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International Journal of Bioprinting Bioprinted stem cell niche regenerates muscles

Figure 2. Bioprinted Notch ligand effectively maintained the undifferentiated status of muscle stem cell. (A) DLL1 (mouse): Fc (human) (rec.) was
bioprinted onto fibrin/protein G-coated coverslips. MPCs from 6-month-old WT mice (WT MPCs) were then seeded on the slides and cultured for
4 days. Immunostaining with Myosin Heavy Chain antibody (MHC-MF20) showed that there were many MHC-MF20-positive cells (myotubes) at the
location in the absence of bioprinted DLL1, whereas there were almost no MHC-MF20-positive cells on printed DLL1 patterns (MHC-MF20: green; DAPI:
blue), indicating that the bioprinted DLL1 prevented the differentiation of seeded muscle stem cells, thus maintaining their undifferentiated stem cell state.
(B) The statistics of proliferation and myogenic differentiation potential.
the differentiation of seeded muscle stem cells, thus pattern persisted throughout the incubation period.
maintaining their undifferentiated stem cell state. Therefore, by performing repeated overprints of the Fc
protein, the system is effective at assuring the persistence of
3.4. Persistence of printed IgG Fc (a model for Fc protein on the DermaMatrix constructs. This indicates
Fc fusion proteins DLL1-Fc we are using) on that when IgG bioprinted DermaMatrix constructs are
3D construct prerinsed, ~70% of initial bound IgG remains bound after
We performed protein binding experiments to demonstrate 11 days of incubation under simulated in vivo condition.
that Fc fusion proteins can be stably bioprinted and In fact, over 18 days after rinsing and incubation, the levels
biopatterned on an artificial 3D DermaMatrix constructs of IgG bound to the DermaMatrix constructs were very
(derived from human acellular dermis). Cy3-labeled consistent (Figure 3B), further validating the retention
human IgG Fc was printed onto DermaMatrix construct, of IgG on the DermaMatrix constructs. GFP-labeled WT
and printed constructs were then rinsed with phosphate- MPCs (GFP-WT MPCs) were seeded in the DermaMatrix
buffered saline (PBS; 2 times for 15 min, 37°C). Fluorescent constructs with DLL1-printed or nonprinted areas, and the
images were taken immediately postprinting (before growth potential of the two groups of cells were compared
washing with PBS for 15 min) and 5 days postprinting, (Figure 3C), demonstrating the cells seeded in the DLL1-
rinsed, and incubated with simulated interstitial fluid (10% printed area grew faster than the cells in nonprinted areas.
serum MEM culture media containing 25 mM HEPES and
0.1% sodium azide) at 37°C (Figure 3A). As it is shown 3.5. More MPCs from DLL1-conjugated constructs
in the images, increasing the number of OP (overprints / were able to migrate through 3D-Matrigel layers in
repeated prints) accordingly increased the amount of IgG an in vitro migration assay
Fc protein printed onto the DermaMatrix constructs. DLL1 (mouse):Fc (human)(rec) was bioprinted onto
Following the removal of unbound IgG Fc, the printed biodegradable DermaMatrix construct (4 × 4 × 1 mm in

Volume 9 Issue 3 (2023) 304 https://doi.org/10.18063/ijb.711