Blastocyst Culture for Deriving Human Embryonic Stem Cells

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Blastocyst Culture for Deriving Human
Embryonic Stem Cells
Ariff Bongso
Summary
Success in producing a human embryonic stem cell line depends largely on the quality of
the 5-d-old embryo (blastocyst) used. Such blastocysts are grown from frozen-thawed d 1–3
surplus embryos left over from infertility clinics and donated for stem cell research with
informed patient consent. Knowledge, therefore, of the culture conditions and embryo scoring
systems that are used to generate high-quality blastocysts are crucial. This chapter describes
an extended microdroplet culture protocol using two-stage sequential culture media with morphological and polarized optical parameters for embryo scoring for each day of embryonic
growth to help produce high-quality blastocysts.
Key Words: Blastocyst culture; embryo scoring systems; human embryonic stem cell
lines.
1. Introduction
The chances of success of producing a human embryonic stem (hES) cell line
depends largely on the quality of 5-d-old embryos (blastocysts) used. Embryos
provided for this purpose are usually surplus embryos left over from in vitro
fertilization clinics and frozen at cleavage stages (two pronuclear, four-cell and
eight-cell stages) on d 1–3 postinsemination. Institutional Review Board
approval and informed patient consent must be sought before such donated
embryos are used for hES cell derivation. There also appears to be a strong
correlation between the stability of the ensuing hES cell line and the quality of
the embryo used to derive it. Knowledge, therefore, of the various markers used
for the daily scoring of embryos from fertilization to blastocyst formation is crucial to select the best quality embryo with the highest cumulative score for hES
cell derivation. Morphological characteristics using polarized parameters (1) and
From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols
Edited by: K. Turksen © Humana Press Inc., Totowa, NJ
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Table 1
Embryonic Stages With Highest Scores Observed d 2–6a
Day
2 (AM)
3 (AM)
Embryonic stage
4 (AM)
Cleavage stage
Cleavage stage
Compacting
Compacting
Compacted
Early cavitating
Late cavitating
5 (AM)
Early blastocyst
5 (PM)
Fully expanded
blastocyst
Hatching blastocyst
6 (AM)
aICM,
Description
4–6 cells, regular blastomeres, no fragments
8 cells, regular blastomeres, no fragments
Blastomeres fusing
Blastomeres fusing
Blastomeres fused
First signs of blastocoele
Distinct blastocoele
ICM and TE not laid down
Distinct ICM, TE, and blastocoele
Embryo diameter same as d 4 or slightly larger
Distinct ICM, TE, blastocoele. Thin zona pellucida
Fully expanded diameter (~ 215 µm)
ICM and TE completely hatched out from zona
pellucida. Empty zona pellucida
inner cell mass; TE, trophectoderm.
cleavage speed are the best markers correlating with quality of embryos (2,3).
The following are the most reliable descriptions of markers used for each day of
human embryonic growth in vitro (Table 1).
1.1. Day 1 (Two Pronuclear and First Cleavage)
Hoffman’s inverted optics are used to determine that normal fertilization
has taken place by visualizing the presence of two distinct pronuclei; monoor multipronuclear embryos also divide to the blastocyst stage and may thus
have the potential of producing hES cell lines (parthenogenotes and
polyploids). There is a strong correlation between two pronuclear (2PN)
scoring and blastocyst development (4,5). The markers for the best embryos
on d 1 include (1) the abutment and proximity of the two pronuclei, (2) the
alignment in vertical rows of the nucleoli of each pronucleus, (3) the appearance of a cytoplasmic clear halo around the pronuclei, and (4) the early
appearance of the first cleavage (two-cell stage) (6) usually at 24–25 h
postinsemination (Fig. 1A).
1.2. Day 2 (Four–Six Cell Stage)
Day 2 cleavage stage embryos (four- to six-cell stage) are usually scored
according to three grades (grades 1, 2, and 3) based on (1) the size and regularity
of the blastomeres and (2) the presence or absence of cytoplasmic fragments
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Fig. 1. Post-thawed d 1–3 human embryos. (A) 2PN (d 1, AM) embryo showing
pronuclear abutment and nucleoli aligned in vertical rows. (B) Five-cell Grade 1 (d 2, AM)
embryo with five regular-size blastomeres and no fragments. (C) Eight-cell (d 3, AM)
embryo with eight regular-size blastomeres and no fragments. (D) Good-quality d
3 (AM) embryo showing compacting blastomeres and no fragments (×200). (Please see
the companion CD for the color version of this figure.)
(Fig. 1B). Grade 1 embryos have regular size blastomeres, no cytoplasmic
fragments, and receive the highest score. Grade 2 embryos have regular size
blastomeres and a moderate number of cytoplasmic fragments, whereas grade
3 embryos have irregular blastomeres and many cytoplasmic fragments.
