Systemic-intraosseous Delivery of Dystrophin-expressing Chimeric Cells to mdx Mice

Dystrophin Expressing Chimeric cells were prepared for systemic-intraosseous injections to the femoral bone of the mdx mice as previously reported [43 (link), 45 (link), 46 (link)]. Briefly, DEC were counted and washed in sterile saline and viability was assessed with 0.4% Trypan Blue. A single DEC dose was suspended in 60 μl of total volume of saline and transferred to tuberculin syringe with 27G needle (ThermoFischer, Waltham, MA, USA). Mdx recipients were anesthetized with 1.5% isoflurane inhalation and injected with subcutaneous buprenorphine SR lab (0.1 mg/kg) for analgesia. Following a 5-mm skin incision over the mid-femoral level, the subcutaneous tissue and the overlying muscles were dissected. The opening was made in the femoral bone with 28G needle passing through the cortex into the medullary cavity of the femur and 60 μl of the recipient bone marrow were aspirated prior to DEC injection. Next, DEC cells and controls were suspended in 60 μl of sterile saline solution and were injected into the femoral bone marrow cavity in the respective experimental groups. After injection, the bone was sealed with bone wax to prevent cell leakage. The muscle and skin were closed using 4–0 monofilament absorbable suture. Animals were allowed to recover in a heated environment and promptly returned to the colony.
After age-matching the recipient mdx mice were randomized into the following experimental groups: vehicle control (n = 3, 60 μl saline), not-fused MB^wt and MB^mdx (n = 3, 0.25 × 10⁶/donor–total 0.5 × 10⁶ in 60 μl saline), fused MB^wt/MB^mdxDEC (n = 4, 0.5 × 10⁶ in 60 μl saline), not-fused MB^wt and MSC^mdx (n = 3 0.25 × 10⁶ /donor–total 0.5 × 10⁶ in 60 μl saline), and fused MB^wt/MSC^mdx DEC (n = 3, 0.5 × 10⁶ in 60 μl saline). Animal follow-up consisted of in vivo assessments of echocardiography at day 0 before DEC injection and at 30 and 90 days post-DEC transplant. At day 90 endpoint the animals were euthanized and heart muscles were harvested for histological and immunofluorescence analysis.