Приматов легких Decellularization и Recellularization помощью специализированного большого органа биореактор
Summary
Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.
Abstract
There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.
Introduction
Bioengineering of tissues and organs is an attractive addition to the field of regenerative medicine. The creation of "lab-grown" organs that are suitable for transplant into patients to replace functionality of diseased organs is highly desirable in order to meet the current and future demand for transplantation needs. The principles of tissue engineering center around the seeding of desired cell types, or progenitors thereof, into a scaffold that supports the shape of the engineered tissue while supplying the appropriate growth factors and culture conditions necessary to mimic developmental or regenerative processes. While synthetic scaffolds have been used for tissue engineering, and the natural extracellular matrix (ECM) may be the best source of organ-specific scaffolds for this purpose. Whole-organ decellularization is a process which allows the removal of cells while leaving the chemical and structural aspects of the native ECM intact. The resulting acellular matrix scaffold can be used as a platform onto which regenerative cells can be seeded and cultured in vitro1,2.
Several rodent models of lung decellularization and subsequent recellularization have been developed to study the feasibility of this technology3-6. While offering essential proof-of-concept, rodent models are not directly translatable to human clinical needs. A recent study pointed out that genomic responses to traumatic injury (and related inflammation) do not correlate well between mice and humans; these findings raise questions of the validity of using mice as models for such complex biochemical processes in humans7. Nonhuman primate (NHP) models offer the advantage of closely resembling the biology of humans at the genomic, anatomic, and physiologic levels and allow more flexible manipulation for greater extrapolation to human use. The rhesus macaque has been used in a variety of preclinical applications and is an excellent model in which to study tissue engineering8-11. We recently described the successful decellularization of rhesus macaque (Macacca mulatta) lungs utilizing a procedure that has minimum impact on the lung ECM12. Lung decellularization is accomplished by treating the tissue consecutively with four decellularization solutions composed of detergents, salts, and enzymes with intermittent washing with deionized water (dH2O) and PBS. We have optimized this procedure by modifying a protocol originally described by Price et al.4 A variety of histological and protein analytical techniques were used to characterize the components of resulting acellular matrices relative to native macaque lungs.
In this report, we demonstrate a detailed protocol for the decellularization of nonhuman primate lungs and the recellularization of the resulting acellular lung scaffolds in a large-organ bioreactor originally demonstrated by Calle et al.13 in JoVE. By modifying their original protocol to accommodate the size, ventilation, and perfusion requirements for large-animal lungs, the technology was successfully moved from the rodent model to the rhesus macaque model. All studies presented in this report were performed in accordance with the Institutional Biosafety Committee (IBC) policies in place at the Tulane National Primate Research Center. Demonstration of this technique is essential because identification of anatomical structures and physical manipulation of larger organs is sometimes difficult without visualizing the steps of the protocol. The studies made possible by these methods provide a basis for essential preclinical studies in decellularized rhesus macaque lungs in which recellularization with rhesus mesenchymal lineage stem cells and rhesus microvascular endothelial cells can be assessed. Our version of this bioreactor simulates the developmental environment and applies forces of mechanical stretch and strain in large-animal lungs and allows the investigation of lung recellularization under conditions known to facilitate pulmonary and endothelial development13-15.
Protocol
Representative Results
Discussion
Tissues can be efficiently and effectively decellularized by a number of methods employing physical, chemical, and enzymatic agents12,20. The challenges of producing 3D biological matrices from large organs include the requirement for large volumes of decellularization solutions, expensive commercial equipment (i.e. bioreactors), and a dizzying amount of methodological perturbations required to achieve the final tissue-derived product. Our method provides a straightforward approach that minimizes phys…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to thank the editors of Tissue Engineering for allowing images from a previous publication to be used in this report.
