The vascularised chamber device significantly enhances the survival of transplanted liver organoids

Reviewer #1 (Public Review):

Summary:
The authors describe their work on finding optimal ways of infusing organoids into mice. They describe five delivery methods and compare organoid survival two weeks after delivery. This work is concluded with the use of a vascularized chamber being the most optimal for organoid viability.

Strengths:
The aim is to have a preclinical, translational model to test methods of organoid infusion. This is important and timely to the field.

Weaknesses:
- A clear aim seems to be missing, although I can extract this from the manuscript. The approach is described a bit cryptically. The manuscript could use a bit more explanation here and there.
- Although the authors themselves argue in the introduction that the use of mice is not optimal, they show a mouse study in which human-derived iPSC organoids are infused in mice.
- As far as I can extract from the Methods section, only one iPSC line was used. Given the huge donor variance, it is essential to repeat the work with multiple iPSC lines.
- I am missing the right control groups, especially for the surgical groups. And the group size is very variable (3 to 7 mice per group). Three per group is then somewhat small in size.

Reviewer #2 (Public Review):

Summary:
In this study, human induced pluripotent stem cell (hiPSC)-derived liver progenitor cell organoids were transplanted into four different transplantation sites in a mouse model of liver disease, using five organoid delivery methods. Organoids were transplanted into the vascularised chamber device established in the groin, or into the liver, spleen, and subcutaneous fat. Results show that the vascularised chamber had the highest organoid survival, 5.1x higher than the site with the second highest survival (p=0.0002), being the intra-hepatic scaffold approach. Animals with the vascularised chamber also had the highest human albumin levels (0.33 {plus minus} 0.09 ng/mL). No organoid survival was observed when delivered into the liver without a scaffold, or when injected into the spleen. Meager survival occurred in transplantations into subcutaneous fat.

Strengths:
A systematic study with a clear line of experiments and well-presented results. The manuscript is well-written and easy to follow. The results and conclusions drawn are convincing.

Weaknesses:
Although the number of organoids and albumin secretion is visibly higher in the vascularised chamber device, the organoids possess relatively higher Sox9+ cells compared to HNFa4a+ cells suggesting higher biliary differentiation than hepatic differentiation. On the other hand, although the intrahepatic scaffold approach, with a relatively smaller number of organoids and less albumin secretion, showed higher hepatic differentiation (although non-significant) suggesting that better scaffolds could be researched further to assess the clinical application of intrahepatic scaffold-based organoid transplantation.