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sorting larger well plates, such as 384-well plates, which required re-stocking of the reservoir

mid-sort.

Pilot study on the Generation of Stem Cell Spheroid Culture Within BB Microscaffolds

In a final proof-of-concept study, the successful deposition of microscaffolds in culture wells
by our optimized microfluidic sorting protocol was applied by deposition of immortalized

human stem cells (hASCs) in culture medium to form scaffolded spheroids (S-SPHs). Cell

deposition was executed via a separate fluid line to minimize the amount of wasted cells and
to increase the repeatability when compared to utilizing the microfluidic device for this task

(Error! Reference source not found.). The repeatability was measured at a constant fluid

pressure of 50 mbar and a valve opening time of 500 ms and 1800 ms, for which the fluid line
showed a dispensed volume of 66.6 ± 7 µL and 245.4 ± 1.8 µL respectively (Error! Reference

source not found. B). Next, the impact of automated deposition on the metabolic activity of
hASCs was assessed. hASCs were dispensed automatically and manually, and their metabolic

activity expressed as RFU units was compared after 24 hours of culture via PrestoBlue assay.
The fluorescence was normalized to the actual number of deposited cells derived from a diluted

standard and shown in Figure 7 c. Overall, cells deposited automatically showed an 8% percent

lower metabolic activity after 24 hours of culture compared to cells that were dispensed
manually. To automate the production of scaffolded spheroids (S-SPHs), the sorting step and

the dispensing step were combined. Initial tests showed that only microscaffolds that were
placed in the center of the well would instigate the formation of round spheroids within. Those

placed off-center would either not receive enough cells to form a spheroid or form an elongated
cell cluster outside of the BB. To account for this, wells were pre-filled with 200 µL of dH20

and an intermediary centrifugation step was added between sorting and seeding the

microscaffolds. Further, the correct choice of well-plate was crucial to the central placement
of the BB and therefore the formation of spheroids. Only plates with a V-shaped bottom were

able to position the BB in the exact center after centrifugation. U-shaped plate bottoms merely

positioned BBs close to the center, whereas flat bottom plates proved completely inept.
Spheroid-laden microscaffolds formed after 24 h of culture using 5000 hASCs. 24h after

seeding the microscaffolds, 95% of the microscaffolds showed the formation of a spheroid in
a V-shaped 96-well plate, regardless of automatic or manual dispensing (Figure 7 d). In U-

shaped 384-well plates, an overall higher percentage of spheroid non-formation was visible,
with automatic deposition showing 11% non-formation, whereas manual deposition resulted in

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