Slide angiogenesis

Medium 200 (M200PRF500), low serum growth supplement (LSGS; S00310), 0.025% trypsin-EDTA (R001100), VEGF (PHC9344), Pierce Protease Inhibitor Mini Tablets (A32953), HisPur™ Cobalt Resin (89964), Detoxi-Gel™ (20339), penicillin-streptomycin (15140122), amphotericin B (15290018), Qubit™ Protein Broad Range kit (A50668) and human umbilical vein endothelial cells (HUVECs; C0035C; RRID: CVCL K312) were purchased from Thermo Fisher Scientific/Fisher Scientific. ToxinSensor™ LAL Endotoxin Assay Kit (L00350) was purchased from Genscript. HisLink™ Protein Purification Resin (V8823) and CellTiter 96^® AQueous One Solution (G3582) were purchased from Promega. Mitomycin C (M4287) was purchased from Sigma Aldrich. Proteome Profiler™ Human Angiogenesis Antibody Array (ARY007) was purchased from R&D Systems. IRDye 800 CW Streptavidin (926-32230) was purchased from LI-COR. Growth factor reduced Matrigel (GFR-Matrigel; 356231) was purchased from Corning. Axitinib (HY-10065) was purchased from MedChemExpress. 0.1 mm glass beads (P000929LYSK0A.0) and soft tissue homogenizing kits (P000933-LYSK0-A) were purchased from Bertin Corp. 4-well culture inserts (80466) and angiogenesis slides (81506) were purchased from ibidi. MycoAlert™ Plus Mycoplasma Detection Kit (LT07-701) was purchased from Lonza.

All of the experiments shown were performed in low throughput 15-well Angiogenesis slides (Ibidi GmbH, Munich, Germany), and growth factor-reduced Matrigel (BD Invitrogen) as the ECM of choice to promote differentiation. Miniaturized 3D cultures were prepared as described previously [6] (link), [7] (link). Bottom wells of ibidi Angiogenesis μ-slides were filled with 10 µl of 50% Matrigel-medium (typically 3–5 mg/ml protein, depending on the batch), and incubated at +37°C for 30–60 min. Cells were placed on top of the polymerised bottom gel at a density of 700–1500 cells/well (depending on the cell line), and incubated at +37°C for 1–2 h. Medium was discarded, and cell layers covered with 20 µl of 25% Matrigel (1.5–2.5 mg/ml depending on the batch). The μ-slides were humidified by adding 15 µl drops of sterile water between the wells. The upper gel was allowed to polymerize at +37°C for 3–4 h or overnight. Wells were then filled with medium, and changed every second day.

Angiogenesis slides (Ibidi, Germany) were coated with 10 μL of Matrigel (BD Biosciences, USA), which was allowed to condense at 37 °C for 2 h. A total of 2000 transfected HTR8/SVneo cells were plated on the top layer. After incubation for 4 h at 37 °C, images were acquired for tube formation analysis. All experiments were conducted in triplicate.

Cells infected with HIV-1 NL4.3DispYFP were purified by magnetic isolation as described above. Control CD4 cells from uninfected cultures were purified correspondingly with anti-CD4 magnetic beads (Miltenyi Biotec, 130-045-101). Infected and uninfected cells were either embedded separately in collagen or were stained with PKH cell dyes as for proliferation assays to enable distinction between infected (green, PKH67) and target cells (red, PKH26) in cocultures. Cells within collagen gels were monitored in angiogenesis slides (ibidi) using bright field, and for stained cells, green and red channels of an inverse light microscope (×10 objective, Nikon Ti-E), equipped with climatisation control maintaining 37 °C and 5% CO₂ (Perkin Elmer). For the duration of up to 3 h, micrographs were taken in 30 s to 1 min intervals.

For independent reseeding of round and aberrant 3D phenotypes, 30–40 spheroids of one class were isolated, dissociated and pooled as described above. A 10 µl Matrigel bed was prepared during dissociation in 15 µ angiogenesis slides (Ibidi). After centrifugation, cells were resuspended in 50 µl assay medium (+5% Matrigel) and added to pre-treated angiogenesis slides. Medium was replaced every 3 days and cells were cultured for up to 6 days.

C166 murine yolk-sac endothelial cells (ATCC) were propagated in flasks coated with 0.1% gelatin (Stem Cell Technologies) using endothelial growth medium (Angioproteomie) and incubated at 37 °C and 5% CO₂ atmosphere. Monocytes were isolated from bone marrow of male CX3CR1^GFP/+ mice and sorted using a FACS-Aria IIIu to discriminate CX3CR1^hi monocytes and CX3CR1^lo monocytes. Endothelial tube forming assays were performed in 15-well angiogenesis slides (Ibidi) as follows. Wells were coated with BD Matrigel^TM and seeded with C166 cells (7500 cells/well) and CX3CR1^hi or CX3CR1^lo monocytes (2000 cells/well). After 20 hours, co-cultures were fixed and labeled with rhodamine-phalloidin. Images were captured using a Zeiss LSM 700 confocal (10x magnification) and Zen software (Zeiss). The centroid of each monocyte was identified and the distance to the nearest endothelial tube formation was calculated using a custom MATLAB code. For each vessel image, a random distribution of “cells” was overlaid on the image (equal to the number of monocytes in the original image) and the distance of the “random cells” to the nearest vessel was calculated. The experimental monocyte distance to vessel was then compared to the computer-generated randomized cell distance to vessel to determine whether the cells preferentially distribute themselves near vessel segments.

Invasion of HUVEC into 3D collagen gels has been elegantly described elsewhere^{43 (link)}. Briefly, thin gels of 2.4 mg/mL bovine collagen I containing 1 µM sphingosine-1-phosphate (Avanti Polar Lipids, 860492P) were formed in angiogenesis slides (Ibidi, 81506). WT and KO HUVEC cells were plated on top of the gel in EGM2 supplemented with 40 ng/mL bFGF and human recombinant VEGF (Thermo Fisher Scientific, 293VE050). After 24 h, wells were fixed in 4% paraformaldehyde and stained with Alexa488-phalloidin. Confocal z-stacks were acquired and the number of invaded cells into the collagen gel were quantified manually in Fiji. Statistical comparisons were performed in Prism.