Exploring Endothelial Expansion on a Chip.

Angiogenesis is the development of new blood vessels from the existing vasculature. Its malfunction leads to the development of cancers and cardiovascular diseases qualified by the WHO as a leading cause of death worldwide. A better understanding of mechanisms regulating physiological and pathological angiogenesis will potentially contribute to developing more effective treatments for those urgent issues. Therefore, the main goal of the following study was to design and manufacture an angiogenesis-on-a-chip microplatform, including cylindrical microvessels created by Viscous Finger Patterning (VFP) technique and seeded with HUVECs. While optimizing the VFP procedure, we have observed that lumen's diameter decreases with a diminution of the droplet's volume. The influence of Vascular Endothelial Growth Factor (VEGF) with a concentration of 5, 25, 50, and 100 ng/mL on the migration of HUVECs was assessed. VEGF's solution with concentrations varying from 5 to 50 ng/mL reveals high angiogenic potential. The spatial arrangement of cells and their morphology were visualized by fluorescence and confocal microscopy. Migration of HUVECs toward loaded angiogenic stimuli has been initiated after overnight incubation. This research is the basis for developing more complex vascularized multi-organ-on-a-chip microsystems that could potentially be used for drug screening.

Versatile and Easily Designable Polyester-Laser Toner Interfaces for Site-Oriented Adsorption of Antibodies.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0-100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

A multilayered cancer-on-a-chip model to analyze the effectiveness of new-generation photosensitizers.

Three-dimensional (3D) cellular models of cancer tissue are necessary tools to analyze new anticancer drugs under in vitro conditions. Diagnostics and treatment of ovarian cancer are major challenges for current medicine. In our report we propose a new three-dimensional (3D) cellular model of ovarian cancer which can mimic a fragment of heterogeneous cancer tissue. We used Lab-on-a-chip technology to create a microfluidic system that allows cellular multilayer to be cultured. Cellular multilayer mimics the structure of two important elements of cancer tissue: flesh and stroma. For this reason, it has an advantage over other in vitro cellular models. We used human ovarian fibroblasts (HOF) and human ovarian cancer cells in our research (A2780). In the first stage of the study, we proved that the presence of non-malignant fibroblasts in co-culture with ovarian cancer cells stimulates the proliferation of cancer cells, which is important in the progression of ovarian cancer. In the next stage of the research, we tested the usefulness of the newly-developed cellular model in the analysis of anticancer drugs and therapies under in vitro conditions. We tested two photosensitizers (PS): free and nanoencapsulated meso-tetrafenylporphyrin, and we evaluated the potential of these drugs in anticancer photodynamic therapy (PDT) of ovarian cancer. We also studied the mechanism of PDT based on the analysis of the level of reactive oxygen species (ROS) in cell cultures. Our research confirmed that the use of new-generation PS can significantly increase the efficacy of PDT in the treatment of ovarian cancer. We also proved that the newly-developed 3D cellular model is suitable for rapid screening of anticancer drugs and has the potential to be used clinically in the future, e.g. in the selection of treatment methods for anticancer personalized medicine.

Studies of anticancer drug cytotoxicity based on long-term HepG2 spheroid culture in a microfluidic system.

Cell-on-a-chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high-throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5-fluorouracil, 5-FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long-term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5-FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5-FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5-FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.

Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer).

Evaluation of biological activity of quantum dots in a microsystem.

The presented work aimed at systematic investigation of biological activity of CdSex S1- x /ZnS and CdSe/ZnS quantum dots (QDs), whose surface was modified with different ligands. For these studies, we used a microfluidic system combined with fluorescence microscopy techniques, which enabled analysis of cells' morphology, viability, and QDs uptake. PDMS and glass-based microfluidic system enabled the precise control of the cell environment, allowed to examine five replications of each tested QDs concentrations (statistically significant number), monitor multiple cellular events, and avoid manual preparation of QDs dilutions. We investigated the influence of the core composition and the type of surface modifiers on QDs toxicity. We also determined whether the examined nanoparticles penetrate into the cells. For all tested nanoparticles, the decrease of cells' viability was observed when increasing nanoparticles concentration. The decrease of live cells' number in microchambers and the accumulation of the nanoparticles around cultured cells were observed. The effect of hydrocarbon chain length of surface modifiers and QDs core composition on the cell viability was confirmed in our tests.

