Apr 28, 2023
Strategies for optimizing the isolation and expansion of sensitive patient-derived duodenoids, ileoids and colonoids .pdf
- 1Boston Children's Hospital
Protocol Citation: Katlynn Bugda Gwilt, Jay R. Thiagarajah 2023. Strategies for optimizing the isolation and expansion of sensitive patient-derived duodenoids, ileoids and colonoids .pdf. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx9d6og8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working as described
Created: April 28, 2023
Last Modified: April 28, 2023
Protocol Integer ID: 81156
Funders Acknowledgement:
NIDDK
Grant ID: RC2 DK118640
NIDDK
Grant ID: F32 DK130267
Abstract
These protocols are an optimization of previous protocols and the original work described in Sato et al., 2009 which provided a foundation for the development of primary duodenoids, ileoids and colonoids from human samples. Here, we optimized methods specifically for use with samples from pediatric patients with genetic intestinal epithelial disorders. These protocols apply specifically to enteroids generated from endoscopic biopsies from patients with Congenital Diarrheas and Enteropathies (CoDE) or Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) or age-matched patients without any intestinal disease and include samples from the duodenum (duodenoids), ileum (ileoids), and colon (colonoids). The duodenoids, ileoids and colonoids from these patients exhibit a range of cellular phenotypes that potentially impact enteroid growth and maintenance including increased apoptosis, defects in proliferation, polarity and vesicular trafficking. These characteristics make it potentially challenging for long-term culture and expansion, limiting the availability of cells for functional experiments. Using a modified culture media, an expanded initial culture time after cell isolation from patient biopsies, and gentle passaging techniques, we were able to successfully culture and expand several patient lines over multiple passages and maintain the cultures for >5 years. The media conditions and protocols described here allow for reproducible phenotypes as well as scaling for larger functional studies on patient lines. These protocols also provide a useful starting point for further optimization for generating and culturing enteroids from patients with novel disease pathophysiology.
Attachments
Before start
Dissolve all reagents to the appropriate concentration according to manufacturer’s protocols. A * indicates that there were no modifications to the reagent.
Materials
| Key resources table | |||
| Reagent | Supplier | Catalog number | |
| TrypLE™ Express Enzyme (1X), phenol red | Thermo Fisher Scientific | Cat#12605010 | |
| Advanced DMEM/F12 | Gibco | Cat#12634-028 | |
| L-WRN Conditioned Media | ATCC (Cite Clevers) | CRL-3276 | |
| Wnt Conditioned Media | ATCC | CRL-2647 | |
| R-spondin-1 conditioned Media | n/a | Hans Clevers | |
| Noggin Conditioned Media | n/a | Hans Clevers | |
| Recombinant Noggin | Peperotech | ||
| Glutamax | Gibco | Cat#35050-061 | |
| HEPES | Gibco | Cat#15630-080 | |
| Primocin | Invivogen | Cat#Ant-pm-2 | |
| Normocin | Invivogen | Cat#Ant-nr-2 | |
| B27 | Gibco | Cat#12587010 | |
| N2 | Gibco | Cat#17502-048 | |
| Nicotinamide | Sigma-Aldrich | Cat#N0636 | |
| N-acetyl-cysteine | Sigma-Aldrich | Cat#A8199 | |
| A83-01 | Sigma-Aldrich | Cat#SML0788 | |
| SB202190 | Sigma-Aldrich | Cat#S7067 | |
| EGF | Peprotech | Cat#315-09 | |
| Gastrin | Sigma-Aldrich | Cat#G9145 | |
| Y27632 | Sigma-Aldrich | Cat#Y0503 | |
| Prostaglandin E2 | Sigma-Aldrich | Cat#P5640 | |
| CHIR99021 | Sigma-Aldrich | Cat#SML1046 | |
| Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mL | Corning | Cat#356231 | |
| Cultrex | R&D Technologies | BME001-10 | |
| Cultrex Organoid Harvesting Solution, 100 mL | R&D Technologies | 3700-100-01 | |
| Corning® Cell Recovery Solution, 100 mL | Corning | Cat#354253 | |
| Collagen IV from human placenta | Sigma-Aldrich | Cat#C5533 | |
| Collagenase, Type 1 | Stem Cell | Catalog # 07416 | |
| 6.5 mm Transwell with 0.4 μm pore polyester membrane insert, TC-treated, sterile, 48/cs | Corning | Cat#CLS3470-48EA | |
| 24 well plate | Costar | Cat#3526 |
Before start
Dissolve all reagents to the appropriate concentration according to manufacturer’s protocols. A * indicates that there were no modifications to the reagent.
Protocol references
Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., & Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262–265. https://doi.org/10.1038/nature07935