procurement-decellularization-rat-hindlimbs-using-an-ex-vivo

Procurement and Decellularization of Rat Hindlimbs Using an <em>Ex Vivo</em> Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

procurement-perfusion-decellularization-porcine-vascularized-flaps

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

Toronto General Hospital Research Institute, University Health Network

University of Toronto

University Health Network, Toronto General Hospital

Tracheal injury, stenosis, and malignancy demand tracheal reconstruction, which often fails due to the lack of a functioning epithelium. We performed an extensive comparative analysis to determine optimal biomaterials for developing tracheal epithelial grafts with mucociliary function. We screened Hyaluronan-Poly(Ethylene Glycol), Chitosan-Collagen, Collagen Vitrigel Membrane, Fibrin Glue, Silk Fibroin, and Gelatin based on various parameters including mechanical strength, bulk degradation, cell attachment, spreading, metabolic activity, focal adhesion formation, and differentiation into ciliated and goblet cells. Silk Fibroin had significantly higher tensile strength (21.23 ± 4.42 MPa), retained 50% of its mass across 5 weeks, allowed 80-100% cell spreading and increasing metabolic activity across 10 days, focal adhesion formation within 2 h, and differentiation into 5.9 ± 2.6% goblet cells. Silk Fibroin, however, led to poor ciliation, producing 5.5 ± 3.9% ciliated cells, whereas Collagen Vitrigel Membrane promoted excellent ciliation. To capitalize on the mechanical and differentiation benefits of its respective components, we developed a composite biomaterial of Silk Fibroin and Collagen Vitrigel Membrane (SF-CVM), which demonstrated enhanced maturation into 20.6 ± 1.7% ciliated and 5.6 ± 1.0% goblet cells. Development of biomaterials-based airway epithelial grafts that provide desirable mechanics and differentiation is a major step towards treatment of airway disease. STATEMENT OF SIGNIFICANCE: Tracheal blockage, injury, and malignancy greater than 50% of the adult tracheal length cannot be safely resected. Tracheal replacement is one approach, but a major cause of transplant failure is the lack of a functioning epithelium. While tissue engineering for tracheal regeneration using biomaterials is promising, there is currently no gold standard. Therefore, we performed a systematic comparative study to characterize relevant materials for generating a biomaterials-based airway epithelial graft. We developed a composite biomaterial intended for surgical implantation providing tensile strength, slow biodegradation, and optimal support for differentiation of mature epithelia. This is a significant step augmenting current state-of-the-art methods for airway surgeries, laryngeal reconstruction, and tracheal tissue engineering.

Optimal biomaterials for tracheal epithelial grafts: An in vitro systematic comparative analysis.

Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

De-epithelialization of porcine tracheal allografts as an approach for tracheal tissue engineering.

Chronic lung diseases remain major healthcare burdens, for which the only curative treatment is lung transplantation. In vitro human models are promising platforms for identifying and testing novel compounds to potentially decrease this burden. Directed differentiation of pluripotent stem cells is an important strategy to generate lung cells to create such models. Current lung directed differentiation protocols are limited as they do not 1) recapitulate the diversity of respiratory epithelium, 2) generate consistent or sufficient cell numbers for drug discovery platforms, and 3) establish the histologic tissue-level organization critical for modeling lung function. In this review, we describe how lung development has formed the basis for directed differentiation protocols, and discuss the utility of available protocols for lung epithelial cell generation and drug development. We further highlight tissue engineering strategies for manipulating biophysical signals during directed differentiation such that future protocols can recapitulate both chemical and physical cues present during lung development.

Current strategies and opportunities to manufacture cells for modeling human lungs.

Successful re-epithelialization of de-epithelialized tracheal scaffolds remains a challenge for tracheal graft success. Currently, the lack of understanding of the bioreactor hydrodynamic environment, and its relation to cell seeding outcomes, serve as major obstacles to obtaining viable tracheal grafts. In this work, we used computational fluid dynamics to (a) re-design the fluid delivery system of a trachea bioreactor to promote a spatially uniform hydrodynamic environment, and (b) improve the perfusion cell seeding protocol to promote homogeneous cell deposition. Lagrangian particle-tracking simulations showed that low rates of rotation provide more uniform circumferential and longitudinal patterns of cell deposition, while higher rates of rotation only improve circumferential uniformity but bias cell deposition proximally. Validation experiments with human bronchial epithelial cells confirm that the model accurately predicts cell deposition in low shear stress environments. We used the acquired knowledge from our particle tracking model, as a guide for long-term tracheal repopulation studies. Cell repopulation using conditions resulting in low wall shear stress enabled enhanced re-epithelialization of long segment tracheal grafts. While our work focuses on tracheal regeneration, lessons learned in this study, can be applied to culturing of any tissue engineered tubular scaffold.

Computational fluid dynamics for enhanced tracheal bioreactor design and long-segment graft recellularization.

Tissue engineering techniques to generate a graft ex vivo is an exciting field of research. In particular, the use of biological scaffolds has shown to be promising in a clinical setting. In this approach, decellularized donor scaffolds are obtained following detergent-based enzymatic treatment to remove donor cells and subsequently repopulated with recipient specific cells. Herein, we describe our bioreactor-based partial decellularization approach to generate hybrid tracheal grafts. Using a short detergent-based treatment with sodium dodecyl sulfate (SDS), we remove the epithelium and maintain the structural integrity of the donor grafts by keeping the cartilage alive. The following will be a step-by-step description of the bioreactor system setup and partial decellularization protocol to obtain a de-epithelialized tracheal graft.

Bioreactor-Based De-epithelialization of Long-Segment Tracheal Grafts.

Topographical cues are known to influence cell organization both in native tissues and . In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.

Golnaz Karoubi

Latner Thoracic Surgery Research Laboratories

Toronto General Hospital Research Institute, University Health Network

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Golnaz Karoubi

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Toronto General Hospital Research Institute, University Health Network

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Latner Thoracic Surgery Research Laboratories