Calcein am

Cells were collected from 1 mL of the culture medium by centrifugation and washed with 50 mM potassium phosphate buffer (pH 6.5). After resuspension with the same buffer, staining of the cells was performed with Hoechist 33258 for observation of all cells, with Calcein-AM for the observation of viable cells and with propidium iodide (PI) (Wako, Japan) for observation of membrane-permeabilized cells (dead cells) for 15 min in the dark at concentrations of 5 µg/mL for Hoechist, 10 µg/mL for Calcein and 0.5 µg/mL for PI according to the supplier’s instructions. Samples were observed using a Nikon E600 microscope with fluorescence capability (Nikon, Japan).

BMSCs cultured in the flask under NPWT or not were fixed in 4% paraformaldehyde (Aspen, China) for 1.5 h and stained with FITC-conjugated phalloidin (Yeasen, China) for 1 h. Finally, samples were stained with diamidine-phenylindole-dihydrochloride (DAPI, Aspen, China) and analyzed with a confocal fluorescence microscope system (Leica SP2, Leica, Germany).
To evaluate cell proliferation under NPWT, on days 1, 3, 6, and 9 after NPWT treatment, a CCK-8 assay was performed. BMSCs cultured with normal pressure served as control. To assess BMSCs viability and death under NPWT, a live/dead assay was performed. On days 1, 3, 6, and 9, the cells in the culture flask were incubated with 1 mM calcein AM (Wako, Japan) for 1 h and then incubated with 1 ug/mL propidium iodide (PI, Invitrogen, USA) for 5 min at 37°C. Next, the cells were imaged using a fluorescence microscopy (IX51, Olympus, Japan). Live cells stained green, whereas dead cells stained red.

Fibroblasts (NIH/3T3) were cultured inside Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich) with 10% fetal bovine serum (FBS, Sigma-Aldrich) for 72 h, inside an incubator (37 °C, 5% CO₂). Prior to experiments, NIH/3T3 cells were collected and all culture medium was removed. Cells were mixed inside phosphate buffered saline (PBS, Wako, Osaka, Japan) to form a PBS cell solution of 5 × 10⁶ /mL cell concentration. The cell experimental solution contains 20% PEGDA and 0.5% photoinitiator, with a cell concentration of about 4 × 10⁶ /mL.
These cell encapsulated microstructures were washed by PBS once, and then immersed in the viability test solution, which was a mixture of 10 μL calcein AM (1 mg/mL, Wako), 15 μL propidium iodide (PI, 1 mg/mL, Wako) and 5 mL PBS. The solution and microstructures were incubated for 30 min. After that, the test solution was removed, and the microstructures were washed by PBS again. UV light (490 nm) was used to observe the samples.

Human urinary bladder transitional cell carcinoma cells (UM-UC-3; American Type Culture Collection, Manassas, VA, USA) were maintained in alfa Minimum Essential Medium (MEM-alfa; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum. For the 3D model, 2 x 10⁷ cells/mL of UM-UC-3 were embedded in collagen-I (Collagen I High concentration, Rat tail; Corning Inc., Belford, MA, USA) according to the previous study [18 (link)]. The collagen solution consisted of 10x MEM-alfa (Sigma-Aldrich), a buffer solution, additional 1 M NaOH (to adjust pH). The final collagen concentration was 3 mg/mL. The cell containing solution was added to 35 mm dish (Nunclon™Delta surface; Thermo Fisher Scientific, Suzhou, China) at a seeding volume of 50, 100, 150, or 200 μL. The solution was incubated to solidify for 30 min at 37°C. UM-UC-3 cells in the 3D model were cultured for 3 days before experiments. In order to measure the thickness of the 3D model, fluorescent microscopic images of cells in z-stuck were obtained by BZ-9000 (Keyence, Osaka, Japan). Cells were incubated with 2 μM Calcein-AM (Wako Pure Chemicals) for 30 min at 37°C prior to the imaging.

ADCC assay was performed using indicated target cells and NK92 cells as the effector cells. Target cells prelabeled with a cell-permeable fluorescent dye (calcein-AM; FUJIFILM Wako Pure Chemical Corporation) for 1 h were seeded into 96-well plates at 1 × 10⁴ cells/well. For blocking assays, target cells were incubated for 30 min with 10 μg/mL antagonistic ZB4 anti-Fas monoclonal antibodies (GeneTex Inc., Irvine, CA, USA) or 10 μg/mL control IgG1 (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan). Specific lysis was assessed at 4 h after exposure to NK92 cells at an E/T ratio 1:1 in the presence or absence of obinutuzumab. Fluorescence intensity of calcein was measured with a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). %ADCC was calculated as follows: (experimental release − background)/(maximum lysis − background) × 100. Here, “background” means labeled target cells only and “maximum lysis” means labeled target cells lysed with 1% Triton X-100 (Sigma-Aldrich). The results of ADCC (%) are presented as mean ± SD. All experiments were performed at least twice, and representative results of one experiment are shown.

To evaluate cell viability, spheroids cultured both in the microfluidic device and in a 96-well plate were fluorescently stained using calcein-AM (Invitrogen) and propidium iodide (PI, Wako) at 37 °C and 5% CO₂ for 2 h. calcein-AM and PI were dissolved in phosphate-buffered solution (PBS, Wako) to adjust their concentrations to 0.5 and 1 mg/mL, respectively.
To embed spheroids, 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan) and 99.5% ethanol (Wako) were used for chemical fixation and dehydration, respectively.
To evaluate protein distribution within the cross-sections of spheroids, the spheroid slices were stained using fluorescent dye-labeled antibodies. In this report, HER2 and integrin were visualized. HER2 is a protein that expresses strongly on the N87 cell surface [23 (link)]. Anti-HER2 antibody conjugated to AF488 (Alexa Fluor^® 488 anti-human CD340 (erbB2/HER2) Antibodies, BioLegend, San Diego, CA, USA) and anti-integrin antibodies conjugated to AF555 (Anti-integrin α_vβ₅ Antibody, Alexa Fluor^® 555 Conjugated, Bioss, Woburn, MA, USA) were used. The concentrations of anti-HER2 antibody and anti-integrin antibody were 5.0 and 3.0 µg/mL, respectively. Slices of a sectioned spheroid were soaked in a mixed solution of the antibodies at 37 °C for 2 h. Dyed slices were placed between the slide and cover glasses.