Effect of downscaling on the linearity range of a calibration curve in spectrofluorimetry.

Interest in the microfluidic environment, owing to its unique physical properties, is increasing in much innovative chemical, biological, or medicinal research. The possibility of exploiting and using new phenomena makes the microscale a powerful tool to improve currently used macroscopic methods and approaches. Previously, we reported that an increase in the surface area to volume ratio of a measuring cell could provide a wider linear range for fluorescein (Kwapiszewski et al., Anal. Bioanal. Chem. 403:151-155, 2012). Here, we present a broader study in this field to confirm the assumptions we presented before. We studied fluorophores with a large and a small Stokes shift using a standard cuvette and fabricated microfluidic detection cells having different surface area to volume ratios. We analyzed the effect of different configurations of the detection cell on the measured fluorescence signal. We also took into consideration the effect of concentration on the emission spectrum, and the effect of the surface area to volume ratio on the limit of linearity of the response of the selected fluorophores. We observed that downscaling, leading to an increase in the probability of collisions between molecules and cell walls with no energy transfer, results in an increase in the limit of linearity of the calibration curve of fluorophores. The results obtained suggest that microfluidic systems can be an alternative to the currently used approaches for widening the linearity of a calibration curve. Therefore, microsystems can be useful for studies of optically dense samples and samples that should not be diluted.

Determination of Acid ß-Galactosidase Activity: Methodology and Perspectives.

Early, accurate diagnosis of lysosomal storage disorders is a major challenge, even for trained specialists. Finding innovative, accurate diagnostic methods, and high throughput, cost-effective tools are crucial to medical progress and will contribute to improved quality of life. The goal of this work was to improve currently used protocols to determine activity of acid ß-galactosidase, and discuss the possibility analysing lysosomal enzymes with microfluidic systems. A principle of the determination of ß-galactosidase activity was fluorometric measurement of a deprotonated form of 4-methylumbelliferone released in the enzymatic reaction. Measurements were performed using Jurkat T cells as a source of the enzyme. We observed the temperature-dependent substrate inhibition effect and determined the substrate (4-MU-ß-d-galactopyranoside) concentration which should be used to determine acid ß-galactosidase activity at 37 °C (0.8 mM) and at room temperature (0.6 mM). We proved that the sample incubation time may be significantly reduced to only a few minutes. We also showed that the amount of alkaline buffer used to stop the enzymatic reaction may be minimized and even, in some cases, eliminated. The presented results show how the sensitivity of the available methods to diagnose patients suffer from gangliosidosis GM1 or Morquio B disease can be improved. The proposed method may be easily implemented with microfluidic systems, which currently are promising tools for point-of-care applications.

Review: 3D cell models for organ-on-a-chip applications.

Two-dimensional (2D) cultures do not fully reflect the human organs' physiology and the real effectiveness of the used therapy. Therefore, three-dimensional (3D) models are increasingly used in bioanalytical science. Organ-on-a-chip systems are used to obtain cellular in vitro models, better reflecting the human body's in vivo characteristics and allowing us to obtain more reliable results than standard preclinical models. Such 3D models can be used to understand the behavior of tissues/organs in response to selected biophysical and biochemical factors, pathological conditions (the mechanisms of their formation), drug screening, or inter-organ interactions. This review characterizes 3D models obtained in microfluidic systems. These include spheroids/aggregates, hydrogel cultures, multilayers, organoids, or cultures on biomaterials. Next, the methods of formation of different 3D cultures in Organ-on-a-chip systems are presented, and examples of such Organ-on-a-chip systems are discussed. Finally, current applications of 3D cell-on-a-chip systems and future perspectives are covered.

