title: scrEXT337
organism: Mus musculus
description: Protocols: Tooth assembloids (2 replicates per condition) were dissociated into single cells using 0.25% Trypsin (Gibco; supplemented with 5M ROCKi) treatment and mechanical trituration and collected in 0.04% BSA (in PBS) using a Flowmi 40 um cell strainer (Sigma-Aldrich). Tooth assembloids were established using a modified protocol as described previously (Hemeryck et al., 2022). Briefly, after dissociating P2-3 organoids and molar DPSCs into single cells, both cell types were combined in round-bottom low-attachment plates (96-well; Greiner). Using a layered approach, first 5,000 DPSCs were seeded by sedimentation (300 g for 1 min at 4 °C), followed by 5,000 molar tooth organoid- or incisor tooth organoid-derived cells (300 g for 1 min at 4 °C) (Nakao et al., 2007). The cells were provided with 10% growth-factor reduced Matrigel and 90% of either a 1:1 mixture of tooth organoid medium (TOM) + pulp medium (PM) or differentiation medium (DM) + pulp differentiation medium (PDM) (see 'treatment protocol' for medium compositions). After 24 h incubation at 37 °C and 5% CO2, the formed structures were collected and plated in 20 L droplets of Matrigel (70%) to generate tooth assembloids. Assembloids were maintained in the 1:1 media mixtures at 37 °C and 5% CO2. Assembloids were maintained for 6 days in 1:1 media mixtures of TOM/PM or DM/PDM before collection. TOM consisted of serum-free defined medium (SFDM; DMEM/F12 1:1, 5 mg/l human transferrin, 5mg/l insulin Hybrimax from bovine pancreas, 35mg/L penicillin, 50mg/L streptomycin, 600uL/L ethanol, 50µl/L catalase, 1g/L NaHCO3, 0.5% BSA) supplemented with 0.5 uM A83-01, 1X B27, 100 ng/mL cholera toxin, 20 ng/mL bFGF, 200 ng/mL FGF8, 100 ng/mL FGF10, 2 mM L-glutamine, 100 ng/mL IGF1, 1X N2, 1.25 mM N-acetyl L-cysteine, 10 mM Nicotinamide, 100 ng/mL noggin, 200 ng/mL RSPO1, 10 uM SB202190, 100 ng/mL SHH, 200 ng/mL WNT3A and 20 ng/mL EGF. PM consisted of α-MEM supplemented with 10% FBS, 103 units/mL ESGRO recombinant LIF protein, 2.5 ng/mL bFGF and 1% penicillin-streptomycin. DM consisted of SFDM supplemented with 1X B27, 100 ng/mL BMP2, 50 ng/mL BMP4, 20 ng/mL EGF, 20 ng/mL bFGF and 4 ng/mL TGF-beta1. PDM consisted of PM supplemented with 100 uM ascorbic acid, 5 mM beta-glycerophosphate and 10 uM dexamethasone. Single cell suspensions were converted to barcoded Drop-seq libraries by using the Chromium Single Cell 3' V2 Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 5,000 cells per library. Single cell libraries were prepared with the Chromium Single Cell 3' V2 Chemistry Library Kit, Gel Bead & Multiplex Kit and Chip Kit from 10X Genomics.
attributes.tag: disease
attributes.value: normal
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: organism
attributes.value: Mus musculus
attributes.tag: cohort
attributes.value: IncisorAssembloid_Control
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: common name
attributes.value: house mouse
attributes.tag: organism part
attributes.value: incisor tooth
attributes.tag: developmental stage
attributes.value: juvenile stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-19
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-19
attributes.tag: growth condition
attributes.value: mixed tooth organoid medium and pulp medium
attributes.tag: cell_type
attributes.value: mixed incisor organoid derived cell and dental pulp cell
attributes.tag: age
attributes.value: 7
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-19
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-19