Blended electrospinning with human liver extracellular matrix for engineering new hepatic microenvironments

Abstract

Tissue engineering of a transplantable liver could provide an alternative to donor livers for transplant, solving the problem of escalating donor shortages. One of the challenges for tissue engineers is the extracellular matrix (ECM); a finely controlled in vivo niche which supports hepatocytes. Polymers and decellularized tissue scaffolds each provide some of the necessary biological cues for hepatocytes, however, neither alone has proved sufficient. Enhancing microenvironments using bioactive molecules allows researchers to create more appropriate niches for hepatocytes. We combined decellularized human liver tissue with electrospun polymers to produce a niche for hepatocytes and compared the human liver ECM to its individual components; Collagen I, Laminin-521 and Fibronectin. The resulting scaffolds were validated using THLE-3 hepatocytes. Immunohistochemistry confirmed retention of proteins in the scaffolds. Mechanical testing demonstrated significant increases in the Young's Modulus of the decellularized ECM scaffold; providing significantly stiffer environments for hepatocytes. Each scaffold maintained hepatocyte growth, albumin production and influenced expression of key hepatic genes, with the decellularized ECM scaffolds exerting an influence which is not recapitulated by individual ECM components. Blended protein:polymer scaffolds provide a viable, translatable niche for hepatocytes and offers a solution to current obstacles in disease modelling and liver tissue engineering.

Figure 1

Mechanical testing. Incorporating human liver ECM into the scaffold produces a significantly stiffer environment for hepatocytes. hRL521 scaffolds are significantly more elastic than both hBTC1 and hFN scaffolds. N = 6, Data shown as mean ± 95% confidence interval with statistics performed using One way ANOVA with Games Howell post hoc analyses.

Figure 2

Immunohistochemistry. Collagen I, Fibronectin and Laminin are all present in their respective single protein scaffolds, as well as in varying degrees in the ECM scaffold, with no false positive staining observed in the control polymer only condition. Stains were performed for Collagen I, Laminin and Fibronectin,post processed using ImageJ. 10x magnification.

Figure 3

Cell viability – Cell titre blue and picogreen DNA quantitation. Cell adherence was assessed by CellTiter-Blue® Cell viability assay (A) and further confirmed by Quant-IT™ Picogreen® dsDNA assay (B). Minimum n = 5. Data shown as mean ± 95% confidence interval with statistics performed using One-way ANOVA with Tukey post hoc testing. *p < 0.05 **p < 0.01, ***p < 0.001. Each condition maintains cell survival and the number of cells increases significantly between each time point (A). A significant increase in fluorescence is observed between the polymer only and hLECM scaffolds at day 5 indicating that the presence of human liver ECM has a positive influence on the early expansion and/or survival of hepatocytes. A consistent pattern is observed in the DNA concentration (B) on the scaffolds.

Figure 4

Cell viability – Live Dead. Live/Dead® Viability/Cytotoxicity staining analyses demonstrates continuing survival of the hepatocytes at the latest time point (16d), with a confluent population of viable cells and a low level of cell death on each scaffold. Results demonstrate the FL is viable at all assessed time points. 10x magnification.

Figure 5

SEM characterization. The scaffolds were assessed for consistency and fibre size via scanning electron microscopy and subsequent image analysis. Fibres diameter was determined by DiameterJ 34, n = 4. 250x magnification. Presence of a dense cell layer is further confirmed by SEM imaging with a carpet of cells visible in each condition at 16d.

Figure 6

Q-PCR of key hepatic genes. Quantitative analysis of gene expression was undertaken on the functional cell layer at five, ten and sixteen days of culture, compared to that of the same culture periods grown on polymer only scaffolds. mRNA levels of Albumin (A), Cyp1A1 (B), Cyp1A2 (C), CYP3A4 (D), Collagen I (E), and Collagen IV (F) and Fibronectin (G) are represented as fold difference relative to polymer only controls and relative to the housekeeping gene GAPDH. One-way ANOVA with Games Howell and Tukey post hoc testing and minimum n = 5. *p < 0.05 **p < 0.01, ***p < 0.001. Error bars = SD.

Figure 7

Albumin production Serum. albumin produced by the THLE-3 cells over 24 hours at 5, 10 and 16 day timepoints. n = 6. Data shown as mean ± 95% confidence interval with statistics performed using One-way ANOVA with Games Howell and Tukey post hoc testing and minimum n = 5. *p < 0.05 **p < 0.01, ***p < 0.001.

Figure 8

Decellularization device. Schematic of decellularization device which maintains pressurized flow to effectively decellularize tissue biopsies.