Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

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 ....

Protocol

1. Whole-organ Macaque Lung Decellularization

1. Preparation of Solutions
   1. Autoclave 10-15 L of deionized water (dH₂O) in 1-2 L bottles.
   2. Prepare "Triton solution" (0.1% Triton X-100 in dH₂O) by mixing 1 ml Triton X-100 in 999 ml of dH₂O while stirring on a magnetic stirrer. Filter the solution through a 0.22 µm filter apparatus. Store at room temperature.
   3. Prepare "SDC solution" (2% SDC in dH₂O) by slowly adding 20 g of sodium deoxycholate (SDC) to ~900 ml dH₂O while stirring. Once SDC is dissolved transfer the solution to a graduated cylinder ....

Results

Results shown below represent separate experiments in which either airway or vascular compartments were seeded with rhesus macaque bone marrow-derived mesenchymal stem cells or rhesus lung-derived microvascular endothelial cells, respectively, that were isolated and characterized as previously described^16-18.

Throughout the decellularization process, macaque lungs displayed a progressive whitening culminating in a translucent appearance at the end of the process; however, the lungs .......

Discussion

Tissues can be efficiently and effectively decellularized by a number of methods employing physical, chemical, and enzymatic agents^12,20. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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