ReviewDecellularization of tissues and organs
Introduction
Biologic scaffolds derived from decellularized tissues and organs have been successfully used in both pre-clinical animal studies and in human clinical applications [1], [2], [3], [4], [5], [6], [7], [8]. Removal of cells from a tissue or an organ leaves the complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM). The tissues from which the ECM is harvested, the species of origin, the decellularization methods and the methods of terminal sterilization for these biologic scaffolds vary widely. Each of these variables affects the composition and ultrastructure of the ECM and accordingly, affects the host tissue response to the ECM scaffold following implantation. The objective of this manuscript is to provide an overview of the various methods that have been used to decellularize tissues, and the potential effects of the various decellularization protocols on the biochemical composition, ultrastructure, and mechanical behavior of the ECM scaffold materials.
Section snippets
Rationale for decellularization of ECM
Xenogeneic and allogeneic cellular antigens are, by definition, recognized as foreign by the host and therefore induce an inflammatory response or an immune-mediated rejection of the tissue. However, components of the ECM are generally conserved among species and are tolerated well even by xenogeneic recipients [9], [10], [11], [12]. ECM from a variety of tissues, including heart valves [13], [14], [15], [16], [17], [18], [19], blood vessels [20], [21], [22], [23], skin [24], nerves [25], [26],
Description of decellularization protocols
The most robust and effective decellularization protocols include a combination of physical, chemical, and enzymatic approaches. A decellularization protocol generally begins with lysis of the cell membrane using physical treatments or ionic solutions, followed by separation of cellular components from the ECM using enzymatic treatments, solubilization of cytoplasmic and nuclear cellular components using detergents, and finally removal of cellular debris from the tissue. These steps can be
Effects of tissue variability upon decellularization
The efficiency of a given decellularization method or protocol is dependent upon the tissue of interest. Despite identical times of exposure and a greater concentration of trypsin (0.5% vs. 0.05%), Grauss et al. [15] found that a trypsin-based decellularization protocol was ineffective at removing the cellular material from the rat aortic heart valve, while Schenke-Layland et al. [19] showed complete removal of cells from a porcine pulmonary valve. It is likely that subtle nuances in the
Verification of cell removal
There are a number of methods available to determine the efficiency of the removal of cellular material from tissues. Standard histological staining with Hematoxylin and Eosin can serve as a first line of inspection to determine if nuclear structures can be observed. Alternative histological stains such as Masson's Trichome, Movat's Pentachrome, or Safrin O can be used to examine tissues for the presence of various cytoplasmic and extracellular molecules. Immunohistochemical methods can also be
Removal of residual chemicals
The decellularization methods described above include a wide variety of chemicals, which are used because of their inherent abilities to damage cells. If the chemicals remain within the tissue in high concentrations after treatment, then it is likely that they will be toxic to host cells when the scaffold is implanted in vivo. There is a need for development of assays to quantify the presence of residual chemicals in the decellularized scaffold material. Similarly, some of the processes that
Conclusion
Complete decellularization of most tissues will require a combination of physical, enzymatic, and chemical treatments, and the protocol will be dependent on the tissue of interest. It is generally desirable to use the mildest protocol possible that yields an acellular material without disruption of the structural and functional component of the ECM. A typical progressive approach would be to start with treatment in a hypotonic or hypertonic solution followed by a mild non-ionic or zwitterionic
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