3-D renal structures generated from human renal cells

Feb 01, 2010

Positive results have been achieved using a novel approach for in vitro reconstitution of human kidney structures.

Chicago-Positive results have been achieved using a novel approach for in vitro reconstitution of human kidney structures, investigators from the Wake Forest University Institute for Regenerative Medicine reported at the American College of Surgeons Clinical Congress.

The technique involves growth of single human renal cells in a 3-D culture system to generate preformed human renal structures. To date, the researchers found that isolated primary human renal cells could be consistently expanded up to 20 population doublings. The expanded cells maintain the expression of a variety of renal cell-specific markers, at least through three passages, and after being seeded into a 3-D matrix, self-assembled into glomerular- or tubular-like structures.

"An increasing demand for renal transplantation, coupled with a donor shortage, has made treatment of end-stage renal disease challenging. Based on our early experience, we are encouraged that this in vitro system for reconstituting single renal cells into kidney structures in vitro may ultimately be developed into an efficient cell-based therapy for patients with end-stage renal disease," said Tamer Aboushwareb, MD, PhD, research instructor at the Wake Forest Institute for Regenerative Medicine and Wake Forest University School of Medicine, Winston-Salem, NC.

The primary renal cells are obtained via enzymatic digestion of homogenized renal tissue. After being grown and expanded, single renal cells are seeded in a 3-D culture system consisting of a matrix of neutralized type I collagen. This matrix is then solidified and cultivated for 10 days.

Cells grow into different renal cell types

Immunohistochemical analysis of the cultivated cells showed they grew into different renal cell types and maintained the phenotypic and functional characteristics of those cells. The analyses were performed using third passage cells and included stains for proximal tubular cells (Neprolisen), distal tubular cells (EMA), collecting ducts (Tamm-Horsfall protein), podocytes (Podocin), and erythropoietin-producing cells (EPO). Several other renal markers were also tested for the study but are not mentioned here.

"From these findings, we can conclude we have a heterogenous culture system," Dr. Aboushwareb said.

Results of cell function studies included demonstration of the uptake of rhodamine-labeled albumin in the NEP positive cells, which indicates proximal tubular cell function, the researchers reported.

Histologic analysis of the structures formed in the 3-D culture environment showed phenotypic resemblance to native kidney structures with positive staining for proximal and distal tubular markers.

"Although vascularity would not be expected to develop in this in vitro system, we are planning to evaluate the cultivated cells to see if they stain positive for vascular markers," Dr. Aboushwareb noted.

Structural morphology of the formed structures was investigated by imaging with confocal microscopy and demonstrated that they were three-dimensional and not just present in a single matrix layer. Albumin uptake was also demonstrated.

Asked about the possibility that the research represents a stem cell experiment, Dr. Aboushwareb acknowledged that progenitor cells were likely present, but added that it was unlikely they were the only source for the reconstituted kidney structures.