Dataset Information

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

ABSTRACT:

Background & aims

Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.

Methods

Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors.

Results

In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures.

Conclusion

Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

SUBMITTER: Wilson SS

PROVIDER: S-EPMC7906999 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Wilson Sarah S SS Mayo Martha M Melim Terry T Knight Heather H Patnaude Lori L Wu Xiaoming X Phillips Lucy L Westmoreland Susan S Dunstan Robert R Fiebiger Edda E Terrillon Sonia S

Frontiers in immunology 20210212

<h4>Background & aims</h4>Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids.<h4>Methods</h4>Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by qua ...[more]

PMID: 33643277

Similar Datasets

Project description:Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560??m (Elp400), 500/700??m (Elp500), and 900/700??m (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Project description:BackgroundOrganoids, which enable disease modeling and drug screening closer to an in vivo environment, can be isolated and grown from organs such as the brain, small intestine, kidney, lungs, and liver. To facilitate the establishment of liver and small intestinal organoids, we developed efficient protocols for cholangiocytes and intestine crypts collecting and organoid culturing.MethodsCholangiocytes were collected from intrahepatic bile ducts, the gallbladder, and small intestine crypts by gravity settling and multistep centrifugation methods. The cells isolated were embedded with Matrigel and grew in three-dimensional spheroids in a suitable culture medium. The stability of organoid cells was assessed by subculture, cryopreservation, and thawing. RNA and DNA extraction of organoids, as well as immunostaining procedure, were also optimized. Hand-picking procedures were developed and performed to ensure similar growth characteristics of organoids.ResultsA large number of cholangiocytes and small intestine crypts were collected under these protocols. Cholangiocytes developed into cyst-like structures after 3-4 days in Matrigel. After 1-2 weeks of cultivation, small intestinal organoids (in-orgs) developed buds and formed a mature structure. Compared to organoids derived from the gallbladder, cholangiocyte organoids (Cho-orgs) from intrahepatic the bile ducts grew more slowly but had a longer culture term, expressed the cholangiocytes markers Krt19 and Krt7, and recapitulated in vivo tissue organization.ConclusionsOur protocols simplified the cell collection procedure and avoided the possibility of exposing tissue-derived stem cells to mechanical damage or chemical injury by gravity settling and multistep centrifugation. In addition, our approach allowed similar growth characteristics of organoids from different mammalian tissue sources. The protocol requires 2-4 weeks to establish a stable organoid growth system. Organoids could be stably passaged, cryopreserved, and recovered under protocol guidance. Besides, the organoids of cholangiocytes and small intestines retained their original tissue characteristics, such as tissue-specific marker expression, which prepares them for further experiments such as preclinical in vitro trials and mechanism research studies.

Dataset Information

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Background & aims

Methods

Results

Conclusion

Publications

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets