U 87 mg atcc htb 14

The glioblastoma cell line U-87 MG (ATCC HTB-14) and the neuroblastoma cell line IMR-32 (ATCC CCL-127) were purchased from the American Type Culture Collection (ATCC, USA). Their clinical status and culture conditions were referenced on the ATCC website (www.atcc.org). Cells (1.25 × 10⁵ cells/well) were cultured in 6-well plates with medium containing 10% FBS for 48 h for IMR-32 and for 24 h for U-87 MG. Both cell lines were then treated with a serum-free medium for different times (24 h for IMR-32 and 48 h for U-87MG) in the absence or presence of the recombinant human insulin-like growth factor 2 (IGF2; I2526, Sigma, St. Louis, MO, USA) at indicated concentrations for a final 24 h incubation.

Primary human astrocytes and Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Lonza (Basel, Switzerland) and maintained in Astrocyte growth medium (AGM) BulletKit (CC-3187 and CC-4123) and Endothelial cell growth medium (EGM)-2 BulletKit (CC-3156 and CC-4176), respectively. Human brain microvascular pericytes were obtained from Sciencell (Carlsbad, CA, USA) and maintained in Pericyte medium (PM) supplemented with 2% fetal bovine serum (FBS), 1% PM growth supplement, and 1% of penicillin/streptomycin solution. Vero (ATCC CCL-81), the human glioblastoma cell line, U87MG (ATCC HTB-14), and 293T (ATCC CRL-3216) cells were obtained from ATCC (Manassas, VA, USA) and maintained in Dulbecco’s modified minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% l-glutamine, and 1% penicillin/streptomycin. All cells were maintained at 37 °C with 5% CO₂.

U87 cells were chosen for development of GB in Yucatan minipig because they were used in previous studies on Landrace pigs and Yucatan minipigs [7 (link),51 (link)]. Cells were obtained from the American Type Culture Collection (U-87 MG, ATCC® HTB-14™, LGC Standards, Molsheim, France). Regarding the ATCC product sheet, mycoplasma contamination was eliminated in September 1975 [52 ]. For inoculation of U87 cells in each subject, U87 cells were plated in 2 T175 tissue culture flasks with a density of 0.8–1.2 × 10⁶ cells per flask. Cells were grown in Minimum Essential Media (MEM) medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-Glutamine and 0.25% g/mL amphotericin B. Cells were maintained at 37°C in a humidified CO2 (5%) atmosphere, and the medium was changed every 3 days. A week later, cells from these flasks were trypsinized with 0.25% trypsin-EDTA and then 35–50 × 10⁶ cells per flask were harvested. Cells were washed twice in PBS before being pelleted 4 min at 3000 g in a 2 ml Eppendorf vial just before the inoculations. The final cell concentration in each Eppendorf vial was about 1.7 ×10⁶ cells/10μl. For each subject, 40 μl and 20 μl of the cells pellet were drawn up into the syringe and injected into the right and left hemisphere, respectively.

Human hepatocellular carcinoma (HepG2/ATCC HB-8065) and human glioblastoma (U-87 MG/ATCC HTB-14)™ were obtained from ATCC (Manassas, VA, USA). Dulbecco’s modified Eagle medium (DMEM, Life Technologies 11965) and fetal bovine serum (FBS, Life Technologies 16000) were procured from Life Technologies (Carlsbad, CA, USA). Penicillin-streptomycin (Cellgro, 30–002-CI) and Trypsin-EDTA (Cellgro 25–053-CI) were manufactured by Cellgro (Manassas, VA, USA). All of the reagents that are required for the microencapsulation of cells, including medium viscosity alginate (Sigma A2033 (μ > 2000 cP, Mv = 900–1000 kDa, M/G ratio 1.6), blue dextran dye (Sigma, D4772), as well as reagent grade salts and solvents were purchased from Sigma Aldrich (St. Louis, MO, USA). Proprietary 3D printing photoresins were provided by Formlabs (Summerville, MA, USA).

Human GBM cell lines, including U87MG (ATCC HTB-14; American Type Culture Collection, Manassas, VA, USA) and A172 (ATCC CRL-1620), and patient-derived primary GBM (Pt#3 and Pt#5) along with their TMZ-resistant and stem-like cells, were cultured in respective media as described previously [16 (link)]. Procedures utilized for establishing TMZ-resistant GBM cells were mentioned previously [16 (link)]. To maintain TMZ-resistant cells, 50 μM TMZ was added into the culture medium, and their resistance characteristics were confirmed using colony-formation assay. Informed consent obtained from the patients followed the protocols (Nos. 201,006,011 and 201,402,018) approved by the Joint Institutional Review Board (JIRB) of Taipei Medical University (Taipei, Taiwan).

The U87 MG (ATCC® HTB-14™) human glioblastoma cell line was obtained from the American Type Culture Collection (ATCC) and was grown in Eagle's supplemented minimum essential medium (EMEM; Lot No. 2062257), further supplemented with heat-inactivated 10 % fetal bovine serum, 1 mM L-glutamine (Gibco Life Technologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37 °C and 5 % CO₂.