Matrigel solution

Prior to cell seeding, scaffolds were sterilized by keeping them for 10 minutes in a 1 ml solution composed of 70% of ethanol (34 963, Sigma-Aldrich), and 30% of sterile Milli-Q water, and then sterilized under ultraviolet light for 1 night. Subsequently, scaffolds were functionalized through pre-incubation with a Matrigel solution (Corning) diluted in DMEM/F12 (Thermo Fisher Scientific) medium and, incubated at 37 °C and 5% CO₂ for 15 min. hiPSCs were seeded onto scaffolds either as single cells (2 × 10⁵ cells/1 cm² scaffold) or small clumps, and cultured in mTeSR1 medium.

MDA-MB-231 cells were plated on the poly(2-hydroxyethyl methacrylate) (pHEMA) coated U-bottom plates at a density of 2 × 10⁵ cells per well with 50 μL RPMI-1640 growth media. After centrifugation, 50 μL of 10% Matrigel solution (Corning, Cat#354234) was gently added to the plate. This was centrifuged one more time and was finally incubated for 72 hours at 37 °C in a humidified atmosphere with 5% CO₂.

Six-week-old male BALB/c nu/nu mice were purchased from Japan SLC (Shizuoka, Japan). LX-2 and Huh7 were cultured separately and, subsequently, mixed in a 2:1 fashion (Huh7: 1 × 10⁷ cells and LX-2: 5 × 10⁶ cells/tumor) in a mixed Dulbecco’s modified Eagle medium (DMEM) (Nacalai Tesque Inc., Kyoto, Japan) and Matrigel solution (1:1) (Corning, Glendale, AZ, USA). The solution was administered subcutaneously to the backs of mice bilaterally, as previously described [27 (link)]. Two weeks thereafter, mice were divided into two groups (n = 6, each) and were injected with PBS or 0.5 mg/kg of LPS intraperitoneally twice a week, respectively. Two weeks later, the mice were sacrificed, and the tumors were harvested. The methods of this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number: 80–23), as revised in 2011. This study and all its experimental procedures were approved by the Animal Ethics Committee of Nara Medical University.

The in vitro invasive capability of GL15, U87 and PriGBM cells was measured using the transwell invasion assays. Briefly, 50 μl of diluted Matrigel solution (1:4; Corning, Cat# 356234) was added to the upper chambers of the Transwell inserts (6.5 mm, 8-μm pore size; BD Biosciences). The inserts were incubated for 1 h at 37°C for gelling. Cells were added at a density of 5 × 10⁴ cells in 100 μl of medium with 2% FBS to the upper chambers, while the lower chambers were filled with 600 μl of DMEM containing 10% FBS. The Transwell inserts were then incubated for 24 h and the subsequent steps were performed as previously described (37 (link)).
The migration ability of cells in different groups was examined by in vitro wound-healing assay. Cells were seeded and were cultured into the 6-well plates untail they reached a 80–90% confluence and then cultured with serum-free DMEM for 24 h. After that, similar size scratches were introduced into the monolayer by a sterile pipette tip. The monolayer cells were rinsed with PBS to remove detached cells, and then replaced and cultured with serum-free DMEM. To analyze the cell migration, the wounded areas were photographed at the indicated 24 h point and then calculated the vacant area of each photo using Photoshop CS5. Percentage of wound healing was calculated as following: [1—(empty area 24h/empty area 0 h)] × 100%.

Migration assay: After re‐suspension in a serum‐free medium, approximately 1 × 10⁵ U87 or U251 cells were seeded into a transwell. Simultaneously, the cell‐culture medium was added to the side beneath the transwell chamber. Next, the cells were fixed and stained with 10% Giemsa in phosphate buffer at room temperature overnight. Then, cell numbers were counted in three random fields under a microscope. Modification for invasion assay: We coated 70 μl of Matrigel solution (Corning) with a density of 50 mg/ml on the upper side of the transwell before seeding the cells.