Graphene oxide internalization into mammalian cells - a review.

Due to the high surface area and convenient functionalization, graphene oxide has a significant potential application in biomedicine. It can serve as multi-purpose platform for bioimaging, gene and drug delivery, photothermal and photodynamic therapy. To implement graphene oxide in diagnostics and therapeutics successfully, it is essential to investigate its mechanisms of uptake into mammalian cells thoroughly. Herein, up to date knowledge about graphene oxide internalization pathways is presented. Nanomaterial lateral dimensions, surface modifications and biotransformation phenomenon as well as a type of the cell may play a pivotal role in a graphene oxide cellular uptake. Hence, the impact of these factors is comprehensively discussed based on so far published studies. Although great progress has been made in elucidating graphene oxide internalization pathway, there are still challenges to overcome. These are discussed along with the prospects concerning further studies in this field of science.

Cellular uptake of biotransformed graphene oxide into lung cells.

Due to its high surface area and convenient functionalization, graphene oxide has many potential applications in biomedicine, especially as a drug carrier. However, knowledge about its internalization inside mammalian cells is still limited. Graphene oxide cellular uptake is a complex phenomenon affected by factors such as the size of the particle and modifications of its surface. Moreover, nanomaterials introduced into living organisms interact with biological fluids' components. It may further alter its biological properties. All these factors must be considered when the cellular uptake of potential drug carriers is considered. In this study, the effect of graphene oxide particle sizes on internalization efficiency into normal (LL-24) and cancerous (A549) human lung cells was investigated. Moreover, one set of samples was incubated with human serum to determine how the interaction of graphene oxide with serum components affects its structure, surface, and interaction with cells. Our findings indicate that samples incubated with serum enhance cell proliferation but enter the cells with lesser efficiency than their counterparts not incubated with human serum. What is more affinity towards the cells was higher for larger particles.

Effect of a high surface-to-volume ratio on fluorescence-based assays.

In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale.

Investigation of the Therapeutic Potential of New Antidiabetic Compounds Using Islet-on-a-Chip Microfluidic Model.

Nowadays, diabetes mellitus is one of the most common chronic diseases in the world. Current research on the treatment of diabetes combines many fields of science, such as biotechnology, transplantology or engineering. Therefore, it is necessary to develop new therapeutic strategies and preventive methods. A newly discovered class of lipids-Palmitic Acid Hydroxy Stearic Acid (PAHSA) has recently been proposed as an agent with potential therapeutic properties. In this research, we used an islet-on-a-chip microfluidic 3D model of pancreatic islets (pseudoislets) to study two isomers of PAHSA: 5-PAHSA and 9-PAHSA as potential regulators of proliferation, viability, insulin and glucagon expression, and glucose-stimulated insulin and glucagon secretion. Due to the use of the Lab-on-a-chip systems and flow conditions, we were able to reflect conditions similar to in vivo. In addition, we significantly shortened the time of pseudoislet production, and we were able to carry out cell culture, microscopic analysis and measurements using a multi-well plate reader at the same time on one device. In this report we showed that under microfluidic conditions PAHSA, especially 5-PAHSA, has a positive effect on pseudoislet proliferation, increase in cell number and mass, and glucose-stimulated insulin secretion, which may qualify it as a compound with potential therapeutic properties.

The 10th anniversary of MXenes: Challenges and prospects for their surface modification toward future biotechnological applications.

A broad family of two-dimensional (2D) materials - carbides, nitrides, and carbonitrides of early transition metals, called MXenes, became a newcomer in the flatland at the turn of 2010 and 2011 (over ten years ago). Their unique physicochemical properties made them attractive for many applications, highly boosting the development of various fields, including biotechnological. However, MXenes' functional features that impact their bioactivity and toxicity are still not fully well understood. This study discusses the essentials for MXenes's surface modifications toward their application in modern biotechnology and nanomedicine. We survey modification strategies in context of cytotoxicity, biocompatibility, and most prospective applications ready to implement in medical practice. We put the discussion on the material-structure-chemistry-property relationship into perspective and concentrate on overarching challenges regarding incorporating MXenes into nanostructured organic/inorganic bioactive architectures. It is another emerging group of materials that are interesting from the biomedical point of view as well. Finally, we present an influential outlook on the growing demand for future research in this field.

