Polymer-Glass Microfluidic Device for Single Cell Photodynamic Therapy Evaluation Krzysztof Maleckia*, Adam Bakalab, Olga Adamowiczc, Nikodem Szymanskic, Michal Chudyc, Artur Dybkoc, Zbigniew Brzozkac, Andreas Schneiderd, Samuel Serrad, Werner Brennera a
Institute of Sensor and Actuator Systems Vienna University of Technology, Austria Division of Pharmacology and Toxicology, Warsaw Agricultural University, Poland c Department of Analytical Chemistry Warsaw University of Technology, Poland d Rutherford Appleton Laboratory EID/Central Microstructure Facility CCLRC-RAL , United Kingdom
Photodynamic therapy (PDT) uses laser, or other light sources, combined with a light-sensitive drug (sometimes called a photosensitising agent) to destroy cancer cells. The utility of 5-aminolevulinic acid (ALA) has been recognized in many different treatment fields over the past decade. Following administration of exogenous ALA, the endogenous photosensitizer, protoporphyrin IX (PpIX), accumulates in tumour tissue. The target tissue is exposed to light. The optical activation of the endogenous photosensitizer causes the generation of singlet oxygen, which is very reactive and results in cell killing. In order to study this process for one or few cells, a miniaturised module, so-called cell docking module was designed and fabricated so as to pick up some cells in controlled way. fully transparent device Cell docking SU-8 photoresist Inlets two PYREX plates different versions of the device (5, 50 and 150 μm width and 30, 60 μm depth of channels) • single photolithographic process • glass / SU-8 / glass multilayer adhesive bonding
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The flowing cell tracks were simulated with FLUENT® and the boundary flow conditions for high efficiency cells trapping (90%) were obtained (inlet: 0,02ml/min, outlet: -3,45Pa).
chip design
SU-8 5μm channels
Cell traps geometry
cells simulation
PYREX / SU-8 chip
– SEM micrograph
conical-ended cell traps
MONOSTRATAL CULTURE OF ISOLATED HEPATOCYTES
2 h
1 h
90% 80% 70% 60% 50 % 40% 30% 20% 10% 1 h
0% 0 ,0 0 0 1
0 ,0 0 1 A L A [mM]
4 h
0 ,0 1
0 ,1
1
10
Ti
e
2 h
0
tim
Once the cells are trapped and docked in the seats the light exposition is run and the cell behaviour can be recorded and analysed using an optical microscope. In this way, a highly precise, digital like, read-out signal, with a single cell as one object (bit), is obtained and allows the determination of the accuracy of the diagnosis.
4 h
Cytotoxic effect of ALA
1 0 0%
hepatocytów
A miniaturised fluidic system, was designed and fabricated in view of an analytical study of the efficiency of photodynamic therapy on live single cells. The cell and therapy effect observation allow the medical doctors to optimise parameters of the patient treatment, i.e. the dose of optical radiation, time of exposition, concentration of the photosensitising agent. Investigations on fabrication processes were performed and optimal process parameters for polymer SU-8 and over glass bonding technologies were stabilised.
Isolated cells were seeded on 96-well plates with black walls The suspension of hepatocytes in the WE medium with addition of: FBS, ampholytic B, penicillin G and streptomycin. Next, the plate with hepatocytes was placed in the incubator in 37oC in 95% O2 and 5% CO2 atmosphere. 5-aminoevulinic acid (ALA) (prepared in animal medium in a concentration range of 0.0001-10 mM ) was introduced to the cells and then the cells were incubated in 37oC. After 1, 2 and 4 hours of incubation the cells had been exposed to the light (λexp625nm, texp30s) generated by laser LED.
hepatocytes viability
CONCLUSIONS
MODELING
TECHNOLOGY
PHOTODYNAMIC THERAPY
b
The highest decrease of the cells viability was observed after 4 hours of incubation in culture medium contained exogenous ALA. The decrease of the cells viability was observed with the increase of ALA concentration applied during incubation.
ACKNOWLEDGEMENTS This work has been financially supported by FP6 Marie Curie Research Training Network “Advanced Methods and Tools for Handling and Assembly in Microtechnology - ASSEMIC”, funded by the European Commission, Contract number: MRTN-CT-2003-504826 This work has been integrated in the European Commission Project „Network of Excellence in Multi-Material Micro Manufacture” - 4M-NoE, contract number: NMP2-CT-2004-500274 CORRESPONDING AUTHORS Krzysztof Malecki - e-mail:
[email protected], phone: +43 1 58801 76639, fax: +43 1 58801 36698, http://www.isas.tuwien.ac.at; Michal Chudy - e-mail:
[email protected], phone: +48 22 660 58 25, fax: +48 22 660 58 25, http://csrg.ch.pw.edu.pl/ * Krzysztof Malecki is a fellow of Marie Curie Research Training Network “ASSEMIC”, of the European Community, Contract no. MRTN-CT-2003-504826
