We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4 ؉ T cells and B220 ؉ B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor-immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-␤ receptorimmunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy. (Blood. 2012;119(6):1570-1580) IntroductionChronic GVHD (cGVHD) is a significant complication of allogeneic HSCT. 1 Progress in developing interventional strategies to counter cGVHD has been hampered by variable onset and pathologic manifestations of cGVHD, now better defined by the National Institutes of Health consensus conference, 2 and a dearth of robust preclinical venues that closely mimic conditions in which cGVHD is generated and manifested. 3 Although the exact causes of cGVHD are unknown, higher antibody levels have been associated with autoimmunity and implicated in cGVHD. 4,5 Studies of newly diagnosed patients with extensive cGVHD showed that they had elevated soluble B-cell activating factor (BAFF) levels and anti-ds-DNA antibodies. 6,7 Increased soluble BAFF in cGVHD was associated with higher circulating levels of pre-germinal center (GC) B cells and post-GC plasmablasts. 8 B cells from cGVHD patients are hyperresponsive to TLR-9 signaling and have up-regulated CD86 levels, 9 which suggests an important participatory role for B cells in establishing cGVHD and emphasizes the need for further investigation into the immunologic role of B cells in cGVHD pathogenesis.Existing murine cGVHD models simulate one or more of the pathologic manifestations, such as increased serum antibodies (typically anti-DNA antibodies), scleroderma, and fibrosis of skin and liver, and the less common immune complex deposition in kidneys and glomerulonephritis. [10][11][12] The type of multiorgan involvement and alloantibodies seen in cGVHD patients often has not been well represented in these preclinical models. Moreover, some models do not involve conditioning regimens, whereas ot...

We have developed a mouse system by which to track the migration and homing of cells in a setting of bone marrow transplantation (BMT)-induced graft-versushost disease (GVHD) after systemic infusion using enhanced green fluorescence protein (

We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering.