All donated embryos in this study were surplus frozen embryos from couples who already had at least a healthy baby after IVF clinic treatment. The informed consent process for embryo donation compiled with International Society for Stem Cell Research (ISSCR) Guidelines for Stem Cell Research and Clinical Translation (2016) and Ethical Guidelines for Human Embryonic Stem Cell research (2003) jointly issued by the Ministry of Science and Technology and the Ministry of Health of People’s Republic of China.
Embryo thawing and zona pellucida removal
1. Before embryo thawing, human embryo culture medium G-2 was equilibrated in a
4-well plate overnight. 0.5 ml of G-2 medium was added to the well, and then 0.25 ml of mineral oil was used to cover the G2 medium.
Note: The G-2 medium must be pre-equilibrated in the incubator overnight or for at least 6 hours.
2. Human blastocysts (day 5 or 6 post-fertilization, d.p.f 5-6) were thawed using a Kitazato Thawing Media Kit by strictly following the manufacturer’s instructions.
3. After culturing in drops of equilibrated G-2 medium for 4 hours, the embryos were transferred to acidic Tyrode’s solution to remove the zona pellucida.
Note: The Pasteur's pipette diameter should be larger than the embryo diameter to avoid damaging the embryos. No more than two embryos were treated in the acidic Tyrode’s solution each time. Once the zona pellucida vanished, the embryos were immediately transferred to G-2 medium to avoid damage to the embryos by the acidic Tyrode’s solution.
4. Embryos were washed in G-2 medium for two times and then transferred to in vitro culture medium.
Evaluation of embryo quality
According to the Gardner’s scoring system6, thawed blastocysts were given numerical scores from 1 to 6 based on their expansion degree and hatching status. The blastocyst with expansion and hatching status above 3 and with visible inner cell mass above grade B were included in the study. Based on morphologies, normal embryos had to meet the two following requirements: obvious expansion during culturing and absence of obviously dead or broken (fragmented) cell mass during development. Otherwise, they were excluded in this study.
In vitro three-dimensional culture of human embryos
1. The culture conditions were as follows: 37.2℃, 6% CO2 and saturated humidity
2. 150 µL/well mIVC1 was added to a low-attachment 96-well plate (3474, Corning) and the culture medium was pre-equilibrated for at least six hours
3. The day 5-6 embryos without zona pellucida were washed in pre- equilibrated mIVC1 three times and then transferred to a new well for each embryo
Note: Embryos should be washed at least 3 times in mIVC1 to remove the G-2 residue.
4. On day 6-8 post-fertilization (d.p.f 6-8), the culture medium was mIVC1
5. At d.p.f 8, 50% of mIVC1 medium was replaced by mIVC2
6. At d.p.f 9, the embryos were transferred to new wells in mIVC2 including 10% Matrigel
Note: Matrigel should be thawed at 4℃, and all operations should be performed on ice.
7. Afterwards, 50% of the culture medium was replaced with new mIVC2 including 10% Matrigel every day.
Note: The time that the embryos are outside of the incubator should be minimized when replacing medium or taking photos. mIVC1 and mIVC2 should be pre-equilibrated in the incubator for at least six hours before use.
Embryo frozen section
1. Embryos were fixed with 4% PFA for 40 mins in room temperature
2. Embryos were washed three times in PBS in room temperature
3. Dehydrated with 15% sucrose for 1 min in room temperature
Note: The dehydration time can be extended appropriately if the sample does not sink
4. Embryos were embedded in a plastic mold with O.C.T.
Note: Embryos should be washed in O.C.T three times before transferred to the mold
5. The embedded embryos were sectioned by a Leica frozen slicer.
6. The slices were baked on a heating plate for 40 mins at 45 degree centigrade
7. The slices were stored in -20 refrigerator
Embryo section staining and taking photos
1. Embryo sections were placed in dark box and washed with PBS three times
2. Samples were permeabilized by 0.2% Triton X-100 for 30 minutes at room temperature
3. Wash with PBS one time
4. Samples were blocked by 3% BSA for 4 hours at room temperature
5. Wash with PBS one time
6. Add primary antibodies with 1% BSA
7. Incubate primary antibodies overnight in a 4℃ refrigerator.
8. Samples were washed with 0.05% Tween-20 in PBS for three times, 3-5mins per time
9. Add secondary antibodies with PBS for 2 hours at room temperature
10. Samples were washed with 0.05% Tween-20 in PBS for three times, 3-5 mins per time
11. Cover slips were covered with 50% glycerine
12. Taking photos by Leica SP8
Note: Samples must be kept moist all the time and without drying out
Whole embryo staining and taking photos
1. Embryos were fixed with 4% PFA in a normal 96-well plate, 200 µL/well, 40 mins in room temperature
2. Embryos were transferred to a new well with 200µL PBS
3. Embryos were transferred to a new well with 200µL 0.5% Triton X-100 for 2 hours
in room temperature
4. Embryos were transferred to a new well with 200µL 3% BSA for 4 hours in room temperature
5. Embryos were transferred to a new well with 100µL primary antibody diluted by
6. Incubate primary antibodies overnight in a 4℃ refrigerator
7. Embryos were washed with 0.05% Tween-20 in PBS for three times, 15 mins per time
8. Embryos were transferred to a new well with 100µL secondary antibody diluted by
9. Embryos were washed with 0.05% Tween-20 in PBS for three times, 15 mins per time
10. Embryos were washed with 50% glycerine and transferred to 8-well IbiTreat µ plates to take photos
11. To make the 3D videos, embryos were mounted in on aqueous solution of 80% glycerol, and a multi-photon microscope (Leica TCS SP8 DIVE) was used to take photos.
Isolation of single cells
1. Three drops of 30 µL 0.25% trypsin were added on a 35mm petri dish (Corning, 351008), 3 mL mineral oil was covered on the trypsin for each dish
2. The dishes were incubated in a 37℃ incubator for 30 mins
3. Embryos were washed in PBS 3 times
Note: Wash the embryo at least 3 times to remove the residue of medium
4. Embryos were washed in 0.25% trypsin drops for 2 times and then transferred to the third drop of trypsin
5. Embryos were incubated in a 7℃ incubator for 15 mins
6. 2 µL DFBS was added to the third drop to terminate the digestion
7. Each embryo was transferred to a drop of G-MOPS in 35mm petri dish covered by mineral oil in room temperature
8. Use different diameters of Pasture pipette to blow the embryo into single cell
9. Each single cell was washed in DPBS
10. Each single cell was pipetted into a PCR tube with 2 µL lysis solution