Ccl 163

Human foreskin fibroblasts (HFF; ATCC® SCRC-1041™) and BALB/c dermal fibroblasts (ATCC® CCL-163) were maintained in Dulbecco's modified Eagle medium (DMEM, high glucose, GlutaMAX™ Supplement, HEPES), and the monkey kidney cell line Marc-145 (ATCC® CRL-12231) was cultured in DMEM without sodium pyruvate and HEPES. Media were supplemented with phenol red, 10% heat-inactivated and sterile filtered fetal calf serum (FCS), 50 U of penicillin/ml, and 50 μg streptomycin / ml. The N. caninum isolates Nc-Liverpool (NC-Liv) and Nc-Spain7 (Nc-Sp7) were maintained by infecting semi-confluent HFF or Marc-145 monolayers and culture at 37°C/5% CO₂, with passages once or twice per week. To obtain tachyzoites for infection of BALB/c mice, infected monolayers were washed in medium without serum, and scraped from tissue culture flasks when tachyzoites were still largely intracellular (>90% of undisrupted parasitophorous vacuoles). They were repeatedly passed through a 25-gauge needle, liberated parasites were suspended in 0.2% trypan blue in PBS and counted in a hemocytometer. They were used for infection experiments when at least >95% viable.

Human foreskin fibroblasts (HFF; ATCC^® SCRC-1041^™) and BALB/c dermal fibroblasts (ATCC^® CCL-163) were maintained in Dulbecco’s modified Eagle medium (DMEM), and the monkey kidney cell line Marc-145 (ATCC^® CRL-12231) was cultured in DMEM without sodium pyruvate and HEPES. Media contained phenol red and were supplemented with 10% heat-inactivated and sterile filtered fetal calf serum (FCS), 100 U of penicillin/mL, and 100 µg streptomycin/mL. The N. caninum-Spain7 (Nc-Spain7) isolate was maintained by infecting semi-confluent HFF or Marc-145 monolayers and cultivation at 37 °C and 5% CO₂, with passages once or twice per week. Tachyzoites were separated from host cells by scraping the cell material from culture flasks when they were still largely intracellular (>90% of undisrupted PVs), and infected cells were repeatedly passed through a 25-gauge needle at 4 °C [26 (link)]. In some experiments, parasite-host cell separation was performed at 21 °C, either in the presence or absence of 5 µm BKI-1294.

NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC^® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC^® CCL-92™) and the inbred BALB/c 3T3 (ATCC^® CCL-163™) [64 (link)]. The NIH/3T3 cells are highly sensitive to sarcoma virus focus formation and leukemia virus propagation. In confluent cultures, NIH/3T3 proliferation is contact-inhibited.

Cell lines 293, HPAC, SW620, CT26, 4T1, and BALB/3T3 were obtained from the American Type Culture Collection (ATCC, cat nos. CRL-1573, CRL-2119, CRL-2638, CRL- 2539 and CCL-163, respectively). E0771 cells were obtained from CH3 BioSystems (cat. no. 940001) and PC9 cells were from Sigma (cat. no. 90071810). B16, glioma 261, and UACC-64 (UACC) cell lines were from the DCTD Tumor Repository at NCI (Frederick, MD). MC38, RENCA, and CHO-PR230 (CHO) cell lines were gifts of Jeffrey Schlom (NCI, NIH), Jonathan M. Weiss (NCI, NIH), and Stephen H. Leppla (National Institute of Allergy and Infectious Diseases [NIAID]), respectively. CHO-TEM8 and CHO-CMG2 cells were previously described^{21 (link)}, as were HCT-116-luc cells⁶⁵ .

To evaluate the in vitro biocompatibility and cytotoxicity of the mesh, a LIVE/DEAD assay was used to assess BALB/c 3T3 clone A31 mouse fibroblasts (American Type Culture Collection®; CCL163™). To prepare conditioned media, 500 mg of Coseal, TachoSil, and the 3D printed tissue adhesive were each incubated in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 v/v % fetal bovine serum and penicillin-streptomycin (100 U mL⁻¹) at 37 °C for 24 h. The supplemented DMEM (without any material incubation) was used as a control. 3T3 cells were plated in confocal dishes (20-mm diameter) at a density of 0.5 × 10⁵ cells cm⁻² (N = 4 for each group). The cells were then treated with either conditioned or control media and incubated at 37 °C for 24 h in a 5% CO₂ atmosphere. Cell viability was determined by a LIVE/DEAD viability/cytotoxicity kit for mammalian cells (Thermo Fisher Scientific). A confocal microscope (SP 8, Leica) was used to image live cells with excitation/emission at 495 nm/515 nm and dead cells at 495 nm/635 nm, respectively. Cell viability was calculated by counting live (green fluorescence) and dead (red fluorescence) cells using ImageJ (version 2.1.0).