A microfluidic device with fluorimetric detection for intracellular components analysis.

An integrated microfluidic system that coupled lysis of two cell lines: L929 fibroblasts and A549 epithelial cells, with fluorescence-based enzyme assay was developed to determine ß-glucocerebrosidase activity. The microdevice fabricated in poly(dimethylsiloxane) consists of three main parts: a chemical cell lysis zone based on the sheath flow geometry, a micromeander and an optical fibers detection zone. Unlike many methods described in literature that are designed to analyse intracellular components, the presented system enables to perform enzyme assays just after cell lysis process. It reduces the effect of proteases released in lysis process on determined enzymes. Glucocerebrosidase activity, the diagnostic marker for Gaucher's disease, is the most commonly measured in leukocytes and fibroblasts using 4-methylumbelliferyl-ß-D-glucopyranoside as synthetic ß-glucoside. The enzyme cleavage releases the fluorescent product, i.e. 4-methylumbelliferone, and its fluorescence is measured as a function of time. The method of enzyme activity determination described in this paper was adapted for flow measurements in the microdevice. The curve of the enzymatic reaction advancement was prepared for three reaction times obtained from application of different flow rates of solutions introduced to the microsystem. Afterwards, determined ß-glucocerebrosidase activity was recalculated with regard to 10(5) cells present in samples used for the tests. The obtained results were compared with a cuvette-based measurements. The lysosomal ß-glucosidase activities determined in the microsystem were in good correlation with the values determined during macro-scale measurements.

Islet-on-a-chip: Biomimetic micropillar-based microfluidic system for three-dimensional pancreatic islet cell culture.

Type 2 diabetes is currently one of the most common metabolic diseases, affecting all ages worldwide. As the incidence of type 2 diabetes increases, a growing number of studies focus on islets of Langerhans. A three-dimensional research model that maps islet morphology and maintains hormonal balance in vivo is still needed. In this work, we present an Islet-on-a-chip system, specifically a micropillar-based microfluidic platform for three-dimensional pancreatic islet cell culture and analysis. The microfluidic system consisted of two culture chambers that were equipped with 15 circular microtraps each, which were built with seven round micropillars each. Micropillars in the structure of microtraps supported cell aggregation by limiting the growth surface and minimizing wall shear stress, thereby ensuring proper medium diffusion and optimal culture conditions for cell aggregates. Our system is compatible with microwell plate readers and confocal laser scanning microscopes. Because of optimization of the immunostaining method, the appropriate cell distribution and high viability and proliferation up to 72 h of culture were confirmed. Enzyme-linked immunosorbent assays were performed to measure insulin and glucagon secretion after stimulation with different glucose concentrations. To our knowledge, this is the first Lab-on-a-chip system which enables the formation and three-dimensional culture of cell aggregates composed of commercially available α and ß pancreatic islet cells. The specific composition and arrangement of cells in the obtained model corresponds to the arrangement of the cells in rodent pancreatic islets in vivo. This Islet-on-a-chip system may be utilized to test pathogenic effectors and future therapeutic agents.

Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System.

Regenerative medicine and stem cells could prove to be an effective solution to the problem of treating heart failure caused by ischemic heart disease. However, further studies on the understanding of the processes which occur during the regeneration of damaged tissue are needed. Microfluidic systems, which provide conditions similar to in vivo, could be useful tools for the development of new therapies using stem cells. We investigated how mesenchymal stem cells (MSCs) affect the metabolic activity of cardiac cells (rat cardiomyoblasts and human cardiomyocytes) incubated with a potent uncoupler of mitochondrial oxidative phosphorylation under microfluidic conditions. A cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to mimic disfunctions of mitochondria of cardiac cells. The study was performed in a microfluidic system integrated with nanofiber mats made of poly-l-lactid acid (PLLA) or polyurethane (PU). The microsystem geometry allows four different cell cultures to be conducted under different conditions (which we called: normal, abnormal-as both a mono- and co-culture). Metabolic activity of the cells, based on the bioluminescence assay, was assessed in the culture's performed in the microsystem. It was proved that stem cells increased metabolic activity of cardiac cells maintained with FCCP.

