1. Isolating human gastrointestinal epithelium from surgical resections or biopsy specimens:

(a) high quality crypt purification

(b) quick and dirty method - no EDTA and non-enzymatic

(c) colorectal cancer colonoids

2. Passaging human epithelium-only intestinal organoids (a.k.a. enteroids/colonoids)

‍ ‍Passaging, thawing, and freezing of human lung organoids (bud tip, AT2, trachea)

‍ ‍Passaging, thawing, and freezing of human esophageal cells

3. Generating LWRN-conditioned media (CM)

4. Assessing LWRN CM Wnt activity

‍ ‍One-Glo luciferase reporter (Promega)

‍ ‍TOPflash luciferase assay (Biotium)

5. Complete gastrointestinal growth media (HCM…)*

‍ ‍Complete lung and esophagus medias (FANY, HEYNAC, SFFF, TATA, 3F/SI, CKAB)

6. Freeze-thawing tissue biopsies and enteroid/colonoid cultures

7. Shipping and receiving live enteroid/colonoid cultures

8. Transwell human colonoid/enteroid monolayers

‍ ‍96 well transwell plate monolayers

‍ ‍Transwell human airway cells in an air-liquid interface

9. IHC/IF of enteroids/colonoids and 2D transwell monolayers

10. Single cell human colonoid-forming efficiency assay

Method A: Human Colonoid-Forming Efficiency Assay (CFA) - new protocol

Method B: Single cell human colonoid-forming efficiency assay - old protocol

11. Human colonoid snap-freeze prep for western-DNA-RNA

12. Preparation of human colonoids for orthotopic transplant into mice.

13. Lentiviral transduction of human colonoids/enteroids

*Comparing Laboratory Prepared Medium and a Commercial Medium Alternative for Expansion of Human Colonoid Cultures