Figure 6.

hBECs can be isolated and expanded in vitro in GMP-compliant conditions

(A) Total number of CD133+ hBECs per gram of liver isolated in non-GMP- and GMP-compliant conditions. ^∗ denotes p < 0.05 (mean ± SEM), Student’s t test (n = 3).

(B) Total percentage of CD133− and CD133+ hBECs isolated using the standard protocol (Ctrol) or a protocol including Percoll gradient isolation. ^∗∗ denotes p < 0.005 (mean ± SEM), Student’s t test (n = 4).

(C) Total percentage of CD133− and CD133+ hBECs isolated using fresh livers (Control) or livers perfused under hypothermic conditions. Student’s t test (n = 3).

(D) CD133+ hBECs isolated in GMP-compliant conditions can be cultured in vitro as organoids in Matrigel spheres. From left, right: bright field depicting morphology of hBECs, FDA (cyan) showing live cells, propidium iodide (PI) (magenta) showing dead cells. Far right: merge. Scale bars, 50 μm.

(E) Heatmap of normalized expression values across genes associated with cholangiocytes, hepatocytes, or progenitor cell populations. These markers were analyzed in hBECs isolated from two donor livers maintained in 2D (GMP-compliant) in vitro culture conditions. P0 represents freshly isolated hBECs. Darker blue represents higher normalized gene expression.

(F) Heatmap of normalized expression values across genes associated with stem cell, cholangiocyte, and hepatocyte proliferation.

(G) hBECs cultured in 3D (GMP-compliant) scaffolds express markers of mature cholangiocytes (EpCAM, K19, CD133, MRP2, and ZO1) but not mature hepatocytes (HNF4α) (n = 5). Scale bars, 100 μm.

(H) Growth kinetics of isolated hBECs from donor livers were cultured on GMP-compliant conditions through a series of passages. Doubling time, purity, and viability of hBECs from isolation to passage 2 (p2) (N = 2).