Engineered Neural Tissue Constructs

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.