Page 23 - manuscript_ijb05583

formation comparing automated and manual deposition modes in U-shaped and V-shaped well

plates. * indicates significance.

Discussion

Unlike state-of-the art microfluidic flow cytometers, the device presented here was developed
for ejecting microscaffolds and not only liquids after the sorting process, which imposed certain

design considerations on the microfluidic device. Typically, microfluidic sorting devices

continuously channel their content into predefined integrated channels within the device. In
contrast, our device necessitated relocating content outside the device to a precise location. To

address this requirement, we developed a system with a temporal interruption, allowing for
content seclusion and ejection without disrupting the flow of the main inlet stream.

Additionally, the synchronization of the ejection process with the availability of an empty well
further justified our departure from a continuous processing approach. A dedicated fluid line

was added to automate cell deposition on top of the microscaffolds and thus produce spheroid-

laden microscaffolds in a scalable manner. Sample focusing was achieved via sheath flow,
which precisely controlled the central sample stream width seen in Figure 4 a-i. In comparison

25
24
to flow focusing via dielectrophoresis and acoustic force , this method maintains a stable
pH and is independent of the to-be focused particle size.

To isolate a single particle and eject it into a single culture well, the developed system split up

the sorting process into secluding and subsequently ejecting. Both processes were combined
into one functional element, by implementing pneumatic on-chip valves that physically

separate the ejection channel from the main channel.

Through combining fluorescent intensity and distance between events as metrics with the

approach of double-flagging this staged sorting process enabled the highest sorting yield,

defined by the number of wells per plate containing a single microscaffold. However, this
comes at the expense of a higher rate of rejected microscaffolds and other challenges such as

BBs trapped in the valve leading to an empty well and BBs crushed by valves leading to debris
dispensing in the system.

22