Compositions and method for establishing organoid cultures from cryogenically preserved tissue

A method of establishing organoid culture from cryogenically preserved tissue, comprising:

• obtaining a cell-containing tissue from an individual, wherein the cell-containing tissue is a whole tissue biopsy comprising epithelial tissue comprising crypt cell, stem cells, and underlying mesenchyme;
• suspending the cell-containing tissue with freezing media in a vial;
• cooling the vial to a temperature of approximately between −80° C. and −196° C. for an amount of time sufficient to cryopreserve the cells or tissue;
• thawing the cryogenically preserved cell-containing tissue;
• washing the thawed cell-containing tissue to remove the freezing media;
• embedding the thawed and washed cell-containing tissue in an extracellular-matrix (ECM);
• incubating the embedded cell-containing tissue for about five to nine days to permit post-thaw tissue repair and recovery of epithelium;
• releasing epithelium from underlying mesenchyme;
• isolating the released epithelium;
• embedding the isolated epithelium in ECM with a recovery medium comprising thiazovivin (TZV), SB431542, Y-27632, and CHIR-99021, wherein Y-27632 is present only during an initial twenty-four-hour period following embedding;
• and
• establishing organoid cultures through adding growth media to the isolated epithelium in extracellular matrix (ECM) following exposure to the recovery medium, wherein the established organoid cultures are capable of expansion beyond 10 days from initiation of organoid culture establishment, wherein the growth medium comprises Wnt3a, Rspondin1, Noggin, and EGF, and one or more of:
• thiazovivin (TZV), SB431542, and CHIR-99021.