Krebs solution

An 800A in vitro apparatus (Aurora Scientific) was used to perform the ex vivo testing of the TA muscles. The testing chamber, along with the Krebs solution (Sigma), was kept at 25 °C using an external water heater. To help ensure tissue viability, a 95% O₂, 5% CO₂ mixture was used to oxygenate the testing chamber along with a petri dish containing Krebs solution. Mice were killed and the lower limbs excised from the ankle to ∼0.5 inches above the patellar tendon to ensure the entire TA muscle was removed. The legs were then pinned down in the petri dish coated with an elastomer (Sylgard-184, Dow Corning). The TA muscles were carefully dissected from the legs under a microscope and held under tension until attached to the force transducer lever. The muscle was attached to the force transducer lever by directly tying a knot around the top of the patellar tendon. The TA muscle was then attached to the force transducer lever and the bottom of the testing apparatus to secure the TA muscle in place. The testing chamber was raised around it to immerse the muscle in Krebs solution. A similar testing procedure to the in vivo protocol was used ex vivo except no voltage recruitment testing was performed. Imaging was performed in blind: the investigators performing the imaging did not know the identity of the experimental conditions for the transplanted cells.

The caudal halves of the uterine right horns were excised and cleaned of adhering fat and mesentery. Whole tissue strips were cut from the area in between the implantation sites into 10-mm length streps and mounted in organ baths (ADInstruments, Sydney, NSW, Australia, ML1110). The bath contained about 20 ml of Krebs’ solution buffer 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄, 1.16 mM CaCl₂, 14.9 mM NaHCO₃, and 5.5 mM dextrose (Sigma Aldrich, St. Louis, MO, United States) (Darios et al., 2012 (link)). The solution was maintained at 37°C and aerated with 95% O₂/5% CO₂ throughout the experimental period with washes every 15 min. The strips were initially tensioned to 1 gm and connected to an isometric force transducer (ADInstruments, Sydney, NSW, Australia, TRI210) by a silk thread. The latter was connected to a bridge amplifier (ADInstruments, Sydney, NSW, Australia, ML221), to amplify and convert the tension force generated by the contractions. The measurements were recorded by Powerlab (ADInstrument, Sydney, NSW, Australia, ML866) and analyzed by using LabChart 8 version. Preparations were allowed to equilibrate for about 60 min to obtain the spontaneous phasic contractions. After that, a continuous curve was recorded for 10 min (Aley et al., 2010 (link)).

On day 7, mice treated with DSS were euthanized with CO₂ and then the luminal contents were cleaned with sterile, cold PBS before the two ends of the luminal tube were ligated to prepare the colorectal intestinal sac described as previously.²⁵ The colorectal intestinal sac was then injected with 100 μl of Evans blue (EB) solution (Cat No E2129, Sigma‐Aldrich) [1.5% (w/v) in PBS] and placed in 20 ml of Krebs solution (Sigma‐Aldrich). After 30 min, the colorectal intestinal sac was washed with PBS until the flushing solution became clear, dried for 24 h at 37°C, and obtained a dry weight. It was then cultured at 55°C for another 24 h with 1 ml of formamide (Sigma‐Aldrich). Finally, the intestinal colorectal sac was removed, and the supernatant was centrifuged. The absorbance was measured at 655 nm.