1.3. Day 3 (Eight Cell to Compacting Stages)
On the morning of d 3, embryos are observed for compaction as evidence of
activation of the genome and the bypassing of the in vitro embryonic block
between d 2 and 3. Embryos that have commenced compacting and have no
cytoplasmic fragments (Figs. 1D and 2A) receive the highest scores, whereas
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Fig. 2. Post-thawed d 3–5 human embryos. (A) Compacting embryo (d 3, AM).
(B) Fully compacted embryo (d 4, AM). (C) Early cavitating embryo (d 4, AM). (D) Late
cavitating embryo (d 4, AM). (E) Early blastocyst (d 5, AM) showing peripheral trophectoderm and inner ICM. Note increase in embryo diameter from cavitating stage in D.
(F) Fully expanded blastocyst (d 5, PM). Note further increase in embryo diameter from
early blastocyst stage in E (×200). (Please see the companion CD for the color version
of this figure.)
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embryos with eight equal-size blastomeres and no cytoplasmic fragments
(Fig. 1C) receive the second highest scores. Poor-quality embryos have many
fragments and lesser blastomeres.
1.4. Day 4 (Compacted to Cavitating Stages)
On the morning of d 4, embryos are scored for complete compaction and the
appearance of an early blastocoele (early cavitation). Fully compacted (Fig. 2B)
and early and late cavitating (Fig. 2C,D) embryos receive the highest scores,
whereas those embryos with slow cleavage, no compaction, and no signs of a
blastocoelic cavity receive low scores.
1.5. Day 5 (Blastocyst Stages)
On the morning of d 5, embryos are expected to have reached the blastocyst
stage. An embryo is defined as a blastocyst if it has a clearly laid down inner cell
mass (ICM), a trophectoderm, and a single blastocoelic cavity. Three categories
of blastocyst can be recognized: (1) early (Fig. 2E), (2) expanding, and (3) fully
expanded (Fig. 2F). The main differences between the three categories are
increases in diameter and thinning of the zona pellucida. Usually on the morning of d 5, only early blastocysts and late cavitating stages are observed, and
these receive high scores. Expanding and fully expanded blastocysts are commonly seen on the afternoon of d 5 or morning of d 6. The graduated embryo
score (7) or highest cumulative score from 2PN to blastocyst stage is used to
select the best blastocyst for hES cell derivation with a high hit rate. The reliable
markers for good quality expanded blastocysts that are used for hES cell derivation are shown in Table 2. Approximately 70–75% of high-quality blastocysts
are usually obtained when good-quality sequential culture media are used.
2. Materials
2.1. Thawing Materials for Frozen Cleavage Stage Embryos (Days 1–3)
1. THAW-KIT 1 (Vitrolife, Goteburg, Sweden; cat. no. 10067). Thaw solution 1
(TS1): 1.0 M PrOH and 0.2 M sucrose in Cryo-phosphate buffer solution (PBS
containing 25 mg/mL human serum albumin [HSA]). Thaw solution 2 (TS2);
0.5 M PrOH and 0.2 M sucrose in Cryo-PBS. Thaw solution 3 (TS3): 0.2 M
sucrose in Cryo-PBS.
2. GIII series medium (Vitrolife G-1 version 3; cat. no. 10091 and G-2 version 3;
cat. no. 10092).
3. HSA solution (Vitrolife; cat. no. 10064).
2.2. Thawing Materials for Frozen Blastocysts (Days 5 and 6)
1. G-ThawKit Blast (Vitrolife; cat. no. 10076).
2. 8, 6, 5, 4, 3, 2, 1% glycerol. Incubation medium (G-2 version 3 and HSA medium,
Vitrolife; cat. no. 10092).
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Table 2
Markers for Good-Quality, Expanded Blastocystsa
Morphological characteristics
Large ICM
Thin zona pellucida
Single, large blastocoele
“Sickle-shape” cells in TE
Cleavage speed
Cavitated, expanding —d 5 (AM)
Fully expanded—d 5 (PM)
Hatching/hatched —d 6 (AM)
aICM,
inner cell mass; TE, trophectoderm.
3. Four-well sterile plastic dishes (Nunclon, Roskilde, Denmark; cat. no. 176740).
2.3. Embryo Culture (Days 1–6)
1.
2.
3.
4.
5.
G-1 version 3 medium (Vitrolife; cat. no. 10091).
G-2 version 3 medium (Vitrolife; cat. no. 10092).
HSA solution (Vitrolife; cat. no. 10064).
Ovoil (Vitrolife; cat. no. 10029).
Four-well sterile plastic dishes (Nunclon; cat. no. 176740).
