# US 12,600,953 B2 · Compositions and method for establishing organoid cultures from cryogenically preserved tissue

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- Number: US 12,600,953 B2
- Document ID: US12600953B2
- Status: Granted
- Kind: B2
- Application number: 16/634,818
- Filed: 2018-07-27
- Published: 2021-03-25
- Granted: 2026-04-14
- Earliest claimed: 2017-07-31
- Assignees: The Regents of the University of Michigan
- Applicants: The Regents of the University of Michigan
- Inventors: Jason Spence; Yu-Hwai Tsai; Michael Czerwinski; John Kao
- Web page: https://patentgrep.com/patent/US12600953B2

## CPC

- C12N 5/0679: {Cells of the gastro-intestinal tract}
- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/415: Wnt; Frizzeled
- C12N 2513/00: 3D culture
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS

## Abstract

The present application is directed to compositions and methods for establishing organoid cultures from cryogenically preserved tissue (e.g., human biopsy tissue), and related methods of use with such established organoid cultures.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The method of claim 1, wherein releasing epithelium from underlying mesenchyme is accomplished with dispase treatment or EDTA treatment.

### Claim 3 (depends on 1)

The method of claim 1, wherein the freezing media comprises DMEM/F12, 10% FBS, and 10% DMSO.

### Claim 4 (depends on 1)

The method of claim 1, wherein the growth media further comprises one or more of
advanced DMEM/F12,
L-Alanyl-L-glutamine,
HEPES,
N2 Supplement,
a supplement comprising: biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, BSA, catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactase, ethanolamine HCl, glutathione, L-carnitine HCl, linoleic acid, linolenic acid, prograsterone, putrescine, sodium selenite, and triodo-I-thyronine,
Pen/Strep,
N-acetylcystine, and
Nicotinamide.

### Claim 5 (depends on 1)

The method of claim 1, wherein the growth media further comprises Y27632.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell-containing tissue is gastrointestinal tissue.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cell-containing tissue is from the colon, the distal ileum, the jejunum, the duodenum, and/or the stomach.

### Claim 8 (depends on 1)

The method of claim 1, wherein the extracellular matrix (ECM) comprises laminin, nidogen, collagen, heparan sulfate proteoglycans, EGF and TGF-β.