Homo sapiens
Protocols: Cells were dissociated as described in section 2.4, but with a prolonged dissociation time of 8 minutes to ensure a fully dissociated cell population. Cells were resuspended in dPBS with 1% (w/v) BSA to prevent intercellular adhesion. Cells were counted within the cell culture facility, using an automated counter, and resuspended at 1 million cells mL-1. A second count was performed within the DeepSeq facility as described below. All subsequent steps were performed with the kind collaboration of Dr Nadine Holmes, Miss Victoria Wright and other members of the DeepSeq facility, and a summary is given below. Organoid culture was performed in either 24-well plate Transwell® inserts using a mixture of Matrigel® and neutralised collagen (1:1) or with drops of Matrigel® formed on a plate. For both methods, 150,000 posterior endoderm differentiated iPSCs were added in a small volume (up to 10% of total volume) of DMEM/F12 medium to cooled hydrogel solution. After rapid, but careful mixing, to avoid both solidification and air bubble formation, the hydrogel/cell mixture was transferred to the Transwell® insert (500 μL) or onto the surface of a well on a 6-well plate (100 μL – 2 drops per well). The 6-well plates containing Matrigel® drops were inverted and incubated for 2 hours at standard cell culture conditions, while plates with Transwell® inserts were incubated at normal orientation for 2 hours before adding culture medium.  Culture medium consisted of basal maturation medium (as described above) with the growth factors EGF 50 ng mL-1, R-spondin 50 ng mL-1 and noggin 100 ng mL-1, and ROCKi at 10 μM. The same media constituents were used throughout culture, except for ROCKi, which was not used in subsequent media changes. Media were changed every 3-4 days, and organoids were passaged into new Matrigel® drops every 10 days. For passage, Matrigel® drops were overlaid with 800 μL Gentle Cell Dissociation reagent per well (StemCell Technologies, Vancouver, Canada) and incubated for 5 minutes. A micropipette was used to disrupt each Matrigel® drop by repeatedly pipetting the drop up and down until disaggregated. The contents of each well were transferred to a 1.5 mL microtube (one per well). The well was washed with 400 μL of dissociation reagent which was then added to the microtube. The organoids were incubated for a further 10 minutes at room temperature, before centrifugation at 5 °C (300g, 5 minutes). Supernatant was discarded and organoids were washed with 1 mL cold culture medium and centrifuged under the same conditions again. The supernatant was discarded and organoids resuspended in thawed 400 μL Matrigel® at 4 °C. New organoid drops were then formed in 6 well plates as described above. At the time of passage, ROCKi was supplemented in medium.  Matrigel® drops were gently disaggregated by incubation in cold PBS followed by tituration. Organoids were then embedded in agarose, using similar principles to those described above and analysed using histological techniques. Single cell 3' whole transcriptome sequencing libraries were prepared from dissociated cell suspensions using the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1, the Chromium Next GEM Chip G Single Cell Kit and the Dual Index Kit TT Set A (10X Genomics; PN-1000147, PN-1000127 and PN-1000215). Cell counts and viability estimates were obtained using the LUNA-II Automated Cell Counter (Logos Biosystems), Trypan Blue Stain, 0.4 % (w/v) and Luna Cell Counting Slides (Logos Biosystems; T13001 and L12001). Live cell counts were used to calculate cell input, rather than total cell count, as visual inspection of cell field on the LUNA II and the gating histogram, showed that > 90% of cells were viable and that the cell counter appeared to be counting some extracellular debris as non-viable cells. The number of input cells targeted was 3,300 cells per sample, with the aim of generating sequencing libraries from ∼ 2,000 single cells. All steps, including GEM Generation and Barcoding, Post GEM-RT Cleanup and cDNA Amplification and Library Construction were performed according to the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 User Guide, Rev B (CG000315). Variable steps of this protocol included using 12 cycles of cDNA amplification and 8-12 cycles of library amplification. Amplified cDNA was quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific; Q32854) and fragment length profiles were assessed using the Agilent 4200 TapeStation and Agilent High Sensitivity D5000 ScreenTape Assay (Agilent; 5067-5592 and 5067-5593).  Completed sequencing libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit and fragment length distributions assessed using the Agilent 4200 TapeStation and the High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584, 5067-5585). Single cell 3' whole transcriptome sequencing libraries were prepared from dissociated cell suspensions using the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1, the Chromium Next GEM Chip G Single Cell Kit and the Dual Index Kit TT Set A (10X Genomics; PN-1000147, PN-1000127 and PN-1000215). Cell counts and viability estimates were obtained using the LUNA-II Automated Cell Counter (Logos Biosystems), Trypan Blue Stain, 0.4 % (w/v) and Luna Cell Counting Slides (Logos Biosystems; T13001 and L12001). Live cell counts were used to calculate cell input, rather than total cell count, as visual inspection of cell field on the LUNA II and the gating histogram, showed that > 90% of cells were viable and that the cell counter appeared to be counting some extracellular debris as non-viable cells. The number of input cells targeted was 3,300 cells per sample, with the aim of generating sequencing libraries from ∼ 2,000 single cells. All steps, including GEM Generation and Barcoding, Post GEM-RT Cleanup and cDNA Amplification and Library Construction were performed according to the Chromium Next GEM Single Cell 3' Library and Gel Bead Kit v3.1 User Guide, Rev B (CG000315). Variable steps of this protocol included using 12 cycles of cDNA amplification and 8-12 cycles of library amplification. Amplified cDNA was quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific; Q32854) and fragment length profiles were assessed using the Agilent 4200 TapeStation and Agilent High Sensitivity D5000 ScreenTape Assay (Agilent; 5067-5592 and 5067-5593).  Completed sequencing libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit and fragment length distributions assessed using the Agilent 4200 TapeStation and the High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584, 5067-5585).
isolate
not applicable
organism
Homo sapiens
organism
Homo sapiens
growth condition
day 8, EpCAM-
cell_line
organoid
collection date
not collected
scientific_name
Homo sapiens
common name
human
cell_type
mesenchymal stem cell
geographic location (country and/or sea)
not collected