Materials
| Sodium nitroprusside | Sigma-Aldrich | 71778-25G | |
| Heparin sodium salt | Fisher | BP2425 | |
| Triton X-100 | Fisher | BP151-100 | |
| Sodium deoxycholate | Fisher | BP349-100 | |
| Sodium chloride | Fisher | 7647-14-5 | |
| Bovine pancreatic DNase | Sigma-Aldrich | DN25 | Prepare stock, aliquot, and freeze |
| Magnesium sulfate | Fisher | 10034-99-8 | |
| Calcium chloride | Fisher | C614-500 | |
| PBS (no Ca/Mg) | Gibco, Life Technologies | 10010-031 | |
| Antibiotic-Antimycotic | Gibco Life Technologies | 15240062 | |
| Cell Culture Media | |||
| Alpha MEM | Gibco Life Technologies | 12561-072 | |
| Medium 199 | Gibco Life Technologies | 11150067 | |
| Premium Fetal Bovine Serum | Atlanta Biologicals | S11150 | |
| L-Glutamine 100x | Gibco Life Technologies | 25030-081 | |
| Endothelial Cell Growth Supplement (ECGS) | ScienCell | 1052 | |
| Antibiotic-Antimycotic | Gibco Life Technologies | 15240062 | |
| Cell Seeding and Bioreactor Culture | |||
| Check valves | Cole-Parmer | EW-98553-20 | |
| Y-connectors | Cole-Parmer | ED-30614-08 | |
| 3-Way stopcocks | Harvard Apparatus | 721664 | |
| MasterFlex L/S 14 tubing | Cole-Parmer | 96420-14 | |
| MasterFlex L/S 16 tubing | Cole-Parmer | 96420-16 | |
| Male lock Luer 1/8 in | Cole-Parmer | EW-45505-04 | |
| Female Luer 1/8 in | Cole-Parmer | SI-45502-04 | |
| Male Luer lock plug | Cole-Parmer | EW-45505-56 | |
| Injection ports | Medi-Dose EPS | IV2004 | |
| Latex tubing | St. Louis Medical Suppy | HN10910 | |
| Hose clamp | Cole-Parmer | EW-06832-02 | |
| 2 L Wide-mouth jar | Fisher | 05-719-276 | |
| 60 ml Syringes | Fisher | NC9035364 |
References
- Ott, H. C., et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat. Med. 14, 213-221 (2008).
- Badylak, S. F., Taylor, D., Uygun, K. Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu. Rev. Biomed. Eng. 13, 27-53 (2011).
- Ott, H. C., et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16, 927-933 (2010).
- Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded. Tissue Eng. Part A. 16, 2581-2591 (2010).
- Petersen, T. H., et al. Tissue-engineered lungs for in vivo implantation. Science. 329, 538-541 (2010).
- Song, J. J., et al. Enhanced in vivo function of bioartificial lungs in rats. Ann. Thorac. Surg. 92, 998-1005 (2011).
- Seok, J., et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc. Natl. Acad. Sci. U.S.A. , (2013).
- Joag, S. V. Primate models of AIDS. Microbes Infect. 2, 223-229 (2000).
- Hu, S. L. Non-human primate models for AIDS vaccine research. Curr. Drug. Targets Infect. Disord. 5, 193-201 (2005).
- Borda, J. T., et al. Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy. Am. J. Pathol. 172, 98-111 (2008).
- Fabbrini, E., et al. Metabolic response to high-carbohydrate and low-carbohydrate meals in a non-human primate model. Am. J. Physiol. Endocrinol. Metab. , (2012).
- Crapo, P. M., Gilbert, T. W., Badylak, S. F. An overview of tissue and whole organ decellularization processes. Biomaterials. 32, 3233-3243 (2011).
- Calle, E. A., Petersen, T. H., Niklason, L. E. Procedure for Lung Engineering. J. Vis. Exp. (49), e2651 (2011).
- Arold, S. P., Wong, J. Y., Suki, B. Design of a new stretching apparatus and the effects of cyclic strain and substratum on mouse lung epithelial-12 cells. Ann. Biomed. Eng. 35, 1156-1164 (2007).
- Huang, H., et al. Differentiation from embryonic stem cells to vascular wall cells under in vitro pulsatile flow loading. J. Artif. Organs. 8, 110-118 (2005).
- Izadpanah, R., et al. Characterization of multipotent mesenchymal stem cells from the bone marrow of rhesus macaques. Stem Cells Dev. 14, 440-451 (2005).
- Craig, L. E., Nealen, M. L., Strandberg, J. D., Zink, M. C. Differential replication of ovine lentivirus in endothelial cells cultured from different tissues. Virology. 238, 316-326 (1006).
- Craig, L. E., Spelman, J. P., Strandberg, J. D., Zink, M. C. Endothelial cells from diverse tissues exhibit differences in growth and morphology. Microvasc. Res. 55, 65-76 (1998).
- Bonvillain, R. W., et al. A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells. Tissue Eng. Part A. 18, 2437-2452 (2012).
- Badylak, S. F., Taylor, D., Uygun, K. Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds. Annu. Rev. Biomed. Eng. , (2010).
