Size-based microfluidic enrichment of neonatal rat cardiac cell populations

Abstract

Native heart consists of myocytes and non-myocytes. We demonstrate here the feasibility of a size-based microfluidic separation of myocytes and non-myocytes from the neonatal rat myocardium. The device consists of a middle channel (50 μm wide, 200 μm tall, and 4 cm long) connected to adjacent side channels by microsieves (80 μm wide, 5 μm tall and 40 μm in length). The side channels increase in width in a flared shape along the length of the device to ensure constant pressure gradient across all sieves. In the first step, non-myoctes were removed from the myocytes by a conventional pre-plating method for 75 min. Subsequently, the non-myocytes were further enriched in a microfludic device at 20 μl/min. We demonstrated that the cells in the middle and side channels maintained viability during sorting and the ability to attach and grow in culture. Upon culture for 48 h cardiomyocytes from the reservoir (control) and middle channel stained positive for cardiac Troponin I, exhibited a well developed contractile apparatus and contracted spontaneously and in response to electrical field stimulation. Most of the cells in the side channel expressed a non-myocyte marker vimetin. Fluorescent activated cell sorting indicated significant enrichment in the side channel (p < 0.001) for non-myocytes. Original cell suspension had a bimodal cell size distribution with the peaks in the range from 7–9 μm and 15–17 μm. Upon cell sorting the distribution was Gaussian in both side channel and middle channel with the peaks in the range 7–9 μm and 9–11 μm respectively, indicating that the separation by size occurred.