Portex

On day 30, anesthesia was induced by intramuscular injection of ketamine 25 mg/kg and xylazine 3 mg/kg. Cannulation of the ear vein with a 24 G catheter (Abbocath, Abbott Medical, Baar/Zug, Switzerland) was performed. After infiltration of the anterior cervical region with lidocaine 1% (Sintetica, Mendrisio, Switzerland), a surgical tracheostomy with a 3.5-mm uncuffed tube (3.5 mm Portex, Smiths Medical, Kent, United Kingdom) was performed. Intravenous anesthesia with propofol 10 mg/kg/h, fentanyl 5 μg/kg/h, and midazolam 0.2 mg/kg/h was administered via the ear vein. The left femoral artery and right internal jugular vein were cannulated with a 20 G catheter for arterial and venous blood sampling and invasive blood pressure measurements.
After confirming adequate anesthesia and analgesia through the absence of movement in response to painful stimuli and cardiovascular monitoring (stable heart rate and arterial blood pressure), neuromuscular blockade was performed with atracurium besylate 0.6 mg/kg/h. Body temperature was monitored with a rectal thermometer and kept between 38 and 39°C with a thermostatic heating pad (Harvard Apparatus, South Natick, MA, United States). Intravenous fluid replacement was administered with Ringer’s acetate 2 mL/kg/h.

The experimental protocol was approved by the institutional ethics committee for experimental research of the University of Geneva and animal welfare committee (Office Véterinaire Cantonal de Genève registration number 1051/3609/1, Geneva, Switzerland). Eight adult New Zealand white rabbits (weighing 2.4-3.1 kg) were anaesthetized by an intramuscular injection of xylazine (5 mg/kg), followed by an iv injection of midazolam (1 mg/kg) and pentobarbital sodium (30 mg/kg) via an ear vein. Following tracheotomy, an endotracheal tube (4 mm i.d., Portex®, Smiths Medical, Kent, UK) was inserted into the distal trachea. Mechanical ventilation was started in volume controlled mode (Servo-i Maquet Critical Care, Solna Sweden equipped with an additional software) with a fixed respiration rate of 40 breaths/min and inspired oxygen fraction (FiO₂) of 0.5. Tidal volume was set to 7-8 ml/kg in order to target an end-tidal CO₂ (E_TCO2) of 5.5-6%. Maintenance of anesthesia was assured by a continuous iv infusion of midazolam (1 mg/kg/h) and fentanyl (100 μg/kg/h) via the ear vein. Muscle relaxation was achieved by atracurium (0.5-1.0 mg/kg/h) after ensuring adequate anesthesia and analgesia level. Airway pressure, heart rate and rectal temperature were displayed and stored on a computer at a sampling rate of 50 Hz via an analogue/digital interface converter (Biopac, Santa Barbara, CA, USA).

The cells were loaded in a cell loading tip pre-filled with mineral oil (Sigma-Aldrich). To manufacture the cell loading tip, a low-retention pipette tip with 200-µl volume capacity (Axygen) was cut at the top, in parallel to the rim and under the filter. A solidified 3-mm-thick piece of PDMS (Dow Corning) was punched from a slab of PDMS with a 5.0-mm sampling tool (EMS-Core). The circular piece of PDMS was then biopsy-punched with a 1-mm-wide biopsy puncher (Kai Medical Laboratory) in the middle. The circular piece of PDMS was pushed inside the tip while remaining parallel to the upper rim of the tip. A 1-ml glass syringe (SGE) was then pre-filled with 1 ml of mineral oil and connected to a 30-cm-long tubing (Portex, Smiths Medical) that can be inserted to a hole in the middle of the circular PDMS piece in the tip. Next, the tip was pre-filled with mineral oil by manually pushing the syringe, and the cell-containing solution was further aspirated with care as to not introduce any air bubble in the system. The tip can then be connected to the cell-encapsulation PDMS device, inlet number 2 (Extended Data Fig. 1c,g), and injection rates are modulated by a Nemesys syringe pump (Cetoni).

After fasting overnight, the mouse was anesthetized by isoflurane with an anesthesia machine air pump (R510-29, Rayward Life Technologies Inc, Shenzhen, China). The posterior end of the mouse was gently pressed to remove the feces that might be present in the distal colon. A 19 G needle with a fine bore polythene tubing (PE-50, 0.58 mm ID, 0.96 mm OD, PORTEX, Smiths Medical, Minneapolis, USA) fastened to its end was attached to a 1 ml syringe. The fine bore polythene tubing was gently inserted intrarectally, reaching approximately 3-4 cm proximal to the anus 22 (link), 23 . 100 μg of CRAMP was slowly injected, and the mouse was positioned head-down for 90 s to avoid CRAMP loss.

After weaning from the ventilator, each spontaneously breathing patient receiving COT underwent RPT with bed head elevation (30°), early sitting position, active mobilization, and the following respiratory maneuvers:(1)

Airways cleaning techniques;

(2)

Manual stimulated cough and sputum induction;

(3)

Manual alveolar recruitment consisting in EzPAP operated a zero PEEP (Portex, Smiths Medical, London, UK) and active cycle of breathing technique.

All the above mentioned protocolized techniques have been previously described more specifically by our group [8 (link)].
This RPT session was performed once daily, from the day of weaning from the ventilator to the day of ICU discharge, for a median duration of each session of 30 min.