Gibco™ DMEM, high glucose, GlutaMAX™ Supplement

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge	Seed different volumes of organoid units to assess survival rate and growth

Upstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge
Protocol tips
Seed different volumes of organoid units to assess survival rate and growth

Publication protocol

Intestinal Organoid Unit Isolation: The OU isolation process was adapted from our previously published protocol (Sala et al., 2011; Grant et al., 2015; Hou et al., 2018). Two-week-old C57BL/6 mice were euthanized in a CO2 chamber and the small intestine was collected and opened longitudinally. The resected tissue was vigorously washed five times with cold Hank's Balanced Salt Solution (HBSS, 14170-112, Life Technology) containing Antibiotic/Antimycotic (Anti/Anti, 15240062, Life Technology) to remove the intestinal content. The intestines were minced into 1 × 1 mm2 pieces, washed twice in cold HBSS/Anti/Anti, sedimented at 164 g (Eppendorf 5810R−1,000 rpm) between washes, and digested with type IV collagenase (800 units/ml, CLS-4, Worthington) and dispase (0.12 mg/ml, 17105-041, Gibco) in HBSS for 20 min at 37°C. The digestion was stopped with 10% fetal bovine serum (FBS, Invitrogen) in high-glucose Dulbecco's modified Eagle's medium (DMEM, 10566-016, Invitrogen), the sediment (pellet) was minced with a pipette and centrifuged at 41 g (Eppendorf 5810R−500 rpm) for 5 min to remove single cells. The resulting pellet containing the OU was resuspended in DMEM supplemented with 10% FBS, non-essential amino acids (NEAA, 11140-076, Life Technology) and Anti/Anti, centrifuged one last time at 105 g (Eppendorf 5810R−800 rpm) and the supernatant was removed.
The OU pellet was resuspended in four times the volume of DMEM/10% FBS/Anti/Anti/NEAA, mixed in equal parts with reduced growth factor Matrigel matrix (354230, Corning) and seeded onto adherent cell culture plates, forming a three-dimensional environment (3D) for OU growth. Two different experiments were carried out to assess the survival rate and growth of the OU. Forty microliters of the OU suspension in Matrigel matrix were plated onto 24-well plates for the survival rate experiment and 60 μL were plated onto 12-well plates for the growth experiments. The plates were incubated at 37°C for 20 min for Matrigel polymerization, and culture medium (DMEM/10% FBS/Anti/Anti/NEAA), containing rhRSPO1 (500 ng/mL) (Levin et al., 2020), or phosphate buffered saline (PBS) (control group), was added to each well. OU cultures were maintained in an incubator at 37°C and 5% CO2 with medium change every 2 days. Preliminary dose standardization of rhRSPO1 was performed by comparing two concentrations: 500 and 200 ng/mL (Supplementary Figure 1). To assess the survival rate of OU in culture, the number of OU found in four random fields per well, identified at the bottom of the well, was counted along the z axis every day over 6 days, under a light microscope. For each condition, the survival rate was indirectly calculated from the ratio between the mean number of OU on each day by the number of OU on the first day (OU number each day/OU number on Day 1). To assess OU growth in culture, eight random fields per well were imaged along the z axis under a Leica microscope (DMI6000B) on days 1, 3, and 6, and the cross-sectional area of each OU was determined with ImageJ software. The average area of all identified organoid units in the eight random fields selected in each well was considered a technical replicate. Three biological replicates, with three technical replicates per condition, were adopted for each experiment.


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