Primary colonic crypts were isolated from proximal colons of WKY and SHR rats with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 50 min, which were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) as described previously [17 (link),18 (link)].
Gentle cell dissociation reagents
Manufactured by STEMCELL
Gentle cell dissociation reagents are a set of specialized solutions designed to disassemble cell-cell and cell-extracellular matrix interactions, allowing for the gentle and efficient dissociation of various cell types from tissues or culture. These reagents are formulated to minimize disruption to cell surface proteins and maintain cell viability during the dissociation process.
Automatically generated - may contain errors
Lab products found in correlation
Organoid growth medium, by STEMCELL (3 mentions)
Matrigel, by BD (3 mentions)
Recombinant human noggin, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by BioLegend (2 mentions)
Y 27632, by STEMCELL (2 mentions)
R spondin 1, by R&D Systems (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Butyrate, by Merck Group (1 mentions)
3 protocols using gentle cell dissociation reagents
1
Isolation and Culture of Colonic Crypts
2
Colonic Organoid Culture and Minocycline Treatment
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The University of Florida Institutional Review Board approved the protocol for this human study (no. 201903360). We received the consent of 16 subjects with high systolic blood pressure (SBP) (156 ± 4 mmHg, high blood pressure, HBP) and 22 subjects with normal BP (110 ± 3 mmHg, normal blood pressure, RBP) for the study. Details of clinical characteristics of these subjects were described in our previous publication [3 (link)]. Within 30 min of the collection of biopsies by colonoscopy, human colonic crypts were isolated from the biopsies (~2 × 4 mm) of colon descending aspects in these subjects with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 90 min at 4 °C. The crypts were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) with recombinant human Noggin [Pepro-Tech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2; BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris], as described previously [3 (link)]. The human colonic organoids were cultured for 8 days, then treated with 1 mM minocycline for 24 h in the following experiments.
3
Isolation and Culture of Colonic Organoids
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Primary colonic crypts were isolated from descending aspects of the colons of REF and HBP subjects with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 90 min. They were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) with recombinant human Noggin [PeproTech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2; BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris], as described previously [17 (link),20 (link),55 (link)].
Colonic organoids were cultured for 8 days, then treated with 3.0 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the following experiments. Selection of the butyrate dose was based on our published data [17 (link)], demonstrating that a 3.0 mM dose was optimal for gene expression without affecting organoid growth [20 (link)]. This is consistent with doses of butyrate in colonic epithelial cells (2 mM) in regulation of the assembly of tight junctions [56 (link)], in Caco-2 cells (10 mM) for apoptosis [57 (link)] and its levels in the gut, portal, hepatic and venous blood [58 (link)].
Colonic organoids were cultured for 8 days, then treated with 3.0 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the following experiments. Selection of the butyrate dose was based on our published data [17 (link)], demonstrating that a 3.0 mM dose was optimal for gene expression without affecting organoid growth [20 (link)]. This is consistent with doses of butyrate in colonic epithelial cells (2 mM) in regulation of the assembly of tight junctions [56 (link)], in Caco-2 cells (10 mM) for apoptosis [57 (link)] and its levels in the gut, portal, hepatic and venous blood [58 (link)].
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