Clinical Trial Evaluation of Stem-cell Based Bioartificial Airway Transplantation for Patients With Benign and Malignant Laryngo-tracheal Diseases

Before transplantation the patients will have the laboratory and instrumental evaluations.

Three days before the transplantation the patient will be underwent bone marrow aspiration. The bone marrow mononuclear cells (MNC) will be isolated from the red blood cells (RBC) in the totally enclosed FDA approved automatic system (Sepax,BioSafe America, Inc.). The final product, re-suspended with cell culture medium (DMEM+10% albumin and 10% autologous plasma) in a volume of 200 mL, will be placed in a 600 mL transfer bag. 2 mL of the product will be taken from the bag before clinical use to test sterility using culture media and immunofluorescent cytometry to characterize cell type and viability.

Two days before the transplant, the patient will begin "boosting" therapy to mobilize cells by means of systemic injections of analogous recombinants of granulocyte colony-stimulating factor (GCSF)(Granocyte, 1 M IU/kg (max. 15 M IU) and Erythropoietin, 400 IU/kg (max.6,000IU). These will be injected for the two days prior to surgery.

InBreath Bioreactor (the special bioreactor for cultivating trachea) The work in the current protocol will involve a bioreactor design previously utilized by Macchiarini P. ang colleges in a successful first-in-man implantation of a tissue-engineered large airway replacement. The device, commercialized under the name, InBreath 3D Organ Bioreactor (Harvard Bioscience, Inc.) is designed for placement within a tissue culture incubator and consists of a modular polysulphone organ chamber, motor unit and remote controller. The chamber is easily detachable from the motor unit and its polysulphone construction permits sterilization with the standard gas plasma sterilization process that is readily available in the operating room in Peoria. The motor unit provides consistent rotation to the tissue holder within the chamber, ensuring controlled application of hydrodynamic shear forces to the developing tracheal construct. A fully enclosed motor housing protects the brushless motor from the corrosive moisture within the incubator. The remote control unit is placed outside the incubator providing a means to adjust rotational speed without disturbing the incubator environment.

The seeded construct was allowed to incubate in the bioreactor for 96 hours prior to removal for implantation. Based on the five previous adult cases using the POSS-PCU (Polyhedral oligomeric silsesquioxane-poly(carbonate-urea) urethane), PET(Polyethylene terephthalate) and PET:PU (Polyurethane) synthetic scaffolds, the internal and external surfaces of the scaffold will be seeded with the freshly isolated bone marrow mononuclear cell fraction. The bioreactor will be started with an initial speed of 0.5 cycles/min for 18 hours (then stepwise increase up to 2.5 cycles/min). Incubation will be during the 48 hours preceding the transplant procedure. This incubation protocol worked very well in the previous cases using the three different synthetic nanocomposite tracheal scaffolds.