Kwik cast sealant

The HTS-24 well transwell (Falcon 351185, Corning, NY, US) membrane was replaced with an electrospun biodegradable scaffold. The scaffold was made of poly(lactic-co-glycolic acid) (PLGA) (50:50, 1.0 inherent viscosity) nanofibers with 900 nm diameter and 4 μm thickness (BioSurfaces, Ashland, MA) to achieve better cell adherence and migration. Its half degradation time was about 6 weeks. A customized biocompatible O-ring (Ultimaker B.V, Netherlands) was glued onto the bottom of the transwell insert using bio-compatible adhesive (Kwik-cast sealant, world precision instrument, Sarasota, FL, USA). The polycarbrolactone O-ring was used to prevent any leakage from the bioprinted hydrogel. The transwell inserts were then treated with 200 μL fibronectin dissolved in deionized water (0.03 mg/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 1 hr at room temperature, followed by aspirating and air-drying overnight. The basal side of the assembled transwell scaffold insert was treated with oxygen plasma for 30 minutes before bioprinting. The final culture area was 0.33 cm² for the HTS-24 well plate.

Mice were anaesthetized with isoflurane, a small craniotomy (around 2 mm in diameter) was performed above the RSP, and a custom head-plate implanted. The dura was gently removed, and then covered with Kwik-Cast Sealant (World Precision Instruments). A skull screw was implanted and secured with cement in the right frontal skull bone. Mice were given 1–2 h to recover. Next, mice were i.p. injected with 50 mg kg⁻¹ ketamine, and head-fixed under an in vivo electrophysiology recording apparatus. To record neural activity, a 32-channel two-shank silicon neural probe (ASSY-37 P-1, Cambridge NeuroTech) was slowly lowered into the RSP. The deepest tip of the electrode was 800–1,000 μm from the brain surface for all recordings.

To measure the tissue’s elastic modulus, we removed the hECT from its bioreactor, and glued it down with Kwik-cast sealant (World Precision Instruments) to the bottom of a petri dish, applying the sealant to polar ends of the hECT, after 1 minute the dish was filled with media for subsequent measurements under AFM. The AFM measurements were performed with Flex-Bio AFM (Nanosurf AG, Switzerland) equipped with an inverted microscope Axio Observer (Carl Zeiss) and an environmental control enclosure. The AFM was operated in static force mode. The cantilever had a spring constant of 0.27N/m and silicon tip radius < 10nm (Stat0.2LAuD, Nanosurf AG, Switzerland). We measured force spectroscopy at 13 different spots (N=8 for each spot) with distances from the edge of the tissue ranging from 200 to 300 μm as shown in Figure 5a. The force curves, as shown in Figure 5b, were analyzed and the elastic modulus was obtained by applying the Hertz model [33 (link), 34 (link)] to the forward approach curves using the ANA control and analysis software (Nanosurf AG, Switzerland) resulting in an average elastic modulus of approximately 10.7 +/− 3.7 kPa for the hECT. This value was later used in the finite element (FEM) model using COMSOL Multiphysics (ver. 5.4, COMSOL, Inc., MA, USA).