important role in disease control. However, fast and accuratediagnosis remains highly challenging for many hospitalized patients. Among variousbacterial diseases, Escherichia coli, (E. coli) a gram-negative bacterium, is the mostfrequently and extensively researched and has become a global concern of food safetyproblems. Traditional cell culture methods are considered as most accurate method forbacterial diagnosis. But some microorganisms like Bartonella spp, Treponema pallidum, etc.are difficult to grow on a culture plate, while some other virus species are unculturable, soeventually the traditional cell culture may take several days to a week for the diagnosticresults. Hence, over several years, different novel methods have been developed, such asoptical methods, electrochemical methods, molecular detection by PCR etc. Even thoughseveral techniques exist, concerns have been raised regarding their sensitivity, specificity etc.In addition, various fluorescence-based methods, and Surface Plasmon resonance (SPR)-based sensors have also been reported for bacterial detection. However, high cost, largeturnaround time, expensive instrumentation, and requirement of skilled personnel are some ofthe disadvantages of these existing techniques.FET biosensors have previously been used in different diagnosis areas. Nowadays, severalFET-based biosensors have been developed, which primarily concentrate on improving theoverall sensitivity of FET devices. However, the charge screening effect observed in aphysiological environment, and additional sample processing increase the complexity of thesensor platform. To overcome this hurdle, an electrical double layer (EDL) gated FETbiosensor has been proposed and explored in the detection of a variety of biological entities.Here, we proposed an EDL-FET sensor platform for the detection of Escherichia coliO157:H7 strain in blood serum. A single-step surface functionalization strategy was used forimmobilizing receptor probes as ssDNA. The probes were designed and immobilized over adisposable chip containing a pair of gold electrodes. A handheld reader which consists of anenhancement mode N-channel MOSFETs was connected to the laptop through a USB port tomeasure the sensor signals. Pulsed gate voltage was applied to the input electrode and whichmodulated the EDL capacitance and potential change eventually resulting in the drain currentof the MOSFET. The probe was immobilized on the gate electrode and tested with differentconcentrations of cDNA spiked in TE buffer. The probe-target binding was observed throughfluorescence change and electrically by measuring the change in drain current. In addition,surface potential change was also examined by performing KPFM test. Similarly, the BioFETdetections were validated for E. coli O157 variants by spiking with short and long DNAtarget sequences simultaneously in serum. The test results confirm selectivity by testingvarious non-complementary sequences, which ensures the potential of this BioFET as apoint-of-care sensing device. Figure 1

Three-dimensional highly porous hydrogel scaffold for neural circuit dissection and modulation.

Biomimetic brain structures and artificial neural networks have provided a simplified strategy for quantitatively investigating the complex structural and functional characteristics of highly interconnected neural networks. To achieve this, three-dimensional (3D) cell culture approaches have attracted much attention, which can mimic cell-cell interactions at the organism level and help better understand the function of specific neurons and neuronal networks than traditional two-dimensional cell culture methods. However, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. To resolve this, 3D hydrogel scaffolds can be fabricated via an innovative thermal treatment followed by an esterification process. A highly porous microstructure was formed within the bulk hydrogel scaffold, which showed a high porosity of 91% and a low Young's modulus of 6.11 kPa. Due to the merits of the fabricated hydrogel scaffolds, we constructed 3D neural networks and detected spontaneous action potentials in vitro. We successfully induced seizure-like waveforms in 3D cultured neurons and suppressed hyperactivated discharges by selectively activating γ-aminobutyric acid-ergic (GABAergic) interneurons. These results prove the advantages of our hydrogel scaffolds and demonstrate their application potential in the accurate dissection of neural circuits, which may help develop effective treatments for various neurological disorders. STATEMENT OF SIGNIFICANCE: While 3D cell culture approaches have attracted much attention and offer more advantages than two-dimensional cell culture methods, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. Herein, we developed a simplified and low-cost strategy for fabricating highly porous and cytocompatible hydrogel scaffolds for the construction of three-dimensional (3D) neural networks in vitro. The cultured 3D neural networks can mimic the in vivo connection among different neuron subgroups and help accurately dissect and manipulate the structure and function of specific neural circuits.

