Study overview.

(A) Schematic diagram of the experimental plan. (B) Summary of overall results.

In vitro iLPC organoids.

(A) Bright-field imaging of iLPC organoids at day 48 post-cell seeding. (B) Corresponding fluorescence imaging of the same organoids showing eGFP expression in all organoids. (C) HNF4α immunohistochemistry, showing robust HNF4α expression in most nuclei (red arrows). (D) Sox9 immunohistochemistry, showing robust Sox9 expression in most nuclei (red arrows). (E) Albumin immunohistochemistry, showing low expression in only a small number of cells within organoids (red arrows). (F) Ki67 immunohistochemistry, showing that >50% of cells were Ki67+ (red arrows) and proliferative. (G) The NovoSorb® polyurethane scaffold used for intra-hepatic organoid delivery, which were circular discs 5mm in diameter, 1mm in thickness, with interconnected pores 300-600µm in diameter. (H) eGFP+ iLPC organoids were suspended in Matrigel and seeded into the polyurethane scaffold and were found well dispersed throughout the pores of the scaffold. Scalebars -20µm for (C), (D), (E), (F), 100µm for (A), (B), (H), 500µm for (G).

Organoid transplantations into the vascularised chamber.

(A) Low magnification haematoxylin & eosin (H&E) staining of the chamber tissue, demonstrating it was encapsulated by a layer of inflammatory connective tissue (black arrows), had large areas of sparsely cellular remnant Matrigel infiltrated with native adipocytes (red arrow), and areas where multiple organoids were clustered together (blue arrow). (B) High magnification H&E staining of thechambe demonstrating multiple organoids which formed ductular structures which contained large cystic spaces with heterogeneous secretions lined by cuboidal-columnar epithelium. (C) Organoids were identified by their expression of eGFP. (D) Only approximately 50% of cells within organoids were HNF4α+. (E) The majority of cells within organoids were Sox9+. (F) The heterogeneous intra-luminal secretions within organoids labelled strongly for human albumin. (G) Up to 40% of cells within organoids were Ki67+ and proliferative. (H) Organoids were often near CD31+ native blood vessels in the chamber. Scalebars -20µm for (C), (D), (E), (G), (H), 100µm for (B), (F), 500µm for (A).

Organoid transplantations into the liver using a polyurethane scaffold.

(A) Low magnification H&E staining demonstrating the scaffold (red arrow) well adhered between two liver lobes. (B) High magnification H&E demonstrating the scaffold directly adjacent and tightly adhered to the native mouse liver (labelled L), with Matrigel, infiltrating host tissue within the pores of the scaffold (labelled with asterisks), and the walls of the scaffold pores appearing as clear triangular material throughout the scaffold area (red arrows). (C) eGFP+ organoids were identified in the scaffold. (D) Many of the cells in the organoid were HNF4α (red arrows). (E) Most of the cells in the organoid were Sox9+ (red arrows). (F) The intraluminal secretions within organoids were strongly positive for human albumin (red arrow), and organoids were often found in the periphery of the scaffold close to native mouse liver (labe l(lGe)dSoLm). e of the cells within organoids were Ki67+ and proliferative (red arrows). (H) Organoids in the scaffold (asterisk) were often located near the native mouse liver (labelled L), and LYVE-1+ blood vessels from the liver extended into the scaffold to surround organoids (red arrows). Scalebars - 20µm for (C), (D), (E), (F), (G), 50µm for (H), 100µm for (B), 500µm for (A).

Organoid transplantations into the liver and spleen using Matrigel alone.

(A) eGFP immunohistochemistry of livers transplanted with organoids in Matrigel alone resulted in no organoid survival. The remnant Matrigel was present on the surface of the liver (labelled L) as a thin sparsely cellular layer (asterisk), with a densely cellular inflammatory layer of cells overlaying the Matrigel (red arrow). (B) Higher magnification of eGFP immunohistochemistry. (C) In some livers the superficial layer of inflammatory cells was much thicker (red arrow), but the same architecture was present, which was a layer of remnant Matrigel (asterisk) on top of the host mouse liver (L), capped by a superficial layer of inflammatory cells (red arrow). (D) Higher magnification of eGFP immunohistochemistry in a liver with a thick layer of inflammatory cells on top of the remnant Matrigel. (E) In spleens transplanted with organoids, no organoid structures were present, demonstrated with eGFP immunohistochemistry. (F) There were isolated flecks of eGFP+ staining scattered throughout the spleen (red arrows), but this was not associated with nucleated cells. Scalebars - 20µm for (F), 100µm for (A), (B), (C), (D), (E).

Organoid transplantations into subcutaneous fat.

(A) H&E staining of the resected tissue demonstrating the remnant Matrigel seen as a pink sparsely cellular area (red dotted line) with some infiltrating adipocytes (red asterisk), clearly demarcated from the surrounding adipose and connective tissue (asterisks). Organoids were found in only 2 animals (out of 6), with each sample only containing 1 organoid. The 1 sample is shown here. (B) In sample 1, only one small eGFP+ organoid was present. (C) Organoids contained intra-luminal human albumin. (D) All cells in the organoid were HNF4α+ (red arrows). (E) All cells in the organoid were Sox9+ (red arrows). (F) Approximately 20% of cells were Ki67+ and proliferative (red arrows). (G) H&E staining of sample 2, showing remnant Matrigel (dotted red line) with areas of infiltrating adipocytes (red asterisk), surrounded by subcutaneous adipose and connective tissue (black asterisks). (H) The eGFP+ organoid in this sample was much larger. (I) Similar to sample 1 the organoid in sample 2 also contained intra-luminal albumin. However, in contrast to sample 1 this organoid had no HNF4α+cells (J) or Sox9+ cells (K). (L) Only a small proportion of cells were Ki67+. Scalebars - 20µm for (B), (C), (E), (F), (H), (I), (J), (K), 50µm for (D), (L), 200µm for (A), (G).

Morphometric & functional analysis.

(A) Organoid survival (number of organoids per sample) (B) Human albumin levels in mouse plasma. (C)Organoid diameter. (D) Organoid circumference. (E)Organoid cross-sectional area. (F)Percentage of HNF4α+ cells per organoid. (G) Percentage of Sox9+ cells per organoid. Analysed with one-way ANOVA with Bonferroni post-hoc analysis. * p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001. N=3-7 per group for (A), (B), (C), (D), (E), N=9-39 per group for (F), and N=16-36 for (G).