Thunder imager 3d live cell microscope

To study the invasiveness of breast cancer cells (BCCs) into the
ELR hydrogel, MDA-MB-231 or MCF7 cells were cultured on top of the
hydrogel, and its invasion toward the hydrogel was monitored by fluorescent
microscopy (N = 3). Briefly, MDA-MB-231 or MCF7 cells
were labeled with Vybrant DiO according to the manufacturer’s
instructions. 10 μL of ELR hydrogels was prepared in a μ-Slide
Angiogenesis chamber (Ibidi). Then, 50 μL of the labeled cells
was added on top of the gel (100,000 cells/mL). Two cell media were
used, one containing 0% FBS and one containing 10% FBS. Gels were
imaged with a Thunder Imager 3D live cell microscope (Leica Microsystems)
after 3 h and 1, 2, and 3 days in culture. A mosaic tile of each XY
conforming the well with zetas of 10 μm of each well was acquired.
To quantify the cell invasion, the volume of migration at each time
was measured with Fiji software.^{72 (link)} To avoid
the effect of any surface defect, the volume of the cells at time
3 h was used to normalize the values.

To study if TGA and
TGAC bioinks enable the 3D printing of several
cell types with a precise location and without the blending of the
layers for an appropriate recapitulation of breast tumors, colored
bioinks were prepared with food coloring. A scaffold consisting of
a core for recreating the tumor site and an outer layer to mimic the
stromal layer in breast tumors was designed (Figure 6A). TGA and TGAC bioinks were prepared as
described before, and then, a drop of food coloring was added. Bioinks
with no food coloring were used for the inner core (tumor core), and
bioinks stained in red were used for the outer layer (stromal layer).
Cell-laden scaffolds with an MCF-7 core and an hAMSC outer layer were
also bioprinted (Figure 6A) to ensure that there was no cell merging when bioprinting. hAMSCs
and MCF-7 were marked with Vybrant DiO and DiD, respectively, according
to the manufacturer’s instructions (Thermo Fisher Scientific)
and then resuspended in the bioink (hAMSCs, 1 ×
10⁶ cells/mL; MCF-7, 1.5 × 10⁶ cells/mL).
Cell-laden scaffolds were cultured in the same media as cancer cells
and kept in culture for only 1 day. Then, hydrogels were fixed with
4% PFA and visualized in a Thunder Imager 3D live cell microscope
(Leica Microsystems).

Cell survival
on the bioprinted and nonbioprinted hydrogels was evaluated with live/dead
staining using calcein AM/PI. Cell-laden hydrogels at different time
points were washed twice with PBS at 37 °C and incubated with
2 μM calcein AM and 4 μM PI in PBS for 20 min. The cell
viability was calculated with a 3D object counter in FIJI software.^{38 (link)} For immunofluorescence images, cell-laden hydrogels
were washed twice with PBS, fixed with paraformaldehyde (20 min, RT),
permeabilized with 0.1% Triton X-100 (5 min, RT), blocked with 10%
goat serum in 3% BSA in PBS (1 h, RT), incubated with an anti-E-cadherin
antibody (1:250, overnight, 4 °C), incubated with goat antirabbit
IgG H&L (1:1000, 1 h, RT), and incubated with phalloidin–tetramethylrhodamine
B isothiocyanate (45 min, RT) and DAPI (10 min, RT). Three washes
of 3% BSA in PBS were done between each step. To study the cellular
morphology in hydrogels, cells were stained only with phalloidin and
DAPI, using the same procedure. Hydrogels were visualized with a Thunder
Imager 3D live cell microscope (Leica Microsystems). Nonbioprinted
hydrogels made of Col1 at 4 mg/mL encapsulating MCF-7 at 1.5 ×
10⁶ cells/mL were used as controls.

Digital images were acquired using an upright DMR6000B microscope (Leica) with a DFC550 camera for immunohistochemical colour pictures; a Thunder imager 3D live-cell microscope (Leica Microsystems) with hardware autofocus control and a Leica DFC9000 GTC sCMOS camera, using HC PL FLUOTAR ×10/0.32 PH1 ∞/0.17/ON257C and HC PL FLUOTAR ×20/0.4 CORR PH1 ∞/0-2/ON25/C objectives; a Zeiss Cell Observer HS with a ×20 and x40 air objective and a Zeiss AxioCam MrX camera; and a Leica SP5 confocal laser-scanning microscope with HCX PL Fluotar ×40/0.75 and ×63/0.75 objectives. The different fluorophores (three or four) were excited using the 405, 488, 568 and 633 nm excitation lines. The acquisition was performed using the Leica Application (v.3.0) or LAS X (v.1.0) software (Leica) or Zeiss LSM software Zen 2 Blue.