A Comparison between Different Machine Learning Approaches Combined with Anodic Stripping Voltammetry for Copper Ions and pH Detection in Cell Culture Media

Recently, the scientific community has shown a great interest about the Organ-on-Chip (OoC) devices, a special kind of micro-fabricated platforms capable of recapitulating the human physiology implementing the traditional cell culture methods and the concept of in vivo studies. Copper ions represent a cellular micronutrient that must be monitored for its potential hazardous effects. The application of electrochemical analysis for heavy metal ions detection and quantification in commercial cell culture media presents several issues due to electrolyte complexity and interferents. In fact, to the best of our knowledge, there is a lack of applications and OoC devices that implement the Anodic Stripping Voltammetry as an ion dosing technique due to the reasons reported above. In fact, considering just the peak intensity value from the measurement, it turns out to be challenging to quantify ion concentration since other ions or molecules in the media may interfere with the measurement. With the aim to overcome these issues, the present work aims to develop an automated system based on machine learning algorithms and demonstrate the possibility to build a reliable forecasting model for copper ion concentration on three different commercial cell culture media (MEM, DMEM, F12). Effectively, combining electrochemical measurements with a multivariate machine learning algorithm leads to a higher classification accuracy. Two different pH media conditions, i.e., physiological (pH 7.4) and acidic (pH 4), were considered to establish how the electrolyte influences the measurement. The experimental datasets were obtained using square-wave anodic stripping voltammetry (SWASV) and were used to carry out a machine learning trained model. The proposed method led to a significant improvement in Cu2+ concentration detection accuracy (96.6% for the SVM model and 93.1% for the NB model in MEM) as well as being able to monitor the pH solution.

Nebulization of Model Hydrogel Nanoparticles to Macrophages at the Air-Liquid Interface.

Nanoparticle evaluation within the pulmonary airspace has increasingly important implications for human health, with growing interest from drug delivery, environmental, and toxicology fields. While there have been widespread investigations of nanoparticle physiochemical properties following many routes of administration, nanoparticle behavior at the air-liquid interface (ALI) is less well-characterized. In this work, we fabricate two formulations of poly(ethylene)-glycol diacrylate (PEGDA)-based model nanoparticles to establish an in vitro workflow allowing evaluation of nanoparticle charge effects at the ALI. Both cationic and anionic PEGDA formulations were synthesized with similar hydrodynamic diameters around ~225 nm and low polydispersity, with expected surface charges corresponding with the respective functional co-monomer. We find that both formulations are readily nebulized from an aqueous suspension in a commercial Aeroneb® Lab Nebulizer, but the aqueous delivery solution served to slightly increase the overall hydrodynamic and geometric size of the cationic particle formulation. However, nanoparticle loading at 50 μg/ml of either formulation did not influence the resultant aerosol diameter from the nebulizer. To assess aerosol delivery in vitro, we designed a 3D printed adapter capable of ensuring aerosol delivery to transwell 24-well culture plates. Nanoparticle uptake by macrophages was compared between traditional cell culture techniques and that of ALI-cultured macrophages following aerosol delivery. Cell viability was unaffected by nanoparticle delivery using either method. However, only traditional cell culture methods demonstrated significant uptake that was dependent on the nanoparticle surface charge. Concurrently, ALI culture resulted in lower metabolic activity of macrophages than those in traditional cell culture, leading to lower overall nanoparticle uptake at ALI. Overall, this work demonstrates that base-material similarities between both particle formulations provide an expected consistency in aerosol delivery regardless of the nanoparticle surface charge and provides an important workflow that enables a holistic evaluation of aerosolizable nanoparticles.

SpheroScan: a user-friendly deep learning tool for spheroid image analysis.

