Hi everyone,
im using this established protocol:
Article Establishment and Culture of Human Intestinal Organoids Deri...
I already optimized everything, written under "troubleshooting" in this protocol. I directly obtain the samples from the endoscopy, its less than 20 minutes between extraction and starting work in the lab. During this time the samples are on ice and in the media recommended in the protocol.
So far i was only able to generate organoids from childrens biopsies, but not from older patients. It seems, as if they needed a stronger Wnt-activation?
I use recombinant human Wnt3a at the correct concentrations. Would you recommend switching to Wnt-surrogate or Wnt-conditioned media?
The protocol should also work with samples from older patients.
Is maybe the tissue piece from the endoscopy too small and i need larger samples from the surgery?
Any other ideas, what i could try?
Thank you,
Marco
Just a quick thought, but perhaps the cells from the older patients are much closer to their Hayflick limit.
Philip G Penketh Thank you. That sure is true, but i also had no success with patients at the age of 40 for example. So i don't think, that's the whole explanation.
Not relevant to to this discussion but somewhat related indirecly. When I was isolating mesenchymal stem cells from patients bone marrow taken from their femur undergoing knee surgery. There was variation among patients. Some patients' bone marrow stem cells did not grew further or very little grew. Whereas some patients bone marrow stem cells provided me millions of cells after 2 passages. I wanted to to go back to history of the patients what medicines and what diseases they had.
May be you should try to relate to patients medical history besides age group
It was the recombinant Wnt. Now with Wnt-surrogate organoids grow efficiently
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