Discarded Livers Find a New Life: Engineered Liver Grafts Using Hepatocytes Recovered From Marginal Livers

Abstract

Treatment for end-stage liver failure is restricted by the critical shortage of donor organs; about 4000 people die in the USA while waiting for a transplantable organ. This situation has been a major driving force behind the rise of tissue engineering to build artificial tissues/organs. Recent advancements in creating transplantable liver grafts using decellularized liver scaffolds bring the field closer to clinical translation. However, a source of readily available and highly functional adult hepatocytes in adequate numbers for regenerative liver therapies still remains unclear. Here, we describe a new method to utilize discarded livers to make transplantable new liver grafts. We show that marginal donor livers damaged due to warm ischemia could be treated with machine perfusion to yield 39 million viable hepatocytes per gram of liver, similar to fresh livers, and these cells could be used to repopulate decellularized liver matrix (DLM) scaffolds to make transplantable liver grafts. The hepatocytes from recovered livers sustained their characteristic epithelial morphology while they exhibited slightly lower protein synthesis functions both in plate cultures and in recellularized liver grafts. The dampened protein synthesis was attributed to residual endoplasmic reticulum stress found in recovered cells. The results here represent a unique approach to reengineer transplantable liver grafts solely from discarded organs.

Keywords: -Endoplasmic reticulum stress; -Hepatocytes; -Liver recellularization; Machine perfusion.

Fig. 1

Recovery of damaged livers for preparation of liver grafts. (A) Schematic of the perfusion setup. (B) Hepatocyte yield after perfusion recovery (*N.S., P = 0.182, n = 8). (C) Gross appearance of DLM scaffold before and after decellularization. (D) H&E staining of DLM scaffold before and after decellularization. (E) Corrosion cast of DLM scaffold to confirm the presence of vascular architecture, blue: venous, red: portal, yellow: bile ducts. (F) Growth factors that are retained in the DLM scaffold. Scale bars (C, E) 1 cm (D) 100 μm. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 2

Hepatocyte characteristics in culture. (A) Phase contrast images of cells in static sandwich culture for 7 days. (B) Albumin and urea secretion by recovered and fresh hepatocytes in static sandwich culture (n= 3). (C) H&E staining of recellularized liver grafts 7 days post perfusion culture. (D) Albumin and urea secretion by recovered and fresh hepatocytes in recellularized liver grafts (n = 6). Scale bars (A, C) 50 μm. ER stress markers in isolated hepatocytes. (E) Ratio of Xbp-1s/Xbp-1u concentrations. (F) Chop and Grp78 expression normalized to fresh cells (n = 5, * P = 0.0012, ** P = 0.001, †P = 0.0006). [Color figure can be viewed at wileyonlinelibrary.com]