In recent years, 3-dimensional (3D) spheroid models have become increasingly popular in scientific research as they provide a more physiologically relevant microenvironment that mimics in vivo conditions. The use of 3D spheroid assays has proven to be advantageous as it offers a better understanding of the cellular behavior, drug efficacy, and toxicity as compared to traditional 2-dimensional cell culture methods. However, the use of 3D spheroid assays is impeded by the absence of automated and user-friendly tools for spheroid image analysis, which adversely affects the reproducibility and throughput of these assays. To address these issues, we have developed a fully automated, web-based tool called SpheroScan, which uses the deep learning framework called Mask Regions with Convolutional Neural Networks (R-CNN) for image detection and segmentation. To develop a deep learning model that could be applied to spheroid images from a range of experimental conditions, we trained the model using spheroid images captured using IncuCyte Live-Cell Analysis System and a conventional microscope. Performance evaluation of the trained model using validation and test datasets shows promising results. SpheroScan allows for easy analysis of large numbers of images and provides interactive visualization features for a more in-depth understanding of the data. Our tool represents a significant advancement in the analysis of spheroid images and will facilitate the widespread adoption of 3D spheroid models in scientific research. The source code and a detailed tutorial for SpheroScan are available at https://github.com/FunctionalUrology/SpheroScan.

Living Biointerfaces for the Maintenance of Mesenchymal Stem Cell Phenotypes

AbstractLiving interfaces are established as a novel class of active materials that aim to provide an alternative to traditional static cell culture methods by enabling users to accurately control cell behaviour in a precise, dynamic, and reliable system‐internal manner. To this day, the only reported biointerface has been a coculture between a biofilm of nonpathogenic genetically engineered bacteria and mammalian cells, where the recombinant proteins produced by the bacteria directly influence cell behaviour. In this work, a biointerface is presented between Lactococcus lactis (L. lactis) and human mesenchymal stem cells (hMSCs). L. lactis have been engineered to produce human C‐X‐C motif chemokine ligand 12, thrombopoietin, vascular cell adhesion protein 1, and the 7th–10th type III domains of human fibronectin, with the aim of recreating the native bone marrow conditions ex vivo. This active microenvironment has been shown to maintain key hMSC stemness markers, preventing their osteogenic and adipogenic differentiation, and maintaining high stem cell viability and physiological cell‐to‐substrate adhesion dynamics. This work presents proof of concept data that hMSC stemness can be regulated by living materials, using a system based on the symbiotic interaction between different engineered bacteria and mammalian cells.

Organ-On-A-Chip: A Survey of Technical Results and Problems.

Organ-on-a-chip (OoC), also known as micro physiological systems or “tissue chips” have attracted substantial interest in recent years due to their numerous applications, especially in precision medicine, drug development and screening. Organ-on-a-chip devices can replicate key aspects of human physiology, providing insights into the studied organ function and disease pathophysiology. Moreover, these can accurately be used in drug discovery for personalized medicine. These devices present useful substitutes to traditional preclinical cell culture methods and can reduce the use of in vivo animal studies. In the last few years OoC design technology has seen dramatic advances, leading to a wide range of biomedical applications. These advances have also revealed not only new challenges but also new opportunities. There is a need for multidisciplinary knowledge from the biomedical and engineering fields to understand and realize OoCs. The present review provides a snapshot of this fast-evolving technology, discusses current applications and highlights advantages and disadvantages for biomedical approaches.

Development of the gut microbiome in early life.

