Porcine kidney decellularization using novel method of tonic cycle to improve cell-ECM interaction

Nafiseh Poornejad, Cory A. Fronk, Westley Kirkham, Grant Holden, Jonathan J.

Wisco, Beverly L. Roeder, Alonzo D. Cook

1. Introduction:

Kidney disease affects 13% of the global population and is one of the leading causes of mortality in the world. Obesity and cardiac disease lead to more patients with hypertension and diabetes. These maladies lead to renal disease and could double the number of affected individuals. Dialysis filters toxins from the blood, substituting for a kidney, but it does not replace other functions such as homeostasis and endocrine functions [1]. A kidney transplant provides the ultimate treatment. However, the number of patients needing kidney transplants far exceeds the number of donors; additionally, those individuals who are fortunate enough to receive a kidney must take immunosuppressive medications for the remainder of their lives to avoid organ rejection [2, 3].
One way to address the challenges of organ transplantation is to grow replacement organs in the laboratory. An artificial organ needs three basic components: a scaffold, cells, and growth factors. The scaffold is the supportive structure of an organ that also carries out many other signaling functions for cell migration and differentiation. Cells are seeded on the scaffold and by aid of growth factors and signaling molecules repopulate the entire scaffold.
The most promising option for scaffolds is to use naturally derived structures. These structures have an inherent vasculature system as well as essential proteins that are required for cell proliferation and differentiation [3]. Harvesting an organ from dead donors or animals and removing the cells from its structure can provide natural scaffolds.
The goal of decellularization (removing the cells from a natural organ) is to remove all cells to avoid any adverse immunological response at the time of transplantation and keep the main components of the extracellular matrix (ECM) such as growth factors and collagen intact. The ECM serves structural, mechanical, and signaling purposes. Depending on the tissue mass, structure, function and biomechanical characteristics of an organ, different methods of decellularization would be effective. The complexity of the structure of the kidney and more than thirty cell types that it has, increase the need for the ECM to be as intact as possible to increase the chance of successful recellularization [1,
2, 4].
A decellularization protocol generally begins with lysis of cell membranes using a physical treatment or ionic solution, separation of cellular components from ECM by action of an enzyme, solubilization of cytoplasmic and nuclear cellular materials by detergents, and finally removal of cellular debris from the ECM [4]. The decellularized ECM should have the following properties: it should be contractile, electro physiologically stable, flexible, mechanically strong and autologous in nature. These properties are required in order to have a construct that is functionally and morphologically similar to the native tissue, integrate with it, stay viable with time and improve the function of the damaged kidney. However, the process used so far leads to ECMs that possess different properties in terms of collagen type, content and density,
and variations in glycosaminoglycans (GAGs) [5]. Efforts need to be taken to improve the decellularization protocol as much as possible so it is possible to grow and differentiate all the kind of cells needed to have a functional organ.
In this study we used a novel tonic cycle method to decellularize porcine kidneys. Also, we used different quantification methods to compare the tonic cycle method with previous published methods that use only detergent wash.

2. Material and Methods

Female porcine kidneys were harvested from a local abattoir, shortly after the animalsâ?? deaths. The kidneys were removed along with the adrenal gland to insure a sufficient length of artery was preserved. Heparinized PBS solution was pumped through the kidney to prevent thrombosis. Afterward, the kidneys were further prepared by removal of fat and excess arterial tissue. To remove remaining thrombus, a hypertonic solution was pumped into the kidney for 30 minutes at a flow rate of 12.5 ml/min to induce crenation. An SDS solution was then pumped into the organ for 30 minutes to disrupt the clots and begin the decellularization. After the initial anti-thrombogenic wash, the experiment followed a strict repetitive cycle of solutionsâ??the tonic cycleâ??until a completely white kidney was achieved. The tonic cycle is as follows: 1 M NaCl solution (hypertonic solution) for 30 minutes, the 0.5%w/w SDS solution for 30 minutes, then DI water (hypotonic solution). This sequence caused the largest osmotic gradient and lysed cells most effectively. In another protocol, this apparatus was used to decellularize with only 0.5% w/w SDS solution. The entire process was performed at 25°C.
Development of the tonic cycle method of decellularization necessitated the construction of a custom bioreactor system. The bioreactor was a 2.2 Liter jacketed glass vessel. The solutions were pumped via a DRIVE MFLEX L/S 1.6-100RPM115V peristaltic pump through â?? ID * 3/16 OD* 1/32 wall (NALGENE 8000-0010) tubing into the organs in the bioreactor. The tubing was connected to the kidney through the artery for continuous antegrade flow.
After complete decellularization, samples of renal cortex were excised from native and decellularized kidneys and were lyophilized, and weighed out. DNA was extracted from samples with the Qiagen DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA) and determined using the Quant-iT PicoGreen (Invitrogen Corp., Carlsbad, CA) dsDNA assay kit according to manufacturerâ??s instructions. Collagen levels were examined using the Sircol Soluble Collagen Assay Kit (Biocolor Ltd., Newtownabbey, UK). Sulfated glycosaminoglycans (sGAGs) were quantified using the Blyscan sGAG Assay Kit (Biocolor, Ltd).

3. Results and Discussion

Preservation of signaling and adhesive components of ECM is a decisive factor that is determining for future recellularization. Then, it is essential to optimize the decellularization procedure to have more intact scaffold. SDS is found as the most effective detergent to remove the cellular compartments and eliminate the immunogenic potential response. However, since SDS is a strong ionic detergent, it can damage the proteins of the ECM. In this study we applied osmotic shock to reduce SDS exposure time while removing all cellular debris that may potentially cause immune response.
The results of the DNA assay showed complete removal of cellular debris (less than 50 ng/mg of tissue dry weight), which eliminates any immune response at the time of transplantation.
Collagen is one of the most significance components of the extracellular matrix that plays an important role in cell signaling and communication which leads to cell proliferation and differentiation. Preserving different types of collagen through decellularization is a critical requirement for future cell growth on the ECM. Collagen content of different decellularized scaffolds shows less damage to the collagenous structure of the scaffold, which increases the possibility of complete recellularization.
Glycosaminoglycans (GAGs) are other components of the ECM that are considered important as adhesion and signaling molecules. One of the challenging parts of the recellularization step is attaching the cells to the ECM, which would be improved by the preservation of GAGs. GAGs determination assay shows that our tonic cycle method also was more successful to preserve the GAGs content of the ECM.
All obtained results showed the advantages of the tonic cycle method over using only SDS for decellularization. The kidney contains more than 30 cell types and it is impossible to culture all types of the cells. To have a completely functional kidney we rely on cell differentiation by the ECM, which depends on intactness of the ECM. Our novel method of decellularization would be the first step to have an ideal scaffold ready for cell culture.

4. Conclusion

In this study, decellularization of porcine kidneys has been successfully completed by two different methods. The combination of osmotic shock and detergent wash is more effective in both removing cellular components and keeping the ECM intact.
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