Separation of spermatozoa from erythrocytes using their tumbling mechanism in a pinch flow fractionation device

Abstract

Men suffering from azoospermia can father a child, by extracting spermatozoa from a testicular biopsy sample. The main complication in this procedure is the presence of an abundance of erythrocytes. Currently, the isolation of the few spermatozoa from the sample is manually performed due to ineffectiveness of filtering methods, making it time consuming and labor intensive. The spermatozoa are smaller in both width and height than any other cell type found in the sample, with a very small difference compared with the erythrocyte for the smallest, making this not the feature to base the extraction on. However, the length of the spermatozoon is 5× larger than the diameter of an erythrocyte and can be utilized. Here we propose a microfluidic chip, in which the tumbling behavior of spermatozoa in pinched flow fractionation is utilized to separate them from the erythrocytes. We show that we can extract 95% of the spermatozoa from a sample containing 2.5% spermatozoa, while removing around 90% of the erythrocytes. By adjusting the flow rates, we are able to increase the collection efficiency while slightly sacrificing the purity, tuning the solution for the available sample in the clinic.

Keywords: Microfluidics; Nanobiotechnology.

Fig. 1. Separation in a PFF chip for different particle types.

Example of a separation in a single chip with positions in the channel for different particle types: theoretical values for 6 and 15 µm beads have been obtained via Comsol simulations. Experimental values for 6 and 15 µm beads, as well as spermatozoa and erythrocytes, are included. Distance from the top wall (the sample side of the device) as measured at the center of the particle (head for spermatozoa)

Fig. 2. Tumbling behaviour of spermatozoa in PFF.

Equivelocity lines and a spermatozoon passing through the pinched section. The tail occupies a part of the fluid that has a higher velocity than the part in which the head resides. The tail then gets pushed faster around the corner, causing a rotation in the x–y plane. Scale bar is 50 µm

Fig. 3. Collection efficienty of the PFF device.

Collection efficiency of spermatozoa (blue) and erythrocytes (red) for different fluid removal ratios. With higher percentage of flow to outlet 3, fewer of both cell types are collected in outlet 4. However, by increasing the flow to outlet 3 with respect to outlet 4, the erythrocytes are more strongly excluded than spermatozoa. Error bars = 1 SD, N = 3

Fig. 4. Extraction purity of the samples.

Extraction purity of the samples. Sample compositions before and after separation for different fluid removal ratios (S = sample, P = product after separation). Error bars = 1 SD, N = 3

Fig. 5. Set-up with microfluidic chip.

Left: outline of the setup, features are not true to size. P1 is the sample pressure, P2 is the buffer pressure, P3 is the waste outlet pressure, and P4 is the product outlet pressure. The chip contains a pinched section and a broadened section. The cells get pushed toward the wall in the pinched section and appear at a distance from the top wall in the broadened section according to their apparent hydrodynamic radius. This distance determines the outlet that the cells will go through. Right, top: PFF chip. Scale bar is 2.5 mm. Bottom: visualization of the flow in the chip using red dye. Scale bar is 50 µm