In vitro generation of three-dimensional renal structures

doi:10.1016/j.ymeth.2008.09.005

Methods

Volume 47, Issue 2, February 2009, Pages 129-133

https://doi.org/10.1016/j.ymeth.2008.09.005 Get rights and content

Abstract

End-stage renal disease is currently being treated effectively by transplantation. However, increasing demand and donor shortage make this treatment challenging. Recent advances in cell-based therapies have provided potential opportunities to alleviate the current challenges of donor shortage. In this study we developed a system to generate renal structures in vitro using primary kidney cells. This system involves the cultivation of expanded primary renal cells in a three-dimensional collagen-based culture system. After one week of growth, individual renal cells began to form renal structures resembling tubules and glomeruli. Histologically, these structures show phenotypic resemblance to native kidney structures. The reconstituted tubules stained positively for Tamm–Horsfall protein, which is expressed in the thick ascending limb of Henle’s Loop and distal convoluted tubules. These results show that renal structures can be reconstituted in a three-dimensional culture system, which may eventually be used for renal cell therapy applications.

Introduction

End-stage renal disease is a devastating condition, which involves multiple organs in affected individuals. Although current treatment modalities, including dialysis and transplantation, can prolong survival for many patients, problems such as donor shortage, graft failure, and other complications remain a continued concern [1], [2], [3], [4]. Numerous investigative efforts have been commenced in order to address these problems [5], [6], [7], [8]. The concept of cell-based approaches using tissue engineering and regenerative medicine techniques has been proposed as a method to improve, restore or replace renal function [9], [10], [11]. Although widespread use of regenerative technology to combat kidney disease has yet to be implemented, some progress has been made [8]. An extracorporeal cell-based system, known as the renal assist device (RAD), uses human renal proximal tubular cells to improve filtration [6]. Despite demonstrations that this device can enhance the dialysis system, it is limited to ex vivo applications.

Another cell-based approach involves implantation of renal cells in vivo for renal function restoration [8]. This approach requires isolation of renal cells from donor tissue, expansion in vitro and reintroduction of renal cells back into the host for renal tissue regeneration [12]. The concept of renal cell therapy was demonstrated in a study in which culture expanded cells were seeded onto an artificial renal device and implanted in vivo. This resulted in the formation of renal structures that produce urine-like fluid [5]. In this particular study single renal cells showed the ability to reconstitute into renal tubules and glomeruli on the artificial renal device. However, the efficiency of the process of structural reconstitution could not be assessed upon implantation in vivo. Reconstitution of renal structure during the culture expansion stage followed by implantation would provide a more controlled assessment of renal tissue in vivo.

Toward this goal, we have developed a culture system that facilitates the formation of three-dimensional renal structures in vitro. We report a method that permits the use of primary renal cells to form tubule- and glomerulus-like renal structures in vitro. Such a system has the potential to eventually allow for the controlled implantation of renal structures and may ultimately serve as an effective method to maximize the outcome of cell-based therapy for renal function improvement.

Section snippets

Isolation of primary murine renal cells

Many techniques, including flow cytometry-based cell sorting and differential sieving, have been employed to isolate and culture individual renal cell types for various experiments [13], [14]. While these approaches may serve specific investigative objectives, formation of renal structures may not be adequately achieved by using single cell types, as multiple factors are believed to be involved, such as epithelial-mesenchymal cell interactions for differentiation [15]. To achieve the goal of

Concluding remarks

The ability to cultivate and expand renal cells in vitro provides an alternative opportunity to treat end-stage renal disease. While cell-based approaches using cultured renal cells are able to form functional kidney tissue structures in vivo, the process of reconstitution is neither well defined nor controlled. In this article, we present a three-dimensional culture system that allows for reconstituting single renal cells into kidney structures in vitro, thus providing a controlled platform

Acknowledgment

The authors thank Dr. Jennifer Olson for editorial assistance and Cathy Mathis for technical assistance. This work was supported, in part, by Tengion, Inc. through a sponsored research agreement.

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Regenerative medicine, which includes tissue engineering, will soon become the most desirable therapeutic option for genitourinary pathologies; including repair, regeneration or replacement of genitourinary system components that are damaged, diseased or defective and cannot be treated using conventional approaches. Engineering these components usually involves use of scaffolds, which could be naturally-derived (decellularized tissue scaffolds) or bioengineered (made using synthetic or naturally-derived polymers). This article describes the need, methods and outcomes of Tissue engineering and other Regenerative medicine approaches that have, and are being used to repair and regenerate the tissues and organs of the genitourinary system; particularly the Testes, Penis, Tunica albuginea, Vagina, Cervix, Urethral sphincters, Urethra, Urinary bladder, and Kidneys. Strategies discussed also include use of stem cells for disease treatment (particularly kidney disease) and 3D bioprinting. There is a section dedicated to reflect on how Tissue engineering is developing as a surgical discipline for the genitourinary system, and examples are presented to show how the clinical landscape for Regenerative medicine of genitourinary system is evolving. Advances in the field of regenerative medicine are resulting in the development of novel techniques and therapies for tissue repair and replacement, and the genitourinary tract system is one of the most studied areas in the field. There are many challenges to address; including vascularization of thick bioengineered tissues and organs; efficient nutrients and oxygen delivery; cell sources; multicellular organization and recapitulating physiology and function. However, the future outlook looks encouraging. Uses of advanced technologies, such as 3D bioprinting, stem cells, gene editing, etc. in the genitourinary system are showing promising results. These approaches may 1 day become a therapeutic alternative to organ transplantation in the future, thereby helping to improve the quality of many lives and even save lives where other treatment options are falling short.
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¹: These authors equally contributed to this article.

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In vitro generation of three-dimensional renal structures

Abstract

Introduction

Section snippets

Isolation of primary murine renal cells

Concluding remarks

Acknowledgment

Kidney Int.

Urol. Clin. North Am.

Kidney Int.

Kidney Int.

Kidney Int.

Differentiation

Kidney Int.

World J. Urol.

Clin. Nephrol.

N. Engl. J. Med.

Nephrol. Dial. Transplant.

Nat. Biotechnol.

Blood Purif.