New Findings What is the topic of this review? The importance of the early life gut microbiome, with a focus on preterm infants and microbially related diseases. Current techniques to study the preterm gut microbiome are appraised, and the potential of recent methodological advancements is discussed. What advances does it highlight? Recent findings in the field achieved by the application of advanced technologies, the applicability of intestinally derived organoid models to study host–microbiome interactions in the preterm gut, and recent developments in enhancing the physiological relevance of such models. Preterm intestinally derived organoids may provide novel insights into the mechanisms underlying preterm disease, as well as diagnosis and treatment opportunities. These models have huge translational potential, offering a step towards precision medicine. Accumulating evidence affirms the importance of the gut microbiome in both health and disease. In early life, there exists a critical period in which the composition of gut microbes is particularly malleable and subject to a wide range of influencing factors. Disturbances to microbial communities during this time may be beneficial or detrimental to short and long‐term health outcomes. For infants born prematurely, naïve immune systems, immature gastrointestinal tracts and additional clinical needs put this population at high risk of abnormal microbial colonisation, resulting in increased susceptibility to diseases including necrotising enterocolitis (NEC) and late‐onset sepsis (LOS). Traditional cell culture methods, gnotobiotic animals, molecular sequencing techniques (16S rRNA gene sequencing and metagenomics) and advanced ‘omics’ technologies (transcriptomics, proteomics and metabolomics) have been fundamental in exploring the associations between diet, gut microbes, microbial functions and disease. Despite significant investment and ongoing research efforts, prevention and treatment strategies in NEC and LOS remain limited. Recent endeavours have focused on searching for new, more physiologically relevant models to simulate the preterm intestine. Preterm intestinally derived organoids represent a promising in vitro approach in the study of host–microbiome interactions in the preterm infant gut, offering new and exciting possibilities in this field.

Perfused Platforms to Mimic Bone Microenvironment at the Macro/Milli/Microscale: Pros and Cons.

As life expectancy increases, the population experiences progressive ageing. Ageing, in turn, is connected to an increase in bone-related diseases (i.e., osteoporosis and increased risk of fractures). Hence, the search for new approaches to study the occurrence of bone-related diseases and to develop new drugs for their prevention and treatment becomes more pressing. However, to date, a reliable in vitro model that can fully recapitulate the characteristics of bone tissue, either in physiological or altered conditions, is not available. Indeed, current methods for modelling normal and pathological bone are poor predictors of treatment outcomes in humans, as they fail to mimic the in vivo cellular microenvironment and tissue complexity. Bone, in fact, is a dynamic network including differently specialized cells and the extracellular matrix, constantly subjected to external and internal stimuli. To this regard, perfused vascularized models are a novel field of investigation that can offer a new technological approach to overcome the limitations of traditional cell culture methods. It allows the combination of perfusion, mechanical and biochemical stimuli, biological cues, biomaterials (mimicking the extracellular matrix of bone), and multiple cell types. This review will discuss macro, milli, and microscale perfused devices designed to model bone structure and microenvironment, focusing on the role of perfusion and encompassing different degrees of complexity. These devices are a very first, though promising, step for the development of 3D in vitro platforms for preclinical screening of novel anabolic or anti-catabolic therapeutic approaches to improve bone health.

Advanced human-relevant in vitro pulmonary platforms for respiratory therapeutics.

Over the past years, advanced in vitro pulmonary platforms have witnessed exciting developments that are pushing beyond traditional preclinical cell culture methods. Here, we discuss ongoing efforts in bridging the gap between in vivo and in vitro interfaces and identify some of the bioengineering challenges that lie ahead in delivering new generations of human-relevant in vitro pulmonary platforms. Notably, in vitro strategies using foremost lung-on-chips and biocompatible "soft" membranes have focused on platforms that emphasize phenotypical endpoints recapitulating key physiological and cellular functions. We review some of the most recent in vitro studies underlining seminal therapeutic screens and translational applications and open our discussion to promising avenues of pulmonary therapeutic exploration focusing on liposomes. Undeniably, there still remains a recognized trade-off between the physiological and biological complexity of these in vitro lung models and their ability to deliver assays with throughput capabilities. The upcoming years are thus anticipated to see further developments in broadening the applicability of such in vitro systems and accelerating therapeutic exploration for drug discovery and translational medicine in treating respiratory disorders.

Organoids to model liver disease.

SummaryThe organoid model represents a major breakthrough in cell biology that has revolutionised biomedical research. Organoids are 3D physiological in vitro structures that recapitulate morphological and functional features of in vivo tissues and offer significant advantages over traditional cell culture methods. Liver organoids are of particular interest because of the pleiotropy of functions exerted by the human liver, their utility to model different liver diseases, and their potential application as cell-based therapies in regenerative medicine. Moreover, because they can be derived from patient tissues, organoid models offer new perspectives in personalised medicine and drug discovery. In this review, we discuss the current liver organoid models for the study of liver disease.

