What do you think about the research that discusses organoid co-culture with endothelial cells? Do you find that the organoids can get adequate nutrients with good culture media?

Answer

At this point in vivo organoids do not have their own vasculature but endothelial cells have been shown to form rudimentary vascular structures in some organoids like liver buds. When they are implanted into immune-deficient mice, the host vasculature fuses with these structures to form functional vascular network and eventually a functional liver bud in these mice. Please refer to this journal article for further details. There is a significant amount of research where endothelial cells and MSCs with iPSCs are being mixed or where vasculature is being bioprinted in 3D structures. In other cases, endothelial cells were included in co-culture spheroids to enhance the functions, e.g. hepatocyte spheroids as described in this article.

With regards to the second questions, once media is optimized for organoid cultures, adequate nutrition and waste removal is achieved by using spinner flasks, perfusion or frequent media changes. Organoids need a continuous feed of fresh nutrients and waste removal as the culture expands in size and since they are not vascularized. There are 3 recommendations that have been used for this purpose:

1. Researchers have been successfully culturing and maintaining organoids embedded in Corning Matrigel matrix droplets which are then suspended in media in Corning Spinner Flasks. Lancaster and Knoblich maintained cerebral organoids of 4 mm in size for up to 15 months. Please refer to the protocol in this paper.
2. We have successfully cultured intestinal organoids by frequently (2 to 3 times a week) changing the media for 2 mm organoids and maintained them for up to 6 weeks. McCracken et. al., passaged these organoids for up to 140 days using this protocol.
3. Perfusion is an option that can continuously replenish the nutrients and remove wastes and is more increasingly utilized in organ-on-chip cultures to maintain all the nutrients, growth factors, metabolites in a constant equilibrium once optimized so cultures can be maintained much longer than static cultures.

The size and compactness of your organoids do play a role in nutrients and oxygen distribution. There is a limitation of relying on passive diffusion to supply nutrients or oxygen within the organoid mass.  As a result, the metabolic status of cells within a spheroid mass could be very different. Take an example of tumoroid studies, necrosis occurs and signs of hypoxic responses inside those larger spheroids. So factors of organoid type, medium and other culture conditions all need to be considered for nutrient and oxygen supply.