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. Th is 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. Th e 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. Th en, 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. Th e 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.

[1]  H. Wolterbeek,et al.  Optimization, application, and interpretation of lactate dehydrogenase measurements in microwell determination of cell number and toxicity. , 2005, Assay and drug development technologies.

[2]  Elisabeth Verpoorte,et al.  Microfluidic biochip for the perifusion of precision‐cut rat liver slices for metabolism and toxicology studies , 2010, Biotechnology and bioengineering.

[3]  Gale Haslam,et al.  Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities , 2004, Cytotechnology.

[4]  V. Adleff,et al.  Enhancement of 5-Fluorouracil Efficacy on High COX-2 Expressing HCA-7 Cells by Low Dose Indomethacin and NS-398 but not on Low COX-2 Expressing HT-29 Cells , 2009, Pathology & Oncology Research.

[5]  John Greenman,et al.  Study of ethanol induced toxicity in liver explants using microfluidic devices , 2011, Biomedical microdevices.

[6]  Andrew L. Niles,et al.  In Vitro Viability and Cytotoxicity Testing and Same-Well Multi-Parametric Combinations for High Throughput Screening , 2009, Current chemical genomics.

[7]  Andreas Zimmer,et al.  A practical note on the use of cytotoxicity assays. , 2005, International journal of pharmaceutics.

[8]  Hannu Uusitalo,et al.  The effects of 5-fluorouracil on ocular tissues in vitro and in vivo after controlled release from a multifunctional implant. , 2009, Investigative ophthalmology & visual science.

[9]  Soo-Jeong Lim,et al.  Cyclooxygenase-independent down-regulation of multidrug resistance–associated protein-1 expression by celecoxib in human lung cancer cells , 2005, Molecular Cancer Therapeutics.

[10]  J. Pfeilschifter,et al.  Cerivastatin inhibits proliferation of interleukin-1 beta-induced rat mesangial cells by enhanced formation of nitric oxide. , 2004, European journal of pharmacology.

[11]  J. Rhee,et al.  Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells. , 2007, World journal of gastroenterology.

[12]  M Balls,et al.  Methods of in vitro toxicology. , 2002, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[13]  Sanjeev Kumar Mahto,et al.  in vitromodels, endpoints and assessment methods for the measurement of cytotoxicity , 2010, Toxicology and Environmental Health Sciences.

[14]  J. B. Griffiths,et al.  Use of lactate dehydrogenase release to assess changes in culture viability , 1990, Cytotechnology.

[15]  Michal Chudy,et al.  PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage , 2010 .

[16]  Laurent Griscom,et al.  Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor , 2011 .

[17]  John Greenman,et al.  A Microfluidic System for Testing the Responses of Head and Neck Squamous Cell Carcinoma Tissue Biopsies to Treatment with Chemotherapy Drugs , 2011, Annals of Biomedical Engineering.

[18]  Brian B. Hasinoff,et al.  The cytotoxicity of celecoxib towards cardiac myocytes is cyclooxygenase-2 independent , 2007, Cardiovascular Toxicology.