Ccd 1112sk

Human foreskin fibroblasts (HFFs; ATCC^® CCD-1112SK™, Manassas, VA, USA), Rhesus macaque foreskin fibroblasts (RMF; isolated in house), and African Green Monkey kidney cells (Vero; ATCC^® CCL-81™, Manassas, VA, USA) were used in this study. The media compositions employed were (1) HFF: minimum essential medium (MEM), 10% inactivated fetal bovine serum (ΔFBS; Atlas Biologicals F-0050-A, Fort Collins, CO, USA), 100 U/mL penicillin-streptomycin (pen-strep; Mediatech/Corning #30-002-CL Tweaksbury, MA, USA), 1% sodium pyruvate (Mediatech/Corning #25-000-CL Tweaksbury, MA, USA), 1% glutaMAX™ (Gibco # 35050-061), and 1% non-essential amino acids (Mediatech/Corning #25-025-CL Tweaksbury, MA, USA); (2) RMF: Dulbecco’s modified essential medium (DMEM; Sigma-Aldrich #D-6046, St. LouisMOUSA), 18% ΔFBS, and 100 U/mL pen-strep; and (3) Vero: DMEM, 10% ΔFBS, 100 U/mL pen-strep. For all experiments, cells were grown at 37 °C in a 95% O₂, 5% CO₂ incubator. B virus (laboratory strain E2490) was propagated in Vero cells. Virus stock titer was determined by standard plaque assay in Vero cells. Propagation and harvesting of B virus isolates were performed in the Georgia State University (GSU) BSL4 Laboratory in accordance with the guidelines of the 5th edition of Biosafety in Microbiological and Biomedical Laboratories.

Fibroblasts (CCD-1112SK, ATCC, Manassas, VA, USA) and HPV18-positive cervical cancer cells (HeLa; KCLB, Seoul, Korea) were cultured with a medium containing 10% FBS and 1% Pen Strep in a 100-mm Petri dish (Nunclon Delta Surface, Thermo Fisher Scientific, Waltham, MA, USA). When cell confluency reached 80%, the cells were washed three times with PBS and treated with 2 mL of trypsin-EDTA (0.25%; Gibco) to produce single-cell units before culturing in 5% CO₂ at 37 °C for 2 min. After culturing, the detached cells were collected in a 15-mL conical tube into which culture medium was added, diluted 10 times to neutralize the trypsin, and centrifuged for 3 min at 1200 rpm. The cells obtained after removal of the supernatant were mixed with the culture medium and then used in the experiment.
The fibroblasts ( $1\times {10}^4,1\times {10}^5$ cells/mL) were seeded on NF with a diameter of 10 mm. After culturing in an incubator overnight, fibroblast-layered NF or bare NF was used as a substrate for inkjet cell printing.
For the cell-laden bioink, cell suspensions ( $3\times {10}^7$ cells/mL) were produced containing 0.5% collagen (collagen type I, Thermo Fisher Scientific) or cell suspension alone.