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.
Highlights
When the factors that maintain the stemness of ES cells are removed, ES cells spontaneously aggregate into a 3-dimensional sphere called embryonic body (EB) which includes all three germ layers of endoderm, mesoderm, and ectoderm, and can be induced to differentiate into different cell types [2,3,4,5,6]
We have developed a microfluidic chip-based system for EB formation in hanging droplets
This method allows for medium exchange during EB culture via the microfluidic channel so it may be used for direct characterization of the cultured EBs on chip
Summary
Owing to their ability to self-renew and differentiate into any cell type in the body, embryonic stem cells (ESCs) have been widely studied for potential uses in tissue engineering and cell therapy [1,2,3]. Despite having a wide selection of the aforementioned methods to choose from, currently the conventional hanging drop method is still the most widely used approach for EB formation because it is easy to perform in the laboratory and has minimal equipment and material requirements [24] This method uses a manual pipette to drip individual small droplets of ES cell-containing medium on a. The throughput of the process is still limited because each droplet has to be individually handled To solve this problem, a modified 384-well plate device has been developed for hanging drop culture which can be scaled up by the assistance of a robotic arm system [26]. We have developed a setup and operation procedure for successful on-chip culture of EBs as demonstrated by their growth curve and expressions of pluripotency markers
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