Functional Evaluation of Primary Renal Cell/Biomaterial Neo-Kidney Augment Prototypes for Renal Tissue Engineering

Abstract

Development of a tissue-engineered neo-kidney augment (NKA) requires evaluation of defined, therapeutically relevant cell and cell/biomaterial composites (NKA constructs) for regenerative potential in mammalian kidney. Previous work identified primary renal cell populations that extended survival and improved renal function in a rodent model of chronic kidney disease (CKD). This study extends that work toward the goal of developing NKA by (i) screening in vivo inflammatory and fibrotic responses to acellular biomaterials delivered to healthy rodent renal parenchyma, (ii) evaluating the functionality of renal cell/biomaterial combinations in vitro, (iii) generating NKA constructs by combining therapeutically relevant cell populations with biocompatible biomaterial, and (iv) evaluating in vivo neokidney tissue development in response to NKA constructs delivered to healthy rodent renal parenchyma. Gelatin and hyaluronic acid (HA)-based hydrogels elicited the least inflammatory and fibrotic responses in renal parenchyma relative to polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) beads or particles and were associated with neovascularization and cellular infiltration by 4 weeks postimplantation. Renal cell populations seeded onto gelatin or HA-based hydrogels were viable and maintained a tubular epithelial functional phenotype during an in vitro maturation of 3 days as measured by transcriptomic, proteomic, secretomic, and confocal immunofluorescence assays. In vivo delivery of cell-seeded NKA constructs (bioactive renal cells + gelatin hydrogels) to healthy rodent renal parenchyma elicited neokidney tissue formation at 1 week postimplantation. To investigate a potential mechanism by which NKA constructs could impact a disease state, the effect of conditioned media on TGF-β signaling pathways related to tubulo-interstitial fibrosis associated with CKD progression was evaluated. Conditioned medium was observed to attenuate TGF-β-induced epithelial-mesenchymal transition (EMT) in vitro in a human proximal tubular cell line (HK2).

Biomaterials for TE/RM products

“…We applied a combination of in vitro functional assays and in vivo regenerative outcomes to functionally screen candidate biomaterials for potential incorporation into a NKA construct prototype. 23 We observed the following: Implantation of acellular hydrogel-based biomaterials into renal parenchyma was typically associated with minimal fibrosis or chronic inflammation with no evidence of necrosis by four weeks post-implantation. Moderate cellular/tissue in-growth and neovascularization was observed, with minimal remnant biomaterial (Figure 3.2, top).…”

Potency evaluation of tissue engineered and regenerative medicine products

“…Additionally, secretion of vascular endothelial growth factor (VEGF) and kidney injury molecule 1 (KIM1) is currently leveraged as a potency bioassay for the bioactive cellular component of the NKA [14]. Finally, secretion of VEGF has been reported as a potency metric for ex vivo-produced oral mucosal equivalent (EVPOME), a tissue-engineered, cell based product prototype for intra-oral grafts [15].…”

“…31,No. 9 by facilitating de novo angiogenesis and amelioration of fibrosis and apoptosis within the diseased kidney (Box 3) [33,14].…”

“…An in vitro model system recapitulating observed disease and regenerative outcomes may be assembled using the human proximal tubular cell line HK2, which is well established as a cellular platform to test the stimulatory or inhibitory effects of small molecules or proteins on TGFb-induced EMT [36]. Modulation of TGFb-induced EMT in HK2 cells has been leveraged as a quantifiable potency assay for NKA candidates [14].…”

Tissue Engineering of Tubular and Solid Organs: An Industry Perspective

“…To this end, we have leveraged principles discussed throughout this chapter, identifying lineage committed, primary renal cell populations as therapeutically bioactive agents capable of mediating functional rescue of aspects of disease phenotype within small animal clinical models of chronic kidney disease (Kelley et al, 2010;Presnell et al, 2010). Similarly, our evaluation of biomaterials compatible with renal parenchyma has led us towards application of gelatin hydrogels as the best tolerated biomaterial scaffold for renal tissue engineering (Basu et al, 2011b). Studies are currently underway to evaluate the ability of bioactive, primary renal cell/hydrogel biomaterial composite constructs (NKA TM ) to catalyze functional rescue of disease in small and large animal clinical models of chronic kidney disease.…”