Photodynamic therapy procedures in the microfluidic system

Evaluation of the effi ciency of photodynamic therapy (PDT) in a hybrid microfl uidic culture system was studied. The geometry of the utilized microsystem for PDT procedures consists microchambers for cell culture and microchannels, which create a concentration gradient generator (CGG). 5-aminolevulinic acid (ALA) as a precursor of the photosensitizer was used. The geometry of the microchip allowed to test diff erent concentrations of ALA in a single assay. Evaluation of the effi ciency of photodynamic therapy was determined 24 hours after PDT procedure (irradiation with light which induced accumulated in carcinoma cells). The performed microsystem contained two independent micropatterns, that enables examination simultaneously various cell lines (carcinoma and normal) or various photosensitizers

Miniaturized device for a cell lysis process

Single-cell studies are crucial for gaining knowledge on complexity of intracellular processes. In many cases, carrying researches into cell ingredients must be proceeded by a lysis process. Cell lysis leads to disintegration of the plasma membrane which is the barrier separating cell contents from the environment. However, investigations at the cellular level would not be possible without proper miniaturized tools, which offer many advantages as low reagents consumption, short reaction time, integration, automation or versatility. The goal of this work was to design and develop a microfluidic chip for a chemical cell lysis process. The geometry of a microsystem presented is based on the hydrodynamic focusing of a cell suspension stream. Applying non-denaturing cell lysis buffer enables to analyze released cell ingredients during next steps of investigations

Microfluidic devices — application in anticancer studies

A rapidly growing pharmaceutical industry requires faster and more efficient ways to find and test new drugs. One of the new method for cell culture and examining the toxic effects of drugs is application of microfluidic systems. They provide new types of microenvironments and new methods for investigation of anticancer therapy. The use of microsystems is a solution that gives the opportunity to reduce not only cost and time, but also a number of tests on animals. In this paper we present designed and fabricated hybrid microfluidic systems which are applicable for cell culture, cell based cytotoxicity assays and photodynamic therapy procedures. Polydimethylsiloxane (PDMS) and sodium glass were used for fabrication of microdevices. The designed geometry of the microdevices includes cell culture microchambers and a concentration gradient generator (CGG). The CGG enables to obtain diff erent concentrations of tested drugs in a single step, which is a significant simplification of cytotoxicity assay procedure. In the designed microsystems three various cell lines (normal and carcinoma) were cultured and analyzed

Novel designs and technologies for cell engineering

Microfluidic devices, such as lab-on-a-chip systems, are highly advantageous for cell engineering and cell based assays. It is a particularly useful approach for development of the in vitro cellular systems mimicking the in vivo environment. In this paper, a novel lab-on-a-chip device for three-dimensional human cell culture and anticancer drug testing is presented. Cells were cultured as Multicellular Tumor Spheroids (MCTS) — the best cancer tumor model developed so far. Diff erent designs were tested and novel technique of microfabrication in poly(dimethylsiloxane) was developed. MCTS were cultured in a system of polymeric microwells, with the network of microfluidic channels for culture medium flow. Design included optimal shear stress and proper nutrients supply for cultured cells. Final design provided MCTS culture for four weeks with the homeostasis-like state achievement, which is characteristic for the in vivo situation

Development of a Point-of-Care system for early diagnostics of genetic diseases

Lysosomal storage disorders (LSDs) represent a group of more than 45 genetically inherited diseases caused by the absence or deficiency of one or more specific lysosomal enzymes. Nowadays, there is a lack of reports on fast, reliable methods for the diagnostics of LSDs. Currently applied diagnostic approaches generate many false-negative and false-positive results, which results in classification of patients to inappropriate therapeutic groups. Moreover, these methods are time-consuming (even 20 hours), and are carried out only in a few laboratories in the world. The goal of this work was to develop a method and a tool, a Point-of-Care system, for diagnostics of LSDs. The polymeric microdevice consists of a cell lysis module, a mixing microchannel and an optical detection module. The system enables to determine the activity of α-galactosidase (deficient in Fabry disease), and to reduce the time of analysis to 10 min. Due to its easy fabrication steps and low price, it seems to be a prospective tool for a point-of-care approach

