Initiation of culture.
Tissues should be regarded as potentially infected with hazard group 3 blood borne pathogens and should be handled with appropriate personal protective equipment.
1 Transfer placental samples into basic wash medium plus penicillin/streptomycin at room temperature and process within one hour.
2 Pre-warm 0.2% trypsin-250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma #E9884)/PBS to 37oc in a water bath.
3 Wash the placental tissue in basic culture medium with gentle stirring for >10 minutes to remove contaminating blood and other maternal uterine cells.
4 Tip into a petri dish. Transfer washed placental pieces with forceps to a clean dish.
5 Scrape villi from the chorionic membrane with a scalpel. Discard membrane.
6 Transfer villi to 50-70 ml pre-warmed 0.2% trypsin-250/0.02% EDTA in a bottle with a magnetic stirrer. Seal the lid and place in a heated shaker at 37 C with gentle shaking to digest for 3-5 min.
7 Filter the disaggregated cell suspension through sterile muslin gauze and wash through immediately with Hams F12/20% FBS to arrest trypsin digestion. (Retain the gauze with partially digested remnants).
8 Centrifuge the filtrate and re-suspend the cell pellet in Hams F12 medium.
9 Retrieve undigested tissue from the gauze and digest further with 10-15 ml collagenase V solution 1.0mg/ml in Hams F12/10% FBS with gentle shaking at 37°C, 5min.
10 Filter the resultant cell suspension and centrifuge to pellet cells as before.
11 Pool the cells from both digests and wash in Advanced DMEM/F12 medium.
12 Resuspend pellet in 1ml Advanced DMEM/F12 medium, transfer to 1.5ml Eppendorf tube and pellet again by centrifugation.
13 Remove the supernatant and estimate the volume of the pellet. Flick the tube to loosen the pellet and place on ice for 2-3 minutes. Add 10 x volume:volume of ice-cold Matrigel and pipette gently to mix. Place tube immediately back onto ice.
14 Plate 20-25μl drops of Matrigel/cell suspension into the centre of wells of Corning 48-well tissue culture plates. Place in the incubator to set for 15 minutes.
15 Overlay each drop with 250μl Trophoblast Organoid Medium (TOM).
16 Maintain cultures in 5% CO2 in a humidified incubator at 37°C. Replace medium every 2-3 days.
17 Examine cultures for the appearance of small organoid clusters. These are usually visible by ~7 days and continue to grow and self-organise. They can be passaged as below when at least 50% reach a diameter of 200-300 µm (usually between another 7-10 days).
1 Without removing the culture medium, using a 1ml pipette tip scrape backwards and forwards across the growth surface of each well to detach the Matrigel drop into the medium. Transfer the contents of the wells into 1.5ml Eppendorf tubes at a ratio of 4 wells per tube.
2 Centrifuge at 600g, 6 minutes to pellet. Remove the supernatant and add approximately 150μl Advanced DMEM F12 to each tube. Pipette up and down 400 times through a small bore pipette tip to break up the organoids and the Matrigel. Do not use excessive force. An electronic pipettor with a ‘mix’ function and its uptake volume set to 150µl is recommended for this step.
3 Add 1ml Advanced DMEM F12 and centrifuge at 600g, 6 min. Remove supernatant. Add 150ul Advanced DMEM F12 and pipette up and down manually a further 80 times using moderate force.
4 Add 1ml Advanced DMEM F12 and centrifuge at 600g, 6 min. Remove supernatant. The pellet at this stage should contain very little Matrigel. If Matrigel is still present, estimate the volume. Flick the tubes to re-suspend the pellets. Place tubes on ice.
5 Working one tube at a time, add ice-cold Matrigel, mix gently and replace the tube on ice. Allow 20-25µl Matrigel per well to be plated, remembering to subtract the estimated volume of any residual Matrigel which remained in the pellet. Pool organoids together and mix gently to distribute them evenly throughout the Matrigel.
6 Dispense 25µl drops carefully onto the centre of the growth area of wells of a 48-well culture plate, taking care not to create bubbles.
7 Place culture plate in the incubator for 15 min to allow Matrigel to set.
8 Add 250µl trophoblast Organoid Medium (TOM) to each well.
1 Place 20% DMSO freeze medium on ice to cool. Label cryovials.
2 Without removing the culture medium, using a 1ml pipette tip scrape backwards and forwards across the growth surface of each well to detach the Matrigel drop into the medium. Transfer the contents of the wells into 1.5ml Eppendorf tubes at a ratio of 3-4 wells per tube.
3 Centrifuge 600g, 6 minutes to pellet organoids.
4 Remove supernatant and add 200µl Advanced DMEM F12 to each tube. Manually pipette gently up and down 80 times with moderate force to partially disrupt organoids. Add 1ml Advanced DMEM F12 and centrifuge again.
5 Remove supernatant and flick pellets to re-suspend. Add 500ul TOM/40% FBS per tube and cool tubes on ice.
6 Slowly add 500ul cold 20% DMSO freeze medium per tube with gentle mixing.
7 Transfer contents to labelled cryovials on ice. Transfer immediately to ‘Mr. Frosty’ or similar cell freezing vessel at minus 800c overnight, then to liquid nitrogen for long-term storage.
Generation of EVT from trophoblast organoids
Differentiation was accomplished using a modification of the protocol used by Okae et al.4.
1 Passage organoids and re-plate in several Matrigel drops into ibidi μ-dishes.
2 Culture for 3-4 days in TOM.
3 Change medium to EVT medium #1. Culture for several days, replenishing medium every 2-3 days.
4 When adherent outgrowths begin to appear (typically 7-10 days) change medium to EVT medium #2.
5 Continue culturing in EVT medium #2 for a further 7-10 days or until sufficient numbers of adherent EVT have been generated.
6 Expression of HLA-G protein by immunofluorescence or flow cytometry can be used to verify differentiation to EVT.