Adenoma-Derived Organoids for Precision Therapy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Tissue Samples
2.2. Isolation of Intestinal Crypts from Human Colorectal Biopsies
2.3. LWRN Complete Medium
2.4. Cryopreservation and Thawing of Colon Organoids
2.5. Immunofluorescence (IF)
2.6. Microscopy and Imaging
2.7. RNA Isolation and RT-qPCR Analysis
2.8. qPCR Primers
2.9. Western Blot Analysis (WB)
2.10. Statistical Methods
3. Results
3.1. Establishing and Characterizing Adenoma-Derived Colonoids
3.2. Enhancing the Growth of Healthy Tissue-Derived Organoids
3.3. G418 Induces APC Restoration Decreasing Canonical Wnt Signaling and Cell Proliferation in Treated Colonoids
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Patient/Isolation | Gender | Genetic Background | Growth (days) | Structure (days) | ||
---|---|---|---|---|---|---|
H | P | H | P | |||
1-1 | F | APC ns S874X | 3 | 21 | U | B (1–15); C (15–21) |
1-2 | 43 | 43 | B (1–43) | B (1–15); C (15–43) | ||
2 | F | APC ns L77X | X * | 15 | X | U (1–15) |
3 | M | APC ns L77X | − | 20 | − | B&C (1–20) |
4-1 | M | APC ns Q341X | 7 | − | C (1–7) | − |
4-2 | 3 | 39 | X | B&C (1–39) | ||
5-1 | M | APC ns Q341X | 3 * | 14 | X | C (1–14) |
5-2 | 27 | − | C (1–27) | − | ||
6-1 | M | APC ns R302X | 6 * | 6 * | C (1–6) | X |
6-2 | 7 | 19 | B (1–7) | B (1–19) | ||
6-3 | 8 | 22 | B (1–8) | B&C (1–22) | ||
7 | F | APC fs Z1379R | 21 | 29 | C (1–21) | B&C (1–29) |
8 | F | APC fs Q264 | 5 | 19 | C (1–5) | B (1–19) |
9-1 | APC fs R332 | 4 * | 7 * | U | B (1–7) | |
9-2 | 8 | 8 | B (1–8) | B (1–8) | ||
10 | F | APC fs 2688delC in exon 15 | 10 | − | B (1–10) | − |
11 | M | MYH G382D −/− | X | 39 | X | B (1–11); C (11–39) |
12 | MYH 1145G>A +/− RAD50 326_329delCAGA +/− | 7 | 21 | B&C (1–7) | B&C (1–21) | |
13 | M | POLD1 V759I +/− | − | 9 | − | 2D (7<) |
14 | PTEN 697C>T +/− | − | 25 | − | 2D (23<) | |
15 | M | Ulcerative Colitis | 7 | − | U (1–7) | − |
16 | M | Unknown | 3 | 36 | U | B (1–11); C (11-36) |
17 | Unknown | X | 36 | X | B (1–18); C (18–36) | |
18 | F | Unknown | 7 | 7 | B (1–7) | B (1–7) |
Gene Symbol | Fw Primer | Rv Primer |
---|---|---|
GAPDH | GCACCGTCAAGGCTGAGAAC | ATGGTGGTGAAGACGCCAGT |
MYC | CTCGGTGGTCTTCCCCTACCCT | TGTCCAACTTGACCCTCTTGGC |
AXIN2 | GCAGCTCAGCAAAAAGGGAAAT | TACATCGGGAGCACCGTCTCAT |
CCND1 | CATCTACACCGACAACTCCATC | TCTGGCATTTTGGAGAGGAAG |
SOX9 | ACTTGCACAACGCCGAG | CTGGTACTTGTAATCCGGGTG |
LEF1 | AGACAAGCACAAACCTCTCAG | TCATTATGTACCCGGAATAACTC |
ACTIN | CCTGGCACCCAGCACAAT | GGGCCGGACTCGTCATACT |
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Evron-Levy, T.; Caspi, M.; Wittenstein, A.; Shorer-Arbel, Y.; Shomron, O.; Hirschberg, K.; Kariv, R.; Rosin-Arbesfeld, R. Adenoma-Derived Organoids for Precision Therapy. Organoids 2022, 1, 54-68. https://doi.org/10.3390/organoids1010006
Evron-Levy T, Caspi M, Wittenstein A, Shorer-Arbel Y, Shomron O, Hirschberg K, Kariv R, Rosin-Arbesfeld R. Adenoma-Derived Organoids for Precision Therapy. Organoids. 2022; 1(1):54-68. https://doi.org/10.3390/organoids1010006
Chicago/Turabian StyleEvron-Levy, Tamar, Michal Caspi, Amnon Wittenstein, Yamit Shorer-Arbel, Olga Shomron, Koret Hirschberg, Revital Kariv, and Rina Rosin-Arbesfeld. 2022. "Adenoma-Derived Organoids for Precision Therapy" Organoids 1, no. 1: 54-68. https://doi.org/10.3390/organoids1010006