Primary Culture of Endometrial Cells

Once an endometrial scraping was obtained and stored in collection media, primary culture was employed to cultivate a population of endometrial epithelial cells which could then be placed in the chimera. Endometrial tissue was removed from ‘Sample Collection Medium’ and transferred to a 10cm culture dish where it was finely dissected using 2 sterile scalpel blades (Swann Morton) to increase surface area for enzymatic digestion until 1 mm3 pieces

61 were achieved. The sample was then transferred to a 30ml universal tube with Dulbecco’s Modified Eagle Medium (DMEM), washing the culture dish with DMEM ensure the entire sample was transferred. To collect the cells the sample was placed in a centrifuge (Sigma Aldrich 4K15) at 500 Relative Centrifugal Force (g) for 5 minutes. The supernatant was removed and the cells re-suspended in 4ml DMEM.

To achieve a single cell suspension enzymes and co-factors were added for an initial digestion:

 Collagenase (20 mg/ml, 500μl) (Sigma Aldrich)  Dispase (10 mg/ml, 500μl) (Roche)

 DNase (4 mg/ml, 100μl) (Invitrogen)  MgCl2 (50μl 100mM)

The cells were transferred to a shaking water bath at 37ᵒC for 1.5 hours.

2.2.1 Crude Fractionation of Epithelial and Stroma Cell Populations

After initial incubation the resulting digest was filtered through a 40 μm cell strainer into a 50ml test tube. The flow-through was washed with DMEM and represented the stromal population. The retentate represented the epithelial population and was transferred to a second 50ml test tube by backwashing with 30-40ml DMEM. Gentle pipetting was used to dislodge adherent cells.

Epithelial Fraction Culture

The Epithelial Fraction (EF) was centrifuged at 500g for 5 minutes to collect the cells after which the supernatant was discarded and the EF re-suspended in 1ml DMEM with further enzyme added to digest the intact epithelial glands:

 Trypsin (1ml 0.25%) (Sigma Aldrich)  DNase (4mg/ml, 100μl)

The EF was then incubated at 37ᵒC for 20 minutes in the shaking water bath. After this final digestion the EF was gently pipetted for 3 to 5 minutes in order to liberate single cells after which the Trypsin was inactivated with 1 ml DMEM. To remove the Trypsin the EF was then centrifuged at 500g for 5 minutes to pellet cells with the supernatant being discarded. The EF was then re-suspended in 10ml ‘Endometrial Culturing Medium’ and plated onto a 10 cm

62 culture dish. The composition of ‘Endometrial Culturing Medium’ is included in the Appendix Section 6.7.

Stromal Fraction Culture

The stromal fraction did not require further enzymatic digestion and subsequently could be centrifuged to discard the Trypsin containing medium and re-suspended in ‘Endometrial Culturing Medium’ before plating onto a 10cm culturing dish.

Selective Adherence

The stromal population would adhere preferentially to the 10cm culturing dish over the epithelial population so after 15-20 minutes the fraction was re-plated to increase isolation of the two individual populations.

2.2.2 Vybrant Labeling of Human Endometrial Cells

In order to identify the human endometrial cells after incorporation into the neonatal mouse kidney chimera they were first labeled using the ‘Vybrant CFDA SE Cell Tracer Kit’ (Invitrogen) which consisted of Carboxy fluorescein diacetate, succinimidyl ester (CFDA SE) and Dimethylsulfoxide (DMSO). This kit marked the cells with green fluorescence, allowing investigators to validate the human specific antibodies used in the study. The protocol was followed according to the manufacturer’s instructions.

The CFDA SE was allowed to warm to room temperature after which a 10mM stock solution was prepared by adding 90ul of DMSO to one vial of 500μg CFDA SE. This solution was then diluted with Phosphate Buffered Saline (PBS) (Sigma Aldrich) to give a final concentration of 20μM CFDA SE.

Labeling Human Epithelial Cells

To label the human cells with Vybrant, the ‘Endometrial Culture Medium’ was removed from the a 10cm culture dish containing adherent cells and replaced with 6ml of a 20μM CFDA SE solution pre-warmed to room temperature. The dishes were then returned to the incubator for 15 minutes at 37ᵒC after which the CFDA SE solution was removed and replaced with pre-warmed Endometrial Culture Medium’ and incubated for a further 30 minutes at 37ᵒC. During this time the CFDA SE underwent acetate hydrolysis after which the fluorescent signal could be observed within the cells.

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2.2.3 Trypsinisation of Human Endometrial Cells

In order to introduce the human endometrial cells into the chimera they first needed to be removed from the culture dishes to which they adhered. To achieve this, the cells were washed twice with 10ml pre-warmed PBS before 10ml 1× Trypsin was added. The plate was then incubated for 10 minutes at 37°C during which time the cells detached from the dish. After incubation the Trypsin was inactivated using 10ml DMEM with 10% Fetal Bovine Serum (FBS) and the cells in suspension were transferred to a 15ml conical tube.

A ratio of 1 in 5 human to mouse cells was used for the study. In order to calculate the volume of human endometrial cell suspension to introduce to the chimera the live cells were counted using an automated cell counter (Biorad) and Trypan Blue (Sigma Aldrich).To count the live cells 10μl of cell suspension was transferred to a cell counting plate and 10μl Trypan Blue added. The two solutions were mixed and 10μl transferred to a haemocytometer which was placed in an automated cell counter.