Decellularization of Porcine Aorta Using Supercritical Carbon Dioxide

Currently, there are over 120,000 individuals in the United States awaiting an organ transplant, but fewer than 30,000 available donors [1].  Fabrication of artificial tissues and organs via tissue engineering (TE) would alleviate the current necessity for tissue and organ donors.  However, the need for functional, biocompatible, and sterile biomaterials for TE scaffolds creates a significant scientific challenge.  There are two primary methods for producing TE scaffolds: synthetic scaffolds and naturally-derived scaffolds.  Naturally-derived scaffolds are produced from the extracellular matrix (ECM) of decellularized animal tissues [2].  These materials offer several benefits, including lower risk of implant rejection and a more natural biochemistry and microstructural environment compared to the native tissue, which promotes post-implant angiogenesis and constructive remodeling [3].  Currently, natural scaffolds are often processed with aqueous detergents, which requires long treatment times, can damage the microstructure, and risks leaving cytotoxic residual material in the matrix [4].    A novel decellularization technique using supercritical carbon dioxide (scCO₂) offers considerably faster treatment of natural scaffold materials, on the order of hours instead of days.  CO₂ is non-toxic, non-flammable, and chemically inert, and has desirable solvent properties and a mild critical temperature (31.1°C), making it viable for use at physiological temperatures.  scCO₂ has been used previously in TE and other biomedical applications, including polymer foaming of scaffolds [5] and sterilization of biomaterials [6, 7].  A study on scCO₂ decellularization was first published in 2008 [8], where scCO₂ was used to decellularize porcine aorta, but dehydration of the scaffold during treatment prevented further progress.  Two years ago, we presented a method to presaturate scCO₂ with water that greatly reduces tissue dehydration during treatment with scCO₂ [9].  In this work, we proceed to examine the extent of decellularization during scCO₂ treatment, using a variety of additives and thermodynamic conditions.   Porcine aorta was obtained from a local butcher and cut into 2 cm x 1 cm rectangles, weighing approximately 200 mg each.  Some tissue samples were treated with scCO₂ for 1 hr at 37°C using two pressure conditions: 10.3 MPa (ρ_CO2 = 0.698 g/mL) and 27.6 MPa (ρ_CO2 = 0.908 g/mL).  Water and ethanol were mixed with scCO₂ prior to contacting the tissue – water to prevent dehydration and ethanol to increase the polarity of the CO₂.  Other samples were treated with sodium dodecyl sulfate (SDS) for 48 hr; this detergent treatment was used as a positive control.  After treatment, DNA was extracted from the decellularized tissues using Invitrogen DNAzol reagent and quantified using UV spectrophotometry.  Other samples were stained with hematoxylin and eosin (H&E) for histological analysis.   In Figure 1, the DNA content found in native tissue is compared to the remaining DNA after each treatment, which helps determine the extent of decellularization.  The amount of DNA significantly decreased in all treatments, though CO₂ at the low pressure condition was much less effective at removing DNA (0.61 μg/mg) than the detergent and high pressure CO₂ condition.  Though the high pressure CO₂ treatment was unable to fully decellularize the tissue, with a residual DNA content of 0.14 μg/mg, the ability to remove almost as much DNA in 1 hr of CO₂ as 48 hr with detergent (0.09 μg/mg) is a promising initial result.  Further development of the method will potentially lead to complete decellularization.   Figure 1 – DNA Content after Treatment   Samples were also stained with H&E and viewed under a light microscope at 4-40x magnification.  In Figure 2, micrographs of untreated, detergent-treated, and high pressure CO₂-treated aortas at 20x magnification are shown.  The detergent treatment removed almost all cellular material (purple/black), but also caused significant misalignment to the elastic fibers (pink/red), which suggests likely damage to the extracellular matrix (ECM).  The CO₂ treatment did not disrupt the elastic fibers, but still left some cellular material intact.   Figure 2 – Aorta Micrographs at 20x Magnification: (a) Untreated, (b) Detergent, (c) scCO₂   Moving forward, the objective is to combine the cell removal of the detergent with the ECM preservation of the scCO₂ treatment.  Ongoing work includes testing at a liquid CO₂ condition (10°C, 27.6 MPa à ρ_CO2 = 1.012 g/mL), using a different additive (Ls-54, a non-fluorinated surfactant with known water and scCO₂ solubility [10]), and tensile testing to further assess how the decellularization process affects the physical properties of the scaffold.  Completion of this study will elucidate the capabilities of this novel decellularization method, which offers a significant reduction in treatment time and potentially less ECM disruption.  

References

1.     The Need is Real: Data. <http://www.organdonor.gov/about/data.html>

2.     Keane TJ, Swinehart IT, Badylak SF: Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance. Methods 2015, 84:25-34.

3.     Brown BN, Badylak SF: Extracellular matrix as an inductive scaffold for functional tissue reconstruction. Translational Research 2014, 163(4):268-285.

4.     Keane TJ, Londono R, Turner NJ, Badylak SF: Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 2012, 33(6):1771-1781.

5.     Bhamidipati M, Scurto AM, Detamore MS: The Future of Carbon Dioxide for Polymer Processing in Tissue Engineering. Tissue Eng Part B-Rev 2013, 19(3):221-232.

6.     Qiu QQ, Leamy P, Brittingham J, Pomerleau J, Kabaria N, Connor J: Inactivation of Bacterial Spores and Viruses in Biological Material Using Supercritical Carbon Dioxide With Sterilant.

7.     Tarafa PJ, Jimenez A, Zhang JA, Matthews MA: Compressed carbon dioxide (CO2) for decontamination of biomaterials and tissue scaffolds. J Supercrit Fluids 2010, 53(1-3):192-199.

8.     Sawada K, Terada D, Yamaoka T, Kitamura S, Fujisato T: Cell removal with supercritical carbon dioxide for acellular artificial tissue. J Chem Technol Biotechnol 2008, 83(6):943-949.

9.     Casali DM, Matthews MA: Processing Tissue Engineering Matrix Materials with Supercritical CO₂, AIChE Annual Meeting, Atlanta, GA, November 2014.  Poster and short oral presentation.

10.  Tarafa PJ, Matthews MA: Phase equilibrium for surfactant Ls-54 in liquid CO2 with water and solubility estimation using the Peng-Robinson equation of state. Fluid Phase Equilibria 2010, 298(2):212-218.