Pdc 001

For the compartmentalised coculture in 2D, Xona microfluidics commercial devices with microchannels of 900 μm (length), 10 µm (width), 5 µm (height) and spaced 50 µm apart (Xonamicrofluidics, #SND900, US) were utilised.
To prepare the devices, a 2 mm perforation was made in the SN seeding side with a punch (Miltex Instruments, #33-31, US), and a 3 mm perforation in the SkM seeding side with another punch (Miltex Instruments, #33-32, US), as shown in Fig. 2B for chambers c1 and c2, respectively. Then, the glasses (Fisher scientific, #12-542-C, US) and the Xona polydimethylsiloxane (PDMS) devices were cleaned sonicating in 100% ethanol for 15 min, drying with a nitrogen gas stream and dehydrating in a drying oven at 60 °C for at least 30 min (Binder, #FD-23). Finally, glass coverslips were permanently bonded to the PDMS pieces exposing both to 1 min under high frequency oxygen plasma (Harrick plasma, #PDC-001) at 700 mTorr, then putting both pieces together and sealing them with at least 30 min dehydration in the oven at 60 °C. At this point, devices could be stored for the beginning of the experiment.

NHC was made following the protocol previously described [22 (link)]. Briefly, nanofibers were electrospun from a PCL solution (16% w/w; Sigma-Aldrich, St. Louis, MO, USA) in a mixture of dichloromethane (Sigma-Aldrich) and dimethylformamide (9/1, v/v; Sigma-Aldrich). Poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT; Sigma-Aldrich), a green fluorescent dye, was added to the PCL solution to enable fiber identification after injection [22 (link)]. Carboxyl groups were introduced to the surface of the fibers in a plasma cleaner (expanded plasma cleaner; PDC-001; Harrick Plasma; Ithaca, NY, USA). These carboxyl groups were initiated by ethyl dimethylaminopropyl carbodiimide (Sigma-Aldrich) and N-hydroxysuccinimide (Sigma-Aldrich) and then converted to maleimide (MAL) groups by N-(2-aminoethyl) maleimide (Sigma-Aldrich). The MAL-modified fibers were cryogenically milled, sterilized with 70% ethanol. All three components of NHC were stored individually at −20 °C, and thawed 30 min prior to use. NHC was prepared by mixing MAL-modified fibers (10 mg/mL) into a mix of HA-SH (4 mg/mL; ESI BIO, Alameda, CA) and PEGDA (2 mg/mL; ESI BIO) in sterile phosphate-buffered saline (PBS) [22 (link),25 (link)] on ice. We mixed and injected NHC or MSC-NHC within 15 min after exposing the contused spinal cord (see below Section 2.4). Once mixed, NHC can be kept on ice for 6 h for injections.