Inhaled isoflurane

Bone marrow was aspirated from the iliac crests of juvenile sheep (n=6, 25–45 kg). Animals were sedated with ketamine (IV, 4 mg/kg, Patterson Veterinary), diazepam (IV, 0.5 mg/kg, Patterson Veterinary), and buprenorphine (IM, 0.015 mg/kg, Patterson Veterinary), anesthetized with inhaled isoflurane (Patterson Veterinary) at 0–5%, vaporized with 100% oxygen at 30–60 mL/kg/min and prepared in sterile fashion. A small skin incision was made over the iliac crest, then a 15 gauge Illinois Sternal/Iliac Bone Marrow Needle (Care Express) was inserted to aspirate a maximum of 2 mL/kg of bone marrow per animal. Syringes were heparinized with 100 IU/mL in 0.9% saline. Aspirated bone marrow was then filtered through a 100-μm cell strainer (Falcon) and collected in sterile 50 mL conical tubes. The BM-MNC fraction was obtained from the filtered bone marrow aspirate with the Purecell Select System for Whole Blood MNC Enrichment (Pall Medical), following the manufacturer’s protocol as previously described [13 (link),14 (link)]. The cell suspension was subsequently vacuum seeded onto the polymeric TETG scaffolds using a previously described vacuum seeding technique (Fig 2A) [14 (link),15 (link)]. After seeding, TETGs were immersed in the post-seeding cell suspension (~140 mL) and immediately delivered to the surgical suite or sampled for further analysis.