Crl 2279

Mouse microvascular endothelial cells (MMECs, CRL-2460, MS1-VEGF) were sourced from American Type Culture Collection (ATCC, Manassas, VA, USA) and serially passaged (p5–p10) for the study in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA), at 11 mM glucose concentration, supplemented with 5% FBS (Sigma-Aldrich, St.Louis, MO, USA), in a humidified atmosphere with 5% CO₂ at 37 °C, as previously described [16 (link),49 (link)]. A value of 11 mM glucose (normal glucose levels for MMECs) is based on random plasma glucose measurements established from C56/BL mice [86 (link)]. Primate VEGF-121 was overexpressed in the MS1 pancreatic microvasculature endothelial cell line (CRL-2279; ATCC, Manassas, VA, USA) to derive the MS1-VEGF/MMEC line [29 (link)]. MMECs reportedly generate well-differentiated angiosarcomas in nude mice [29 (link)]. Additionally, these cells have been used to investigate signal transduction pathways involved in tumorigenesis and angiogenesis and are, therefore, considered appropriate for the proposed studies on the putative anti-angiogenic effects of metformin [29 (link),87 (link),88 (link)].

For the in vitro biological tests, two differently shaped specimens were tested: Bulk and print-ring specimens. MC-based hydrogels were prepared using a filter-sterilized solution (0.22 μm pore size filters, VWR International, Milan, Italy) and UV light sterilized MC powder. Bulk specimens (n = 3) were prepared by adding MC solutions in a tissue culture multiwell plate (24 wells TCPS, TCPS-24). The print-ring specimens were printed with the Kiwi 4D printer under a laminar flow cabinet; the printer was moved under the cabinet and disinfected with 70% v/v ethanol solution and sterilized by UV light for 30 min before MC printing. In the in vitro tests, murine embryonic fibroblasts (NIH/3T3, CRL-1658, ATCC, Manassas, VA, USA) modified with a gene for the expression of green fluorescent protein (GFP) and endothelial murine cells (MS1, CRL-2279, ATCC, Manassas, VA, USA) were selected to investigate the feasibility of using different cell phenotypes to produce CS. For both cell lines, Dulbecco Modified Eagle’s Medium (DMEM, Sigma-Aldrich, Saint Louis, MO, USA) supplemented with fetal bovine serum (FBS, 10% v/v), L-glutamine (1% v/v), Hepes (1% v/v), sodium pyruvate (1 mM, 1% v/v), and a solution of penicillin/streptomycin (1% v/v) was used.

MILE SVEN 1 mouse vascular endothelial cells of wild-type (MS1_WT) were obtained from American Type Culture Collection (ATCC; CRL2279) and stably transfected with human B7-H3 expression vector to generate MS1_B7-H3 cells as described previously [61 (link)]. DMEM (Corning Inc., Glendale, AZ, USA) cell culture media containing 5% fetal bovine serum and 100 units/mL of penicillin and 100 µg/mL of streptomycin was used to culture MS1 cells. Similarly, 4T1 (ATCC; CRL2539) mouse breast cancer cells and MDA-MB-231 (ATCC; HTB-26) human breast cancer cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 µg/mL of streptomycin.

Wildtype MILE SVEN 1 (MS1_WT) mouse vascular endothelial cells (CRL2279; American Type Culture Collection (ATCC)) and MS1 cells engineered to stably express human Thy1 protein (MS1_Thy1) (selected using puromycin antibiotic marker) were cultured under sterile conditions in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 100 U/mL penicillin and 0.1% streptomycin and maintained in 5% CO₂ at 37 °C and used for Thy1-scFv binding assays as stated below.