Matrigel matrix

One hundred microliters of the diluted Matrigel matrix (Corning, Wiesbaden, Germany, #356234) was carefully added to the center of each Transwell^® insert (Corning, Wiesbaden, Germany, #3464) for invasion assays and incubated at 37 °C for 1 h to allow the Matrigel matrix to form a gel. Then, 200 μL of serum-free medium per single cell clone of HT-29 (2.5 × 10⁵/mL) was seeded into inserts and put in cell culture wells. The media were removed after 24 h and washed with PBS. Cells were fixed with 4% PFA, and the remaining buffer in upper chamber was removed, washed twice with PBS and the membrane was stained in 0.1% crystal violet for 30 min at room temperature. Cells were viewed underneath an inverted microscope and six visual fields were randomly chosen to calculate the number of migrated cells.

Porcine jejunum crypts were isolated and cultured in a 3D organoid culture system with the support of Matrigel matrix (Corning, USA) and IntestiCult organoid growth medium (StemCell Technologies, Canada) following previously published protocols with minor modifications (31 (link), 37 (link)). Briefly, jejuna were collected and opened longitudinally to remove luminal contents. Jejuna were cut into segments which were digested in 5 mM EDTA for 30 min on ice. After disassociation, the digestate was passed through a 70-μm filter and washed several times with cold PBS. The isolated crypt pellets were counted, and approximately 500 organoids were seeded into wells of a 24-well tissue culture plate (Corning, USA) and then incubated with Matrigel matrix in IntestiCult organoid growth medium. Growth medium was replaced every 2 to 3 days, and organoid morphology was observed daily under a microscope.
The differentiated organoids were used for development of 2D porcine intestinal monolayer organoids. Well-developed organoids were collected from Matrigel and dissociated with TrypLE Express (GIBCO, USA) for 10 min at 37°C, seeded into 48-well plates, and then incubated at 37°C in a humidified 5% CO₂ atmosphere; the intestinal monolayer organoids reached confluence after 3 days.

The 3D scaffolds were made into transwell cell culture inserts (8 μm pore size, Corning), using 50 μl of Corning Matrigel™ Matrix (10 mg/ml, Corning) according to standard procedures. Once gelled, 3000 hBM-MSCs were added to each transwell and incubated at 37°C in 5% CO₂ atmosphere. After 48 h the hBM-MSCs medium inside the insert was replaced with 200 μl parasite complete medium containing 500,000 stage II gametocytes, while 1 ml of hBM-MSCs medium was added into the bottom of the plate wells. Every 48 h, cells from Matrigel™ Matrix were recovered using Corning Cell Recovery Solution (Corning), to monitor cell growth and viability by Giemsa staining.

Corning® Matrigel® Matrix (356230) was thawed overnight on the ice at 4°C according to the guidelines in the manual. All pipets and procedures were previously kept on ice. Then, 289 μl chilled Corning Matrigel® Matrix into 24-well culture plates to avoid air bubbles. Plates were incubated at 37°C for 30–60 min. The medium remaining was removed carefully without disturbing the matrix layer, and the plates were ready to use. HUVEC cells were previously cocultured with 10 μg/ml non-irradiated-EVs or irradiated-EVs for 48 h (n=3). Then, 300-μl cell suspensions were collected and added to each well and incubated at 37°C, in a 5% CO₂ atmosphere. Tube formation was observed 3 h later under microscopy at 100-fold magnification and 10 fields per section were randomly selected for quantitative counting. The photo was further analyzed by ImageJ.

HNSCC patient tumor derived organoids were generated as previously described with some modifications^{45 (link)}. Briefly, tumors were first cut and minces into small pieces, which were then incubated in a digestion medium (including 1 mg/mL Collagenase XI, 10 μg/mL DNAase I, 10.5 μmol/L Y-27632 in human complete medium) at 37 °C for 30 min, the supernatant was collected and filtered through 70 μm filters and centrifugated at 800 rpm for 5 min. The pellet was then resuspended in Matrigel matrix (Corning) and seeded onto a 24-well plate for 15 min inside a CO₂ incubator at 37 °C. Finally, each well of the 24-well plate was incubated with 1 mL of tumor organoid culturing medium, which included advanced DMEM/F12 medium containing HEPES 10 mmol/L, 1X Glutmax, A83–01 500 nmol/L, hEGF 50 ng/mL, mNoggin 100 ng/mL, hFGF10 100 ng/mL, hGastrin I 0.01 μmol/L, nicotinamide 10 mmol/L, N-acetylcysteine 1.25 mmol/L, PGE2 1 μmol/L, B27 supplement, R-spondin1 conditioned media, and Afamin/Wnt3A conditioned media. For co-culture experiment, sCAFs or rCAFs were first seeded onto a 24-well plate overnight, while organoids mixed with Matrigel matrix (Corning) were then placed on top of these cells in the presence or absence of DDP -/+ cetuximab.

All in vivo experiments were conducted according to protocols approved by the Animal Research Committee at Case Western Reserve University.
Subcutaneous tumor formation: Overall, 2 × 10⁷ cells were injected subcutaneously in a 1:1 mixture of DMEMF12 media and Matrigel matrix (Corning). For each group (FT237 p000, FT237 p181a, FT237 p181a-antimiR) a total of five mice were used per group with two injection sites per mouse for a total of ten tumor injections per group. Tumors were measured biweekly for 25 weeks. At 25 weeks all mice were euthanized and tumors were recovered for end-point measurements and histological analysis.
Intraperitoneal (IP) tumor formation: Overall, 2 × 10⁷ cells were injected intraperitoneally in a 1:1 mixture of DMEMF12 media and Matrigel matrix (Corning). For each group (FT237 p000, FT237 p181a, FT237 p181a-antimiR) a total of ten mice were used per group with one injection per mouse for a total of ten tumor injections per group. Mouse weights were monitored biweekly for 22 weeks. At 22 weeks, all mice were euthanized and IP nodules were recovered for end-point measurements and histological analysis.