A Microfluidic Chip For Cell-based Assays

Abstract Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line, mesenchymal stem cells, HeLa cells, bovine endothelial, human breastdenocarcinoma cell. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.In vitro mammalian cell culture is an essential and powerful tool in biological science. It has made substantial contribution to the understanding of many phenomena, such as intracellular enzyme activities, intracellular flux, and cell-cell interactions, since it originated over one century ago. In recent years, cell culture in high-density array formats, including microfluidic arrays, has attracted tremendous interest due to the potential for rapid large scale cell-based assays. Many microfluidic devices have been developed for cell culture and to study intracellular enzyme activities, cellular responses to chemical gradients, cellular differentiation, dynamic gene expression and cell cytotoxicity screening. Some microfluidic devices were designed for cell culture purpose in a high-throughput manner. However, few devices can establish a uniform distribution of cells in whole microfluidic cell array. As the research emphasis shifts from bulk cell culture, where the behavior of tens of thousands of cells are assayed in individual microwells, to research at the single cell level, cell culture viability and standarized, uniform growth conditions become increasingly important. In bulk culture formats, variations in cell seeding density, nutrient delivery and waste removal are sources of stress on cell cultures that introduce intracellular variations in subsequen assay procedures. Moreover, traditional culture methodology requires technical expertise and specific facilities to handle cells harvesting, media exchanging and cell sub-culturing procedures. In this paper, we describe the development of a continuous microfluidic cell culture platform, in which cells can be repeatedly grown to confluence, trypsinized and recovered. Integrated arrays of U-shaped sieves within round microchambers promote uniform spatial seeding of small clusters of cells (~10/sieve; ~100/chamber), while the ability to tightly regulate the media delivery and removal from the chambers promotes cell viability. The main research results are as follows:1. The silicon wafer is O2 plasma modificated for 10s before spinning; SU-8-10 photoresist spinning at 750 rpm(20μm)for 1min; Soft bake the wafer for 5min at 60℃and 8min at 90℃, moderate bake 1min at 60℃and 3min at 90℃; time of exposure is 18s, light intensity: 40mW/cm2; develop for 3min;drying at 90℃.2. U-shaped sieves within round microchambers promote uniform spatial seeding of small clusters of cells (~10/sieve; ~100/chamber).3. Cell loading within the individual sieves of the microfluidic array was quite uniform and the deviaton of cell number in per sieve can be controlled within 10%.4. With this platform,we also successfully cultured BALB/3T3 cells,mesenchymal stem cells,HeLa cells, and bovine endothelial cells,and Cells spreading and maintenance of morphology in the micro-fluidic cell culture device was similar to that observed on tissue culture plastic.5. Cells spreading and maintenance of morphology in the micro-fluidic cell culture device was similar to that observed on tissue culture plastic, and cell viability was over than 98% after five subculture cycles measured with a LIVE/DEAD viability stain .6. The FACS Assay and on-immunofluorescence assay indicated that UC-hMSCs expressed CD44, CD73, CD105, but no hematopoietic lineage markers, such as CD34, CD45, HLA-DR.