Bioartificial Renal Epithelial Cell System (BRECS): A Compact, Cryopreservable Extracorporeal Renal Replacement Device

Abstract

Renal cell therapy has shown clinical efficacy in the treatment of acute renal failure (ARF) and promise for treatment of end-stage renal disease (ESRD) by supplementing conventional small solute clearance (hemodialysis or hemofiltration) with endocrine and metabolic function provided by cells maintained in an extracorporeal circuit. A major obstacle in the widespread adoption of this therapeutic approach is the lack of a cryopreservable system to enable distribution, storage, and therapeutic use at point of care facilities. This report details the design, fabrication, and assessment of a Bioartificial Renal Epithelial Cell System (BRECS), the first all-in-one culture vessel, cryostorage device, and cell therapy delivery system. The BRECS was loaded with up to 20 cell-seeded porous disks, which were maintained by perfusion culture. Once cells reached over 5 × 10⁶ cells/disk for a total therapeutic dose of approximately 10⁸ cells, the BRECS was cryopreserved for storage at -80°C or -140°C. The BRECS was rapidly thawed, and perfusion culture was resumed. Near precryopreservation values of cell viability, metabolic activity, and differentiated phenotype of functional renal cells were confirmed post-reconstitution. This technology could be extended to administer other cell-based therapies where metabolic, regulatory, or secretion functions can be leveraged in an immunoisolated extracorporeal circuit.

Keywords: Acute renal failure; Bioreactor; Cryopreservation; End-stage renal disease; Extracorporeal cell therapy; Progenitor.

Figure 1

Bioartificial Renal Epithelial Cell System (BRECS) design, including depiction of internal components (top). Cells were grown on niobium-coated carbon disks (A), which were held in place within the BRECS by tuning forks. Baffles were used to critically space tuning forks and direct flow through disks. A flow separator was positioned at the inlet to maintain steady and homogeneous flow throughout the BRECS. Cells shown in (B) were stained with DAPI (blue), and cells shown in (C) were stained for zona occludens-1 (ZO-1; green) and acetylated tubulin (AT-1; red).

Figure 2

Flow chart of BRECS cryopreservation and reconstitution. HTS, Hypothermosol; CS5, CryoStor 5; P (or H)REC, porcine (or human) renal epithelial cell.

Figure 3

Cell retention on Starwheel cultured disks (A) and BRECS cultured disks (B) are, respectively, shown by nuclear stain (DAPI). Of note, the significantly lower renal epithelial cell (REC) density on the Starwheel cultured disk (A) does not allow for visualization of the trabeculated disk structure, as opposed to the well-defined trabeculation observed on the BRECS cultured disk, due to high REC density (B). Scale bars: 100 µm.

Figure 4

Lactate production and estimated cell number over the time course of PREC-seeded BRECS perfusion culture (n = 4) as averages ± standard error.

Figure 5

Immunohistochemical study of HRECs grown on collagen IV-coated carbon disks from BRECS units, post 4 weeks culture, before cryopreservation (A–C) and 2 weeks postreconstitution (D–F). In (A) and (D), tight junctions (ZO-1, green fluorescence), centralized cilia (AT-1, red punctate fluorescence), and nuclei (DAPI, blue) can be seen in cells across the surface area of the porous disk. In (B) and (E), prominent CD13 (red) distribution indicates presence of brush border enzyme for Na⁺-dependent amino acid transport, a marker for renal tubule cell phenotype, with nuclei stained (DAPI, blue). In (C) and (F), presence of Gamma-glutamyltranspeptidase (γGT; red) suggests glutathione metabolism capability of the cells, with nuclei stained by DAPI, also shown in blue). Scale bars: 100 µm.

Figure 6

PREC (A) and HREC (B) viability after cryopreservation demonstrate a high density of living cells (fluorescein diacetate, green) and relatively low density of dead cells (propidium iodide, red). Scale bar: 100 µm.

Figure 7

Evaluation of PREC metabolism in seeded BRECS prior to and after liquid nitrogen gas phase cryopreservation (−140°C ) for 3 months and cryopreservation in an ultralow −80°C freezer for 1 month. Blue background section designates the precryopreservation culture period, and the pink background section marks the postconstitution culture period for both cryostorage methods. Note that the representation of cryostorage has been truncated to allow for direct comparison of metabolic parameters while in culture.

Figure 8

Proposed extracorporeal circuit for future clinical treatment. Pre- and post-BRECS filters allow immunoisolation of the therapeutic cells from the patient's body. UF = ultrafiltration.