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 microm wide, 200 microm tall, and 4 cm long) connected to adjacent side channels by microsieves (80 microm wide, 5 microm tall and 40 microm 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-myocytes were removed from the myocytes by a conventional pre-plating method for 75 min. Subsequently, the non-myocytes were further enriched in a microfluidic device at 20 microl/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 microm and 15-17 microm. Upon cell sorting the distribution was Gaussian in both side channel and middle channel with the peaks in the range 7-9 microm and 9-11 microm respectively, indicating that the separation by size occurred.