Simulation of hypoxia of myocardial cells in microfluidic systems.

The paper presents a newly designed microfluidic system that allows simulation of myocardial hypoxia by biochemical method. The geometry of the microsystem was designed in such a way, that quantitative fluorescent measurements using a spectrofluorometric plate reader was possible. Biochemical simulation of hypoxia was carried out using potent mitochondrial oxidative phosphorylation uncoupler-Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). Two cardiac cell lines were used in the study-rat cardiomyoblasts (H9C2) and human cardiomyocytes. The effectiveness of biochemical simulation of hypoxia was studied using two fluorescent dyes: carbocyanine iodide (JC-1) and Fluo-4. Changes in the mitochondrial membrane potential and concentration of intracellular calcium ions were tested. The major novelty of this research was the applying the microfluidic system to create hypoxia conditions for cardiac cells using the biochemical approach. In further studies, the presented hypoxia model could be used to develop new methods of treatment of ischemic heart disease for example in cell therapy based on stem cells.

Synergistic effect of the combination therapy on ovarian cancer cells under microfluidic conditions.

Ovarian cancer belongs to the group of gynecological cancers and indicates the high resistance to many drugs used in standard anticancer therapy. The treatment of ovarian cancer is a big challenge for the present medicine. In our report we tested the effectiveness of the combination anticancer therapy against ovarian cells: human ovarian carcinoma (A2780) and human ovarian fibroblasts (HOF). Two different types of drugs were used: doxorubicin (DOX) and a new-generation photosensitizer, nanoencapsulated meso-tetraphenylporphyrin (nano-TPP). The aim of the research was to compare the effect of the sequential combination therapy (chemotherapy with DOX and photodynamic therapy with nano-TPP) carried out in static and dynamic conditions. To achieve dynamic culture conditions, similar to in vivo environment, we designed a new microfluidic system in which the simultaneous, independent cultures of two cell lines (non-malignant and cancer cells) and their one-step analysis were possible. We observed that the sequential combination of photodynamic therapy (PDT) with chemotherapy allowed to obtain the synergistic effect of the treatment with using low doses of drugs. We also confirmed that the use of microfluidic conditions significantly increased the effectiveness of combination therapy and allowed for maintaining a high selectivity of the action of drugs on cancer cells. To the best of our knowledge, for the first time the microfluidic system was used to carry out sequential combination therapy against ovarian cancer.

Combinations of regenerative medicine and Lab-on-a-chip systems: New hope to restoring the proper function of pancreatic islets in diabetes.

Cases of type 2 diabetes mellitus have significantly increased in recent years. Researchers worldwide are combining their knowledge of biology, medicine, tissue engineering, and microtechnology to develop new effective treatments. An important aspect of current research is to develop of a complete model of three-dimensional pancreatic islets to test various factors that affect disease development and evaluate new therapies and drugs. Several methods have allowed the development of three-dimensional research models. The use of Lab-on-a-chip systems with appropriate microstructure geometry is a promising solution to macroscale problems. Such a device allows the development of a complete platform reflecting conditions that prevail in the body. Organ-on-a-chip platforms are successfully used mainly in studies of lung, heart, and liver diseases. This review presents the current state of knowledge on the creation of three-dimensional pancreatic islet structures in both microscale and microfluidic systems. We highlight the most important aspects of developing the geometry of such devices. We also discuss analytical detection methods that are suitable for detecting hormones that are secreted from pancreatic islets and, in combination with appropriate Lab-on-a-chip systems, can be used as a Micro Total Analysis System (µTAS).