A PDMS-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation

Abstract

The conventional hanging drop technique is the most widely used method for embryoid body (EB) formation. However, this method is labor intensive and limited by the difficulty in exchanging the medium. Here, we report a microfluidic chip-based approach for high-throughput formation of EBs. The device consists of microfluidic channels with 6 × 12 opening wells in PDMS supported by a glass substrate. The PDMS channels were fabricated by replicating polydimethyl-siloxane (PDMS) from SU-8 mold. The droplet formation in the chip was tested with different hydrostatic pressures to obtain optimal operation pressures for the wells with 1000 μm diameter openings. The droplets formed at the opening wells were used to culture mouse embryonic stem cells which could subsequently developed into EBs in the hanging droplets. This device also allows for medium exchange of the hanging droplets making it possible to perform immunochemistry staining and characterize EBs on chip.

Keywords: embryoid body; embryonic stem cell; microfluidic hanging drop.

Figure 1

Illustration of the microfluidic hanging drop chip, and its setup and operation. (A) Photo of the PDMS μHD chip (openings facing up) with opening diameter of 1000 μm. Scale bar is 1 cm; (B) Schematic illustration of the μHD chip operation. The microchannel is firstly filled with 75% ethanol, followed by introducing cell suspension into the microchannel by hydrostatic pressure-driven flow, and allowing cells to descend to the bottom of the droplets. The docked cells centralized and aggregated at center of droplet bottom due to the concavity of the droplets and grow into spheroids; (C) Illustration of μHD chip setup. The μHD chip was placed in a 10 cm dish containing 1× PBS in the bottom of the plate to prevent medium evaporation from the hanging droplets.

Figure 2

Measured droplet heights under various hydrostatic pressures in μHD chip with 1000 μm diameter opening wells. (A) Side-view photographs of a portion of the μHD chip showing protruding droplets of different heights (top: average height = 142.8 μm at 98 N/m²; middle: average height = 215.6 μm at 127.4 N/m²; bottom: average height = 281.2 μm at 147 N/m²; (B) the relationship between the droplet height and hydrostatic pressure. Scale bar is 500 μm.

Figure 3

Demonstration and characterization of solution exchange in μHD chip. (A–C) Photographs showing the solution replacement process (blue dye replacing DI water) at 0 min, 60 min and 100 min, respectively. Scale bar = 1 mm; (D) photograph of the whole chip taken at 80 min after the replacement process started. Scale bar = 4 cm; (E) the relationship between the normalized intensity of the blue dye solution and the time after the replacement process started.

Figure 3

Demonstration and characterization of solution exchange in μHD chip. (A–C) Photographs showing the solution replacement process (blue dye replacing DI water) at 0 min, 60 min and 100 min, respectively. Scale bar = 1 mm; (D) photograph of the whole chip taken at 80 min after the replacement process started. Scale bar = 4 cm; (E) the relationship between the normalized intensity of the blue dye solution and the time after the replacement process started.

Figure 4

EB formation after 1 day. (A) Images of the top-view of the chip after 1 day culture; (B,C) The closed view of one EB; (D) the comparison between the number of single EB and EBs. The photo shows the satellite EBs are less than 40 μm; (E) the size distribution of the EBs.

Figure 4

EB formation after 1 day. (A) Images of the top-view of the chip after 1 day culture; (B,C) The closed view of one EB; (D) the comparison between the number of single EB and EBs. The photo shows the satellite EBs are less than 40 μm; (E) the size distribution of the EBs.

Figure 5

EB growth observation for 3 days. (A–D) Images of EBs taken at day 0 (A); day 1 (B); day 2 (C) and day 3 (D) after cell seeding; (E) the growth curves of the EBs cultured in the μHD chip and conventional hanging drop system. The scale bar is 200 μm.

Figure 6

Immunochemistry staining of EBs. (A) Confocal images of SSEA-1 and DAPI stained EBs formed by using the conventional hanging drop method and the μHD chip. Scale bar = 50 μm; (B) images of on-chip immunochemistry stained EBs. Scale bar = 100 μm.