Endogro vegf medium

HUVECs (Promocell) were cultured in EndoGRO-VEGF medium (Millipore). For the bio-ink we used 10⁶ HUVECs per ml suspended in Basal medium (EndoGRO, Millipore), supplemented with fibrinogen (13.24 µM) (F8630-5G; Sigma-Aldrich) and aprotinin (7.68 µM) (10820–25MG; Sigma-Aldrich) that facilitated the gelation processes post printing. A red food dye, Allura red AC (458848-100G, Sigma-Aldrich), was also added to a final concentration of 10 mΜ to enhance light absorption by the bio-ink.

Freshly prepared bio-ink (~100 μL) was loaded to the squared capillary using the syringe pump. The laser beam was focused in the middle of the capillary, 500 µm far from its distal end. The receiving substrate was fixed on an xyz translation stage and placed 500–700 µm far from the capillary tip. Laser energies at the sample varied from 90 to 130 μJ. Printing patterns consisted in arrays of individual droplets separated by a 500 μm gap. After printing, samples were placed in an incubator (37 °C, 5% CO₂) for 5–10 minutes. Next, we rinsed the samples twice with EndoGRO-VEGF medium (Millipore) to dilute the light absorbing red dye and put them back in the incubator till further analysis.

All colon cancer cell lines (Caco-2, Lovo, SW480, HCT116, DLD-1, HT29, SW620, HCT15 and SW837) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cell line authentication was assessed using a short tandem repeat DNA profiling method in our laboratory, and the latest verification was done in July 2013. The mycoplasma detecting kit (catalogue no. MD001), purchased from Shanghai Yise Medical Technology was performed to test whether there are mycoplasma contamination in all cells. HUVECs were maintained as described previously42 (link) and cultured in EndoGRO-VEGF medium (Millipore, MA, USA).
Antibodies used in this study were as follows: SARI (ab157466, Abcam, London, UK), SARI (241887, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SARI (Zhengneng bio, Chengdu, China, for detecting mouse SARI protein), Cp (ab48614, Abcam), HIF-1α (H6536, Sigma, MO, USA), HA (04–902, Millipore), VEGF (ab46154, Abcam), CD31 (550274, BD Biosciences, CA, USA), CD31 (ZM-0044, ZSBIO, Beijing, China, for detecting human CD31), Histone H3 (4499P, CST, MA, USA), GAPDH (sc365062, Santa Cruz Biotechnology) and β-actin (sc-47778, Santa Cruz Biotechnology).