ARRAY PLATFORM FOR HIGH-THROUGHPUT ORGANOID PROFILING
Organoid culture and image-processing methods and systems are described. Such methods and systems provide the ability for high-throughput characterization of a variety of phenotypes at single-organoid resolution.
1 . A method of monitoring organoid development in parallel for a plurality of organoids, each arising from one or more organoid progenitor cells, said method comprising:
(i) distributing organoid progenitor cells from a population of organoid progenitor cells into array compartments to provide an open planar array comprising a plurality of organoid growth compartments in which each organoid growth compartment comprises one or more organoid progenitor cells;
(ii) incubating cells in the organoid growth compartments of the open planar array under growth conditions in which the one or more organoid progenitor cells in each organoid growth compartment divides;
(iii) imaging compartments of the open planar array at multiple time intervals;
(iv) analyzing at least one phenotypic parameter of multi-cell structures growing in at least a portion of the organoid growth compartments imaged in step (iii).
2 . The method of claim 1 , wherein distribution of organoid progenitor cells into the open planar array provides a plurality of organoid growth compartments, each comprising a single organoid progenitor cell.
3 . The method of claim 1 , wherein organoids in each grow compartment of the open planar array are cultured under the same conditions.
4 . The method of claim 1 , wherein the population of organoid progenitor cells is genetically distinct across the population.
5 . The method of claim 1 , wherein (iii) and (iv) occur simultaneously.
6 . The method of claim 1 , further comprising distributing additional co-culture cells into at least a portion of the organoid growth compartments containing organoid progenitor cells or a multi-cellular structure arising from the organoid progenitor cells.
7 . The method of claim 6 , wherein the co-culture cells are immune system cells, optionally wherein the immune system cells are T cells, dendritic cells, macrophages, or natural killer cells.
8 . (canceled)
9 . The method of claim 1 , wherein the at least one phenotypic parameter is rate of cell division, cell polarity, or cell migration.
10 . The method of claim 1 , wherein step (iv) comprises detecting a signal from a cellular molecule labeled with a detectable label, optionally wherein the detectable label is a fluorescent label.
11 . (canceled)
12 . The method of claim 10 , wherein the population of organoid stem cells is genetically modified to introduce the cellular molecule into cells.
13 . The method of claim 10 , wherein the fluorescent label is attached to an agent that specifically binds the molecule.
14 . The method of claim 1 , wherein the population of organoid progenitor cells comprises epithelial cells.
15 . The method of claim 1 , wherein the population of organoid progenitor cells comprises cells having a mutation at one or more genetic loci associated with a cancer and/or cells having mutations at two or more genetic loci associated with the cancer, optionally wherein the cancer is gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, esophageal cancer, head and neck cancer, or renal cancer and/or one or more mutations at genetic loci associated with cancer are introduced into the cell by gene editing.
16 .- 18 . (canceled)
19 . The method of claim 1 , wherein:
the compartments are microwells;
step (i) comprises loading cells using centrifugation; or
imaging occurs at least once a day following distribution of single organoid stems cells into compartments.
20 - 21 . (canceled)
22 . The method of claim 1 wherein the method further comprises retrieving cells from at least a portion of the organoid growth compartments for genetic analysis, optionally wherein genetic analysis comprises RNA sequence, ATAC sequence analysis, whole genome sequencing, or methylation profiling.
23 . The method of claim 22 , wherein retrieving cells comprises aspirating cells.
24 . (canceled)
25 . A platform for monitoring organoid growth comprising (i) an open planar array comprising at least 100 organoid growth compartments, wherein each organoid growth compartment is a microwell comprising a three dimensional multi-cellular structure arising from one or more organ progenitor cells and (ii) an imaging system.
26 . The platform of claim 25 , wherein each of the three dimensional multi-cellular structures in a plurality of microwells comprises cells comprising a molecule labeled with a fluorescent label.
27 . The platform of claim 26 , wherein the fluorescent label is attached to an agent that specifically binds the molecule.
28 . The platform of claim 25 , wherein each three dimensional multi-cellular structure in a plurality of microwells comprises epithelial cells.
29 . The platform of claim 25 , wherein each three dimensional multi-cellular structure in a plurality of microwells comprises cells having a mutation at one or more genetic loci associated with cancer.
30 . The platform of claim 29 , wherein the three dimensional multi-cellular structure arose from a single cell that was genome edited to comprise the mutation; and/ or the cancer is gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, esophageal cancer, head and neck cancer, brain cancer, or renal cancer.
31 . (canceled)