Organs-on-chips: into the next decade.

Organs-on-chips (OoCs), also known as microphysiological systems or 'tissue chips' (the terms are synonymous), have attracted substantial interest in recent years owing to their potential to be informative at multiple stages of the drug discovery and development process. These innovative devices could provide insights into normal human organ function and disease pathophysiology, as well as more accurately predict the safety and efficacy of investigational drugs in humans. Therefore, they are likely to become useful additions to traditional preclinical cell culture methods and in vivo animal studies in the near term, and in some cases replacements for them in the longer term. In the past decade, the OoC field has seen dramatic advances in the sophistication of biology and engineering, in the demonstration of physiological relevance and in the range of applications. These advances have also revealed new challenges and opportunities, and expertise from multiple biomedical and engineering fields will be needed to fully realize the promise of OoCs for fundamental and translational applications. This Review provides a snapshot of this fast-evolving technology, discusses current applications and caveats for their implementation, and offers suggestions for directions in the next decade.

(Invited) Nanomaterials and Anticancer Therapies in-Vitro Studies Using Lab-on-a-Chip Microsystems

IntroductionNanomaterials due to their physicochemical properties arouse interest of researchers representing various fields of science, including chemical, biological and clinical analysis. Research in these areas has led to a foundation of nanomedicine, in case of which, both diagnosis and therapy of various diseases are based on nanoparticles (e.g. treatment of infectious or cardiovascular diseases and cancer) [1-3]. Nanoparticles made of biodegradable polymers and lipids, gold- and silver-based nanoparticles, carbon nanotubes, semiconductor-based nanoparticles and many others allow the delivery of therapeutic compounds directly to diseased/altered cells.Lab-on-a-chips for cell cultures studies Lab-on-a-chip microsystems are valuable analytical tools that can be used during both in vitro tests of nanomaterials and development of new therapeutic procedures [4]. Lab-on-a-chip systems enable to precisely control the cells’ microenvironment and overcome the limitations of traditional cell culture methods. In microfluidic systems, a transport of necessary substances (oxygen and nutrients) in appropriate concentrations is ensured. In addition, a flow of a culture medium generates shear stresses, the presence of which affects the functioning of cells and can significantly change the diffusion kinetics and interactions of nanomaterials with a cell membrane. By using the microchannel system, it is possible to obtain concentration gradients of the tested compounds, which better mimic conditions found in living organisms. In such systems, the channels and chambers are characterized by a high surface area to volume (SAV) ratio, which reflects the in vivo conditions (gas diffusion, nutrient transport). The consequence of all the mentioned parameters of microsystems is that we can observe phenomena, which do not occur in the macroscale. Method The microsystems that we used during in vitro studies were made of polymer or polymer and glass. The application of biocompatible and transparent materials for microsystems fabrication allows microscopic observation during tests. Microchambers and microchanels in polydimethylsiloxane (PDMS) were obtained using photolithography and molding method. Both elements (PDMS/PDMS or PDMS/glass) of microsystems were permanently bonded after the use of oxygen plasma. Cell culture in the form of a monolayer (2D model) was obtained when a hybrid system was used, while spherical aggregates (3D model) were obtained using a system made of PDMS. Results and Conclusions In our studies, microsystems were used to assess the cytotoxicity of nanomaterials and test the effectiveness of therapeutic procedures. Biological activity of quantum dots was evaluated and their accumulation inside the cells was monitored. It should be underlined, that the application of microsystems made possible to perform and evaluate the effectiveness of two different therapeutic procedures: electrochemotherapy (ECT) and photothermal therapy (PTT). ECT studies were conducted on a cell monolayer (the most commonly used model in biological studies) in a microsystem with integrated electrodes whereas, the PTT studies were conducted on spheroids (model of the early stage of an avascular tumor).