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International Journal of Bioprinting Osteogenic differentiation of hMSCs by PBF-LB

with a secondary antibody and Hoechst staining The supernatant was removed, and 1 mL of PBS was added to
aforementioned as well as subsequent manipulations were the suspension. The cell concentration was adjusted to 2 ×
performed while keeping the cells away from light. The 10 cells/mL in PBS. After that, the sample cell mixture for
5
cells were then washed thrice for 10 min each time in PBST ICELL8 cx aliquot was prepared.
and incubated in PBST containing phalloidin (Invitrogen, The recovered cDNA was enriched using the DNA
Carlsbad, CA, USA). After that, the stained samples were Clean & 5-kit (Zymo Research). To the cDNA product,
mounted in ProLong™ Gold Antifade Mountant (Thermo 0.6× volume of AMPure XP beads was added, mixed by
Fisher Scientific, Carlsbad, CA, USA). pipetting, and incubated at room temperature for 5 min.
2.5. Cell orientation analysis They were then placed on a magnetic stand and allowed
Immunofluorescence staining was performed to to stand for 2 min. The supernatant was removed with a
quantitatively analyze the morphological changes in MSCs pipette, and 200 µL of 70% ethanol was added to wash the
on culture substrates. For each sample, images of F-actin, beads and allowed to stand for 1 min. The tube was then
vinculin, and nuclei were taken at multiple points with a rotated and allowed to stand for 10 s on one side of the
20× objective lens using a fluorescence microscope (BZ- tube, and then reversed and allowed to stand again for 10
X710, Keyence). Images were captured by focusing on cells s on the other side. This procedure was repeated twice,
adhering from the top of the groove shape. Single cells and then the supernatant was removed with a pipette,
were analyzed, excluding those whose cell morphology and the procedure was repeated twice. Subsequently, the
was difficult to accurately distinguish, and 40–60 stained ethanol was removed with the tube still in place, the lid was
cells were analyzed per sample. Images of the nuclei and opened, and the beads were allowed to stand for less than
F-actin/vinculin-stained images were gray scaled using 2 min to dry. Next, 13 µL of DNase-RNase-free water was
Photoshop 6.0 (Adobe Inc., Mountain View, CA, USA) added, mixed by pipetting, and incubated for 5 min. After
and further binarized. Both images were loaded into allowing it to stand for 1 min, 12 µL of the supernatant
CellProfiler (Broad Institute Cambridge, MA, USA) to was collected and transferred to a new tube. The resulting
recognize the shape of the cell body based on the position cDNA was quantified using the Qubit dsDNA HS Assay
of the cell nucleus. The cell shape was approximated as Kit (Life Technologies), and a Qubit 3.0 Fluorometer,
an ellipse. The angle between the major axis of the ellipse High Sensitivity DNA Kit, and Agilent 2100 Bioanalyzer
and the groove direction on the substrate was defined as 0, were used to confirm the quality of the cDNA libraries.
and was represented in a histogram at 10° intervals. To Library length was measured and quantified using a
designate the flat substrate, is the angle between the major High Sensitive DNA Kit and Agilent 2100 Bioanalyzer for
axis of the ellipse and the scanning direction during sequence analysis. The library was sequenced using the
modeling. The degree of cell orientation was defined as a HiSeq2500 for Read1:26 cycles and Read2:101 cycles, and
value representing the degree of cell orientation using the approximately 250 million reads were obtained.
following equation : 2.7. Alkaline phosphatase assay
26
1 To verify osteogenic differentiation, alkaline phosphatase
2
R 2 cos (I) activity was measured using the Alkaline Phosphatase
k 2 Colorimetric Assay Kit (Abcam) according to the
1 n cos k manufacturer’s protocols. Cells were exposed to the culture
2
medium for 3 days, after which samples were washed
cos (II) twice with PBS. Then, 50 μL of cell lysate with assay buffer
2
k
n was added to a 96-well plate, and 50 μL of p-nitrophenyl
Under this definition, R=1 when all cells are aligned phosphate was added. Samples were light-shielded and
perfectly parallel to the groove direction (θk = 0°); incubated at 25°C for 60 min. Finally, 20 µL of stop
conversely, R = 0 when perfectly unoriented (θk… at solution was added to the wells, and the absorbance was
random). analyzed at 405 nm and measured at 650 nm as a reference
(MULTICKAN FC, Thermo Fisher Scientific).
2.6. Single-cell RNA sequencing
The culture medium was removed, and the cells were 2.8. Statistical analysis
washed twice with PBS. Then, 250 µL of each of the The data obtained from the experiments are expressed as
cell detachment agents (accutase: collagenase = 3:2) the mean ± SD. Student’s t-test was used for comparisons
was injected and kept in the incubator for 10 min. between two groups, and Tukey’s test was used for
Mesenchymal stem cells were then filtered through a cell comparisons among three or more groups. A difference
strainer to obtain single cells. After centrifugation, the was considered significant if the p-value was less than 0.05

Volume 10 Issue 1 (2024) 408 https://doi.org/10.18063/ijb.1425