Re-epithelialization of whole porcine kidney scaffold
Evan M. Buckmiller, Advisor: Alonzo D. Cook, Department of Chemical Engineering
An estimated 20 million adults (approximately 10% of all individuals over the age of 18) in the United States suffer from some level of chronic kidney disease (CKD), making CKD the 9th leading cause of death in the United States (1). The only successful treatment for end-stage renal failure is transplantation from a donor with similar blood antigen makeup, while living the remainder of their lives receiving immunosuppressive therapy. Unfortunately, individuals awaiting a kidney transplant outnumber viable donations. Scientists have been tasked with discovering and developing methods to answer this growing need. One promising method is the decellularization of porcine (pig) kidneys, followed by seeding patient specific cells onto these organ scaffolds. After cell proliferation on these scaffolds, these recellularized scaffolds are then transplanted into patients. This method has been successful with flat and tubular-shaped organs, but mixed success has been observed with more complex organs such as kidney. The largest obstacle in dealing with whole organ recellularization is ensuring the seeded cells are evenly distributed throughout the entire kidney scaffold. We present a novel method of altering the pressure within the recellularization apparatus (bioreactor) to generate a more homogeneous seeding of cells throughout the kidney structure.
Kidney collection, decellularization, and sterilization
Kidneys were collected from healthy pigs, visually inspected, and palpated to detect abnormalities. They were then immersed in a 1% phosphate buffered saline (PBS) solution and perfused with heparinized (10 U/ml) 1X PBS solution to reduce the formation of thrombus within the kidneys. The kidneys were then frozen for storage at −20℃. Kidneys were later thawed and perfused with 0.5% (w/v) sodium lauryl sulfate (SDS) for 5-7 hours at constant low pressure. Removal of porcine renal cells was accomplished once the kidney exhibited a uniform, white appearance. Kidneys were then perfused with deionized (DI) water for 2 days at a constant flow rate. Decellularized kidney scaffolds were then visualized with scanning electron microscopy to ensure sufficient removal of porcine cellular components. Decellularized kidney scaffolds were sterilized by recirculating 70% ethanol (v/v) through the kidney for approximately 2 hours. The scaffolds were then aseptically transferred to a sterile (autoclaved) bioreactor and 70% ethanol was perfused through the kidney and bioreactor for an additional 15 minutes. The ethanol was then removed and replaced with sterile DI water and rinsed several times to remove the ethanol from the scaffold. The scaffold was then perfused for 48-72 hours with nutrient-rich media, and antifungal/antibacterial agents to protect from potential contamination.
Recellularization/seeding of the kidney scaffold
Four techniques were employed in developing the most effective method of distributing labeled human renal cortical tubular epithelium (RCTE) cells throughout the entire kidney. First, varying concentrations of cells were perfused through the renal artery at high pressure for 30 minutes, followed by a moderate pressure for 7 days. In the second technique, cells were perfused through the ureter at a high pressure for 30 minutes, followed by a moderate pressure for 7 days. In the third technique cells were injected into the ureter at a moderate pressure while the kidney was under a negative pressure (vacuum) for 30 minutes, followed by perfusion at a moderate pressure for 7 days. In the final technique, cells were perfused through the ureter at a high pressure while the kidney was under a high negative pressure for 30 minutes, followed by perfusion at a moderate pressure for 7 days.
The porcine kidneys were decellularized until they were observed to be sufficiently “white”, and the accompanying staining with imaging confirmed the absence of cellular material. DNA quantification of acellular kidney scaffolds suggests a thorough and sufficient removal of porcine genetic material from the original porcine kidneys. The concentration of the cellular media injected was critical to obtaining even dispersion. Injecting a low concentration of cells was not conducive to good cell coverage, however, injecting a high concentration of cells led to cell agglutination. We found the optimal cellular concentration to be 1 million cells/mL. Perfusing the kidney with high arterial pressure (the first of the four methods) led to the injected cells being retained mainly within the vasculature and the glomeruli. Little evidence of cell proliferation was observed within the tubular regions of the scaffold. Most of the cells stayed within the media, suggesting that most of the cells did not adhere to the scaffold. Perfusing the kidney with high ureteral pressure caused a majority of the cells to adhere to the scaffold within the medulla region, but very few of the cells reached the cortex region. However, combining high ureteral pressure with high vacuum pressure caused nearly all of the cells to accumulate within the cortex region, leaving the medulla region without detectable cells.
Optimal cellular coverage through the kidney scaffold was obtained by injecting cells through the ureter while the kidney was under negative pressure. High pressure during the first 30 minutes also produced the most even cellular dispersion with nearly 50% coverage. Nephric components (glomeruli, tubules, Bowman’s capsules, etc.) were also observed.
Many labs have successfully decellularized and recellularized many different organs (heart, pancreas, lung, liver, kidney) from various animal sources (canine, monkey, pig, etc.) (2). Successful decellularization of whole kidneys been demonstrated across many different species, including mouse/rat (3), small primate (2), as well as deceased human (4). Promising results have been observed when recellularization attempts have been performed on mouse/rat derived renal tissue (5), but little has been published related to the recellularization of whole porcine kidney.
Future studies should test the functionality of these recellularized kidneys. They should also focus on the incorporation of induced pluripotent stem cells in order to generate patient specific kidney constructs (6). Patient specific kidneys derived from their own induced pluripotent stem cells would effectively eliminate the chance for rejection, negating the need for immunosuppressive therapy for patients receiving these kidney transplants. The use of stem cells could also be the solution to generating the numerous, diverse cell types found within the kidney.
The novel method presented herein obtains broad dispersion of RCTE cells throughout the entire kidney by altering the conditions of recellularization, in particular, the pressure within the recellularization chambers and the flow rate of cellularized media. In order to obtain a broad dispersal of the different cell types, we tested 4 different variables: introducing the cells through the renal artery or the ureter, injecting vs perfusing the cells into the kidney scaffold, varying the flow rates and cell concentrations, and altering the pressure within the recellularization apparatus. The concentration of the cellular solution was also critical to the success of this project. The pressure within the recellularization apparatus, combined with the incremental increase in cellular media flow rate helped to obtain the desirable results.
1. National Center for Chronic Disease Prevention C, PromotionDivision of Diabetes Translation H (2015) Chronic Kidney Disease Initiative—Protecting Kidney Health.
2. Nakayama KH, Lee CCI, Batchelder CA, Tarantal AF (2013) Tissue specificity of decellularized rhesus monkey kidney and lung scaffolds. PLoS One 8(5):e64134.
3. Peloso A, et al. (2015) Creation and implantation of acellular rat renal ECM-based scaffolds. Organogenesis 11(2):58–74.
4. Orlando G, et al. (2013) Discarded human kidneys as a source of ECM scaffold for kidney regeneration technologies. Biomaterials 34(24):5915–5925.
5. Robertson MJ, Dries-Devlin JL, Kren SM, Burchfield JS, Taylor DA (2014) Optimizing recellularization of whole decellularized heart extracellular matrix. PLoS One 9(2):e90406.
6. Orlando G, et al. (2011) Regenerative medicine as applied to solid organ transplantation: Current status and future challenges. Transpl Int. 24(3), 223-232.