‘Lab-on-a-chip’ for cell engineering: towards cellular models mimicking in vivo

One of the main scopes of modern cell engineering is development of cellular models that can replace animals in drug screening and toxicological tests, so called alternative methods. Construction of the alternative model is a very challenging task due to a richness of factors creating the in vivo environment. The monolayer cell culture — cultivation of adhesive cells on artificial surfaces such as glass or polymer — lack most of the in vivo-like interactions, but still is the only tool for the majority of applications. One of the most prospective approaches on mimicking in vivo environment is “Lab-on-a-chip” technology. Microfluidic devices offer lots of advantages over traditional in vitro culture, e.g. much higher cell volume-to-extracellular fluid volume ratio or possibility of regulation of hydrodynamic stress. This presentation aims to introduce latest advances of our team in microfluidic cell culture devices. Our novel approach is to cultivate three dimensional multicellular aggregates (spheroids) in microenvironments arranged in a microfluidic system. The geometry and materials of the system allow for cultivation, observation and analysis of multicellular spheroids. The results presented concern multicellular tumor spheroids (MCTS) rising from human cancer cells, which are considered to represent most of the conditionings of cancer tumor in vivo. The fully developed MCTS microdevice will be a reliable tool for anticancer drug screening, as the results most likely will be in a close accordance with the results obtained in vivo

New microfluidic device for lactate dehydrogenase (LDH) activity analysis

In this paper, we present cytotoxicity analysis (determination of lactate dehydrogenase — LDH activity performed in a designed and fabricated microfl uidic system. This method allowed for analysis of a supernatant collected from A549 (human lung cancer) and HT-29 (human colon cancer epithelial) cells, which were incubated for 24 h with selected compounds. LDH is an intracellular enzyme present in tissues, which is released into the supernatant caused by membrane damage or cell lyses. In our tests, LDH-Cytotoxicity Assay Kit (BioVision) was used. The toxic eff ect of drugs was measured in the developed microsystem made of PDMS (poly(dimethylsiloxane)). Analytical reaction took place in the special designed microchannel geometry. Then, the LDH activity was measured at 490 nm using spectrophotometer. In subsequent experiments, appropriate conditions for measurements using a microfl uidic system were chosen. It was found that the selected reagent is sensitive to temperature changes and light exposure. Reaction time in the microsystem was determined by changes of fl ow rates of reagents. Afterwards, for the various reaction time, the toxic eff ect of 5-fl uorouracil, celecoxib and 1,4-dioxane was performed. The obtained results were compared with the results carried out in 96-well plates. Based on these results, it was noted that the enzymatic reaction time in the microsystem is shorter than in 96-well plate. Moreover, the advantage of using microsystem is also the small amount of reagents

An integrated gas removing system for microfluidic application. Design and evaluation

Microfluidic systems are used in a wide range of applications, including medical diagnostics, cell engineering and bioanalytics. In this work we focused on “Lab-on-a-chip” microsystems for cell cultivation. A troublesome problem of gas bubbles entering microdevices causing signal interferences and cells damage was emphasized. A novel, integrated debubbler in the form of cylindrical traps covered with thin PDMS membrane was designed and manufactured. Demonstrated debubbler was successfully applied in a long-lasting culture of HT-29 cell aggregates

Potentiometric detection of the metabolic activity of human tumor cells

Monitoring of cellular viability is a key part of toxicological assays in vitro. On-line monitoring of metabolic activity would be particularly useful for evaluation of responses to potential therapeutic compounds. Current assays are mostly based on fluorescent dyes and optical detection methods. These methods offer high sensitivity and specificity, however are not suitable for long-term on-line observations. Electrochemical methods can be an alternative for current protocols. Electrochemical detection is low cost and label-free, therefore suitable for long-term cell culture monitoring. In this work investigations on human cancer cells viability will be presented. Cells were cultured as two-dimensional monolayer or three-dimensional spheroids. Different cell culture media were examined. Potentiometric detection was used for continuous monitoring of cell culture as well as end-point investigations. Different growth phases were identified using applied method. Finally, response to an anticancer drug was successfully observed