Fluidic Culture and Analysis of Pulmonary Artery Smooth Muscle Cells for the Study of Pulmonary Hypertension

Abstract

There is an urgent need to develop novel in-vitro models to mimic the disease conditions in pulmonary hypertension (PH). We developed a microfluidic cell culture device for PH studies that withstood high shear stress. Techniques were also developed for cell recovery from the microchannel and mRNA isolation from the collected cells. Using this device, we found that shear stress caused a 7.5-fold increase in the transcription levels of a PH-related molecule, Cyclin D1.

Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel

“…We have also reported the effects of various culture conditions of vascular endothelial cells, vascular smooth muscle cells, and lymphatic endothelial cells in microchannels. 1,2,[20][21][22] We confirmed that the number of immortalized human microvascular endothelial cells (HMEC-1) was increased day by day in a microchannel with a depth smaller than 100 μm, 22 whereas there was no such increase in the numbers of human dermal microvascular endothelial cells-neonatal (HMVEC-dBINeo) and human dermal lymphatic microvascular endothelial cells-neonatal (HMVEC-dLyNeo). Moreover, although these primary cells proliferated in a conventional polystyrene Petri dish culture, no proliferation was observed in our 65-μm-deep microchannel.…”

“…Microchannels were fabricated by a molding process as described previously. 2,24 In brief, the degassed PDMS mixture was poured onto a master to a thickness of 4 mm, which has channel structure of 0.065 × 0.3 × 20, 0.25 × 1 × 10, or 0.5 × 1 × 10 mm (H × W × L), and then baked at 65 C for 1 h. The PDMS replica was peeled off from the master and adhered to a glass slide (26 × 76 mm), and then baked further at 100 C for 1 h. Through-holes were made at both ends of the microchannel with a 1.0-mm biopsy punch. Both bonding surfaces of the PDMS sheet and a cover slip were exposed to oxygen plasma at 100 W for 35 s.…”

“…In general, cells in a shallow microchannel are seeded at a higher density compared to that used in a cell culture flask to achieve a sufficient density for confluence. 1,2 To investigate the effect of a lower seeding density, the cells were seeded in the microchannel at the normal density used in the conventional culture method (∼50 cells/mm 2 ).…”

“…A microfluidic device is a new cell culture tool that can provide complex microscale structures with well-controlled parameters to mimic organ structures and the physical environment in the body. [1][2][3][4][5] Moreover, the development of a microfluidic organ model, named organ-on-a-chip, has become a widely used tool for bioassays, and has proven to be especially useful for drug development as an alternative approach to animal testing, emerging as a major topic in bioanalytical chemistry. [6][7][8][9][10][11][12][13][14][15][16] Since a microchannel has a higher culture area-to-volume ratio compared to a conventional cell culture dish, traditional cell culture techniques cannot be directly transferred to a microfluidic cell culture system.…”

“…These findings indicated the importance of seeding primary cells at a sufficient density, i.e., at near confluence, in a shallow microchannel. 1,2 Alternatively, a microchannel cell culture is commonly performed in a sub-millimeter-deep microchannel. For example, the depth of commercially available microchannels provided by ibidi (Germany) ranges from 200 to 800 μm.…”