8 well cell culture slide

Equal amounts of the samples were inserted in MG-63 cells cultivated on 8 well cell culture slides (SPL, Seoul, South Korea) at a density of 10⁴ cells/well. After 72 hours of incubation, the cells were stained with dual stains of acridine orange (100 μg/mL) and ethidium bromide (100 μg/mL) that were dissolved in phosphate buffer saline (PBS) at equal volumes to examine the mode of cell death. The stained cells were examined using fluorescence microscope (AxioImager Z2, Zeiss, Jena, Germany). Green-colored cells were classified as ‘living’, whereas yellow, orange, or red cells were classified as ‘early apoptotic’, ‘late apoptotic’, or ‘necrotic’, respectively (Leite et al., 1999 (link)). Three separate experiments were conducted (n = 3).

SK-N-BE(2) cells were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA) and expanded in a growth medium based on Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, Thermofisher), supplemented with 10% fetal bovine serum (Thermofisher), 1% Insulin-Transferrin-Selenium G Supplement (Thermofisher), Plasmocin (0.2%) treatment ant-mpt (1/10) (InvivoGen) and 1% penicillin/streptomycin (Thermofisher) at 37 °C and 5% CO₂ atmosphere. 2D cell cultures were grown in 8-well Cell Culture Slides (SPL Life Sciences) until they reached confluence before immunocytochemistry (ICC) analysis.
To create the bioinks, cells were cultured and trypsinized. The resulting pellet was resuspended with the prepolymer solution at 37 °C to a 2.5 × 10⁶ cell density. The bioink was loaded in a bioprinting syringe and gelified at −20 °C for 3 minutes before printing.

SK-N-BE(2), SH-SY5Y human NB cell lines were chosen from a variety of available cell lines, since MYCN-amplified and ALK-mutated tumors represent 64% of high risk neuroblastoma (50 and 14%, respectively). SK-N-BE(2), SH-SY5Y, and SW10 mouse Schwann cell lines were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). NB cells were expanded in supplemented IMDM medium (Gibco, Life Technologies, Waltham, MA, USA) and SW10 in supplemented DMEM (Gibco, Life Technologies, Waltham, MA, USA) at 37 °C and 5% CO2 atmosphere. Two dimensional cell cultures were grown in 8-well Cell Culture Slides (SPL Life Sciences, Waltham, MA, USA), until they reached 60% confluence before immunocytochemistry (ICC) analysis. Bioinks for 3D culture were formed by mixing SK-N-BE(2) or SH-SY5Y cells with the prepolymer solution at 37 °C to a final solution of 2 × 10⁶ mL⁻¹, as previously reported [21 (link)], including in some cases, an additional 2 × 10⁵ mL⁻¹ of SW10 cells. Hydrogels were cultured from 2 to 12 weeks in supplemented IMDM medium replaced every 2 or 3 days before immunohistochemistry (IHC) analysis.

Human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany) were grown in 25 cm² flasks, coated by 1% gelatin solution according to the manufacturer’s instructions. The fourth to sixth passages were used for the experiments. Co-culture of HUVECs with malaria sera was performed based on previous study with minor modifications [14 (link)]. ECs were grown on an 8-well cell culture slide (SPL Life Sciences Co., Ltd., Gyeonggi-do, Korea) until confluence. When cells reach > 90% confluence, complete EC media was removed and replaced with new serum-free media containing malaria sera (10%) from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Control groups included incubation of HUVECs with serum-free media alone and serum-free media mixed with non-malaria patient sera (10%). Incubation periods were at specific times (T) of 0, 30, 60 and 90 min.

To confirm the morphological change of MC3T3-E1 cells, the cells (2 × 10⁴ cells/mL) were cultured in an 8-well Cell Culture Slide (SPL Life Sciences, Pocheon, Korea) containing 100 μL of the culture medium in an incubator. Aqueous dispersions of the only DMEM (control), NDs, and Alen-NDs (100 μg/mL) were added to each well and allowed to grow for 7 days. The culture slides were gently washed with PBS, fixed with formaldehyde solution (4%) for 15 min at room temperature, and rinsed 3 times with PBS. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and stained with rhodamine for 1 h in dark. The fluorescence was visualized by confocal microscopy (LSM710, Carl Zeiss, Oberkochen, Germany), and the cellular dimensions were analyzed using ImageJ® software (National Institutes of Health, Bethesda, USA).
The osteogenic differentiation was confirmed by fluorescence-activated cell sorting analysis (FACS, FACS Canto II, BD Biosciences, San Jose, USA). MC3T3-E1 cells (1 × 10⁵ cells/mL) were cultured after treatment by NDs and Alen-NDs (100 μg/mL) in the medium for 7 days. Then, anti-CD44, anti-CD51, and anti-CD45 antibodies (eBioscience) were treated to each well for 30 min on ice. After staining, cells were fixed with paraformaldehyde (2%) and analyzed with a FACS. Data were analyzed with FlowJo software (v. 10.1, FlowJo LLC, USA).