Publication protocol
Isolation of epithelial organoids and stromal cells: Endometrial glands and stromal cells were isolated by a modification of the method described by Laird et al. (1997). To remove blood and debris the tissue was first rinsed in culture medium consisting of phenol red-free DMEM/F12 (Gibco BRL) supplemented with 2% dextran-coated, charcoal-treated fetal calf serum (Gibco) and antibiotic/antimycotic agents as described above. The tissue was then minced finely with a sterile scalpel and after rinsing and removal of the medium by centrifugation at 50 g for 1 min, the tissue pieces were incubated in culture medium containing 0.1% collagenase A (Roche, Germany) for 1 h at 37 °C. To aid digestion, the tissue was gently pipetted several times during the incubation period. The digest was thereafter passed through a wire sieve to remove any undigested material and centrifuged at 250 g for 2 min. After resuspending the pellet in culture medium, glandular structures were separated from stromal cells by centrifugation at 50 g for 1 min. The red layer, mainly of vascular organoids on top of the white glandular sediment, was carefully removed by pipetting. The supernatant was collected and further centrifuged at 250 g for 2 min to pellet the stromal cells. The stromal pellet was freed of red blood cells by exposing it to 1 ml distilled water for not longer than 30 s, after which time 9 ml culture medium was added and the stromal cells were repelleted at 250 g. Further purification of the isolated glandular and stromal cells was done by gently pipetting the fractions, both suspended in 1 ml culture medium, onto 9 ml medium each and letting them sediment at unit gravity for 5 (epithelial glands) or 30 min (stromal cells). The procedure was repeated once or twice for organoids. Stromal cells were harvested from the top 9 ml and used for cell culture. The viability of the stromal cells thus obtained was between 80 and 95% as assessed by Trypan Blue exclusion. Only the sedimented epithelial glands were collected for organoid culture.
Organoid culture: Ten organoid/stromal cell co-cultures and in three cases parallel organoid cultures without stromal cells were prepared from freshly isolated epithelial organoids and stromal cells (Figure 1). The co-cultures always consisted of organoids and stromal cells from the same individual. The organoids were rinsed with serum-free medium and suspended in undiluted Matrigel basement membrane matrix (BD Biosciences, USA). Aliquots of 200 ml suspension were pipetted into pre-cooled tissue culture inserts (3 μm pore size, 10 mm diameter; Nunc, Denmark) and allowed to gel for 30 min at 37 °C. To prevent the organoids from sedimenting on the insert filter, Matrigel with sufficiently high protein content (>10 mg/ml) was chosen. Epithelial cells growing on the filter exhibited uncontrolled growth characteristics and were excluded from this study. Depending on the size of the sample, four or eight inserts were prepared from each tissue specimen. Stromal cells were plated into four wells of a 24-well tissue culture plate at a density of 30 000 viable cells/well in a volume of 500 μl culture medium. After gelling, the Matrigel-coated inserts were transferred into the wells containing stromal cells and 200 μl medium was added into the inserts. When sample size allowed, a parallel set of four inserts was prepared without stromal cells.
The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. After 2 days, the culture medium was renewed and after another 24 h the cultures were subjected to hormonal treatment. One insert of each set was removed and fixed in 4% paraformaldehyde to serve as the day 0 control. The other three were cultured for a further 7 days with 10 nmol/l 17β-E2 (Sigma) or 10 nmol/l E2 together with 100 nmol/l MPA (Sigma); the day 7 control insert received a similar amount of 100% ethanol used to dissolve the steroids. The medium was renewed every 2 days and fresh steroids were added. Seven days after treatment with hormones, the inserts were collected, fixed in 4% paraformaldehyde overnight and thereafter immersed in 70% ethanol. Under a stereomicroscope, the insert membrane was partially detached from its housing with an injection needle and the fixed Matrigel bed was carefully detached from the membrane. The gel was cut with a razor blade into four pieces, which were then closed into an embedding cassette and automatically infiltrated with paraffin (Tissue-Tek V.I.P.; Miles Inc., USA). The gel pieces were thereafter manually aligned with their cut sides up in a base mould and embedded in paraffin.
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