Gentamicin amphotericin

High-glucose DMEM supplemented with 10% FBS (Gibco), MEM non-essential amino acids, 100 mM sodium pyruvate and 100 U/mL penicillin-streptomycin was used as “Standard Medium”. CnT-Prime was purchased from CELLnTEC. “Uromedium” was prepared according to Osborn et al.^{6 (link)} with slight modifications. Briefly, “Uromedium” was comprised of EpiLife medium with 60 µM calcium (Gibco; MEPI500CA) supplemented with 60 μg/mL bovine pituitary extract (Gibco), 5 μg/mL human recombinant insulin (Diagnocine), 500 ng/mL hydrocortisone (Tokyo Chemical Industry), gentamicin/amphotericin (Gibco), 2% FBS, 0.1 ng/mL human recombinant epidermal growth factor (EGF) (RSD) and 100 μM 3-Isobutyl 1-methylxanthine (IBMX) (Sigma-Aldrich). “UCM” was comprised of EpiLife medium with 60 µM calcium supplemented with 60 μg/mL bovine pituitary extract, 5 μg/mL human recombinant insulin, gentamycin/amphotericin, 2% FBS, 0.01 ng/mL human recombinant EGF, 1 mM IBMX and 1 μM tranylcypromine (Abcam).

Isolated SpC were cut in three sections (apical, central, and caudal; ~1 × 1 × 2 mm) and cultured within 5 μL drop of 100% Matrigel Growth Factor Reduced (Corning 354 230) casted onto glass coverslip. For oSpC 3D culture onto decell muscles, scaffolds were cut in ~1 × 2 × 4 mm sections and put on a glass coverslip; then a single oSpC section was added onto each scaffold section and covered by 10 μL Matrigel droplet. To evaluate neural attractant effects of decell muscles, SpC sections were placed side by side with scaffold or with a sterile hydrophilic cotton gauze (~1 × 1 × 2 mm) onto a glass coverslip; 15 μL Matrigel droplet was used to embed the samples. The inert and decell scaffolds were placed approximately the same distance from each other (~1.5 mm) using a millimeter grid that was located under the petri dish at the moment of seeding. Samples were cultured in Neurobasal medium (Gibco 21 103 049), B‐27 supplement (Gibco 17 504 044) 1X, 2% Horse serum (Gibco 16 050 122), 0.5 mM GlutaMAX Supplement (Gibco 35 050 038), 25 μM 2‐Mercaptoethanol (Gibco 31 350 010), 25 μM L‐Glutamic acid (Sigma G5889), Gentamicin/Amphotericin (Gibco R01510), 10 ng/mL ciliary neurotrophic factor (CNTF, PeproTech 450‐13), and 10 ng/mL glial‐cell‐line‐derived neurotrophic factor (GDNF, Peprotech 450‐10). oSpC sections were maintained in culture for 14 days. Half medium was changed every 4 days.

For rat fetal spinal cord cultures, E14 fetuses were obtained from pregnant Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA). Rat fetal spinal cords were isolated following the ethical approval (D2784.N.I6Q) and culture as previously described^{12 (link)}. Briefly, isolated spinal cords were cut in three sections and cultured within 15 μL drop of 100% Matrigel (Corning) casted onto glass coverslips. Samples were cultured in Neurobasal medium (Gibco 21-103-049), B-27 supplement (Gibco 17-504-044) 1X, 2% Horse serum (Gibco 16-050-122), 0.5 mM GlutaMAX Supplement (Gibco 35-050-038), 25 μM 2-Mercaptoethanol (Gibco 31-350-010), 25 μM L-Glutamic acid (Sigma-Aldrich, G5889), Gentamicin/Amphotericin (Gibco R01510), 10 ng/mL ciliary neurotrophic factor (CNTF, PeproTech 450-13), and 10 ng/mL glial-cell-line-derived neurotrophic factor (GDNF, Peprotech 450-10).

Blood was dispensed into polypropylene tubes (BD Falcon™) and diluted 1:2 in sterile filtered phosphate-buffered saline (PBS). This solution was added to polypropylene tubes (BD Falcon™) containing Ficoll-Hypaque PLUS™ (Amersham Biosciences) (1:3). Cells were separated by centrifugation for 30 min at 900 x g 20°C, and the PBMC ring formed between the Ficoll interface and plasma was collected. Cells were washed twice by centrifugation and resuspended in DMEM medium (SIGMA™) supplemented with 10% FBS and 1% gentamicin-amphotericin (GIBCO^®). Cells were counted in a Neubauer chamber using Trypan Blue dye (Sigma™).