Karina Ziółkowska

Microfluidic devices as tools for mimicking the in vivo environment

One of the major branches of microfluidic development is cell engineering. A number of devices for cell cultivation, lysis, single-cell analysis and cell-based toxicity tests have been reported in the literature. The variety of structures that can be created leads to devices more closely mimicking the in vivo environment than classic cell cultures. Studies on this topic will have an effect on the evaluation of methods that can replace animals in biomedical research. The aim of this review is to present latest advancements of “lab-on-a-chip” for cell cultivation and engineering. The authors focus on the achievements leading to in vivo-like methods. The materials and fabrication methods in silicon, glass, PDMS and other polymers were briefly characterized. Microfluidic devices were applied for mimicking the in vivo environment at various levels of mammalian body organization—from the surroundings of single cells to interactions between functional organs. Solutions for “human-on-a-chip”, perfusion cell cultures, extracellular matrix analogues, microscaffolds, spheroid formation and co-cultures were reviewed in this paper. The presented solutions have the potential to become new cellular models for toxicology, drug development and biomedical research.

Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip.

In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The three-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for four weeks. After two weeks of the culture growth slowdown and stop were observed and high cell viability was determined within next two weeks. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concentrations. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diameter can be observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-fluorouracil. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. The presented microfluidic system is a solution for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free and easy-to-conduct mean of a quantitative determination of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture.

Miniaturized tools and devices for bioanalytical applications: an overview

This article presents an overview of various miniaturized devices and technologies developed by our group. Innovative, fast and cheap procedures for the fabrication of laboratory microsystems based on commercially available materials are reported and compared with well-established microfabrication techniques. The modules fabricated and tested in our laboratory can be used independently or they can be set up in different configurations to form functional measurement systems. We also report further applications of the presented modules e.g. disposable poly(dimethylsiloxane) (PDMS) microcuvettes, fibre optic detectors, potentiometric sensors platforms, microreactors and capillary electrophoresis (CE) microchips as well as integrated microsystems e.g. double detection microanalytical systems, devices for studying enzymatic reactions and a microsystem for cell culture and lysis.

Substrate inhibition of lysosomal hydrolases: α-Galactosidase A and β-glucocerebrosidase

OBJECTIVE We have investigated the kinetics of α-galactosidase A and β-glucocerebrosidase deficient in Fabry and Gaucher diseases, respectively. DESIGN AND METHODS We have performed spectrofluorymetric measurements of the activity of enzymes using a derivative of 4-methylumbelliferone as a substrate and a human T-cell line as a source of enzymes. RESULTS We have observed the substrate inhibition effect, which is related to temperature. CONCLUSIONS The diagnostic procedures for Fabry and Gaucher diseases used now in laboratory practice neglect temperature-dependent substrate inhibition, which may significantly reduce the sensitivity of enzyme activity determinations.

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

AbstractIn 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. FigureDependence of the linear range on surface-to-volume ratio in microfluidic detection.

7.1.2 A novel tool for biochemical diagnostics of rare genetic disorders: an integrated microfluidic system with optical detection

In this work, an integrated microfluidic system for biochemical diagnostics of lysosomal storage disorders is presented. The polymeric microsystem consists of a zone for hydrodynamic focusing of cell suspension, a mixing microchannel, and an optical detection module. The system was used for determination of the activity of enzymes deficient in Fabry and Gaucher diseases. On the contrary to the currently used protocols of determination of lysosomal enzymes’ activities, the microdevice enables significant reduction of the time of analysis. Moreover, the experimental set enables to avoid termination of enzymatic reaction and sample dilution, what increases sensitivity of the developed method. Due to easy fabrication steps and their low cost, the system seems to be a prospective tool for a point-of-care approach.