MICROFLUIDICS-BASED SINGLE CELL ISOLATION AND TRAPPING OF CANINE CUTANEOUS MAST CELL TUMOR CELLS

Abstract

In this research, the primary objective was to develop a microfluidic platform utilized for the size-based single cell sorting of canine cutaneous mast cell tumor (MCT) cells which were categorized into 4 groups; I, II, III and IV, upon their individual sizes ranged from 5-9, 10-14, 15-19, and equal to or over than 20 mm respectively. And the secondary aim was to establish the microfluidic-based entrapping microdevice which had a capacity to ensnare single MCT cells into its triangular microwell array at the ratio of one cell per one microwell. The study consequence consistently indicated the accomplishment of device developments. All microfluidic devices were designed and fabricated under the standard soft-lithography. Finally, two microfluidic devices were constructed; the Archimedian spiral microchannel (ASM) and the triangular microwell array. The configuration of ASM consisted of 5-turned, 500mm-wide x 130mm-high, rectangular spiral microchannel with 720mm-long conical exit microchannel separated into 10 asymmetric exit slots. Meanwhile, the entrapment microdevice were composed of the array of 40mm-high x 15mm-deep triangular microwell with 160mm-high main flow channel. The study result indicated that, at the flow rate 1 ml. min-1, the spiral microchannel had a capability to separate single MCT cells in group III and IV, which were the targeted cells, out of the MCT cells group I and II, based upon their sizes. However, the detrimental effects of fluid shear stress and extensional stress in the spiral microchannel, exhibited by the computational simulations, were required an amendment. Because, under hemocytometry quantification, trypan blue and TMRM staining, light-microscopy and SEM morphological assessments and leaky DNA assays, they suggested that the amount of sorted MCT cells was significantly reduced and the cell vitality was only 40 percent. Meanwhile, the morphological assays also confirmed cell elongation and/or wreckage causing the 2.4-fold increment of leaky DNA. For single MCT cell entrapment, the study consequence exhibited that, under pulsatile flow (0. 01 ml. h-1 inflow rate for 30 sec and 3min pausing interval), the trapping microdevice had an ability to stochastically entrap MCT cells in its triangular microwell array. And the efficacy of the microdevice for single and multiple MCT cell entrapment was 35% and 15% respectively. In the epilogue, this study is the first report on the attainments in microfrabication engaged in veterinary field for size-based single MCT cell sorting and entrapment. In addition, there are a tendency to merge these two microdevice components together to form a complete microfluidic-based single cell sorting, trapping and culturing on a chip which is essentially benefit to biomedical engineering researches in the future.