Raft absorbers

Following differentiation, CTX cells were used to create EngNT according to methods described previously^{21 (link)}. All gels were prepared using 80% v/v Type I bovine dermis collagen (3 mg/ml; Koken, diluted to 2 mg/ml using 1 mM HCl) mixed with 10% v/v 10 × minimum essential medium (Sigma) and neutralised using RAFT Neutralising Solution (Lonza Bioscience) before addition to 10% v/v CTX cell suspension to give a cell density of 2 × 10⁶ cells/ml of gel. Gels were allowed to set in tethering moulds at 37 °C for 15 min and then immersed in culture medium and incubated at 37 °C in a humidified incubator with 5% CO₂/95% air for 24 h, during which time the cells contracted the tethered gels and become aligned^{39 (link)} (Supplementary Figure 2). Using RAFT absorbers (Lonza Bioscience) the aligned gels were stabilised for 15 minutes, a process whereby a biocompatible absorbent material is placed upon the gel and absorbs interstitial fluid to generate a dense robust hydrogel with a 50 fold increase in cell and collagen density. The resulting sheets of EngNT were rolled to form rods (12 mm length) and each construct was secured within a NeuraGen™ sheath (13 mm long) using Fibrin glue (TISSEEL, Baxter), ready for implantation.

Collagen-fibrin blend gels were prepared as follows: 10× MEM and Collagen I (rat tail; First Link) were mixed on ice in a ratio of 1:8. Fibrinogen reconstituted in aprotinin was added to the solution v/v ratios of 10%, 20%, 30%, and 40%. Subsequently mixture was neutralized with 7% v/v sodium hydroxide. One unit thrombin in CaCl₂ was added to polymerize fibrinogen. Two hundred forty microliter of the resulting collagen-fibrin solution was pipetted into 96-well plates and placed at 37°C. Gels were stabilized using RAFT absorbers (Lonza, Germany) for 15 min. Original height before and after stabilization, as well as potential swelling of the gels in 300 μL medium, was evaluated with side elevation view imaging (KSV CAM 200) on day 0, 1, 3, and 5.

Differentiated CTX0E03 cells (dCTX0E03) were used to create stabilized cellular collagen scaffolds. These were used to mimic the conditions in engineered neural tissue constructs. All gels were prepared using 80% v/v type I rat tail collagen (2 mg/ml in 0.6% acetic acid; First Link) mixed with 10% v/v 10× minimum essential medium. The mixture was then neutralized using sodium hydroxide (NaOH) and 10% v/v cell suspension was added to give cellular collagen at a series of cell densities (0.5–1.5 × 10⁶ cells/ml of gel). These cell seeding densities were based upon the range used within NRCs (Coy et al., 2020 (link); Georgiou et al., 2015 (link); O'Rourke et al., 2018 (link)) (Table 1).
Next, 240 μl of the cellular collagen mixture was added to individual wells of a 96 well plate and the gels were allowed to set at 37°C for 15 min. Using RAFT absorbers (Lonza Bioscience) the gels were stabilized using plastic compression for 15 min, a process whereby a biocompatible absorbent material is placed upon the gel and absorbs interstitial fluid to generate a dense, robust hydrogel (Brown et al., 2005 (link)). The resulting compressed gels were then immersed in culture medium and incubated at 37°C in a humidified incubator for 24 h under different oxygen concentrations, chosen to reflect the range of oxygen concentrations in which cells would reside in vivo.