3. Methods
3.1. Thawing Protocol for Frozen Cleavage Stage Embryos (Days 1–3)
1. Prepare all paperwork. Identify name of patient and location of straw in liquid
nitrogen (LN2) Dewar. Keep the straws immersed in LN2 until actual thawing.
Label dishes for thaw solutions and pipette appropriate amounts of TS1, TS2, TS3,
and Cryo-PBS. Thaw straws containing embryos one at a time. Perform all steps
at room temperature.
2. Remove straw and air thaw for 30 s (see Note 1).
3. Place straw in a 37°C water bath for 30 s. Remove and carefully wipe. Cut the plug
end of the straw with sterile scissors and attach it to a 1-mL syringe. Then cut the
other end carefully without shaking straw or making air bubbles.
4. Gently expel the embryos into TS1. Observe the embryos coming out of the straw;
if you do not see them all, quickly refill the straw and gently flush, because
occasionally the embryos will stick to the sides of the straw.
5. Incubate the embryos for 5 min in TS1.
6. Gently transfer the embryos to TS2 for 5 min (see Note 2).
7. Transfer the embryos to TS3 for 5–10 min (see Note 3).
8. Transfer the embryos to Cryo-PBS for 5–10 min (see Note 4).
9. Place embryos into G-1 version 3 and HSA medium if embryos were cryopreserved
on d 1 or 2 and in G-2 version 3 and HSA medium if cryopreserved at the eight-cell
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stage on d 3, and incubate at 37°C in a 5% CO2 in air atmosphere (see Note 5).
Assess and record survival and morphology compared to prefreeze. Score embryos
according to markers described earlier for d 1–3 (see Subheadings 1.1.–1.3.).
10. Human embryos are cultured in G-1 version 3 and HSA medium on d 1 and 2 and
in G-2 version 3 and HSA medium on d 3–6. The medium is changed every 48 h.
Each embryo is monitored and scored daily in the morning. See embryo culture
protocol described in Subheading 3.3.
3.2. Thawing Protocol for Frozen Blastocysts (Days 5 and 6)
1. Label each well of four-well dishes as 8, 6, 5, 4, 3, 2, 1% glycerol, and two wells
as incubation medium.
2. Pipet appropriate volumes (0.5 mL) of the thawing solutions into each well.
Prepare also a separate dish with incubation medium.
3. Equilibrate the dishes at 37°C in a 5% CO2 in air atmosphere for 1 h.
4. Fill a large beaker with warm water (37°C). Identify name of patient and location
of straw in LN2 Dewar. Prepare all paperwork. Keep the straws immersed in LN2
until actual thawing. Thaw straws containing blastocysts one at a time and
perform all steps at 37°C.
5. Remove straws from LN2, and wipe the straw quickly with a clean tissue. Air-thaw
straw for 30 s and watch the straw for leaks.
6. Gently plunge the straw into the beaker of water (37°C) for 40–50 s.
7. Remove and gently dry the straw with tissue. Cut off the heat-sealed end with
scissors and attach to a 1-mL syringe. Cut the cotton-plug end with a sterile
scissors and expel the blastocysts into the 8% glycerol solution. If the embryos
are not seen, flush the straw a few times. Leave the embryos in the 8% dish for
5 min.
8. Transfer the embryos to the 6% glycerol solution for 10 min at 37°C in 5% CO2
in air.
9. Transfer the embryos to the 5% glycerol solution for 12 min at 37°C in 5% CO2
in air.
10. Transfer the embryos to the 4% glycerol solution for 12 min at 37°C in 5% CO2
in air.
11. Transfer the embryos to the 3% glycerol solution for 14 min at 37°C in 5% CO2
in air.
12. Transfer the embryos to the 2% glycerol solution for 14 min at 37°C in 5% CO2
in air.
13. Transfer the embryos to the 1% glycerol solution for 16 min at 37°C in 5% CO2
in air.
14. Rinse the embryos twice in incubation medium.
15. Transfer the embryos to the dish with fresh equilibrated incubation medium (0.5 mL)
and incubate at 37°C in 5% CO2 in air.
16. Assess each embryo under the microscope. Compare the blastocyst quality with
appearance at the time of freezing. Score the blastocyst according to the markers
described earlier (see Subheading 1.5. and Tables 1 and 2).
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3.3. Embryo Culture (Days 1–6)
3.3.1. Microdroplets Under Oil
Culture of human embryos in microdroplets of medium (20–100 µL) under
oil can save on costs, help to maintain optimum osmolarity and pH, and control
fluctuations in temperature. Daily monitoring and scoring of individual
embryos are also easy, quick, and reliable. In vivo in the human fallopian tube,
embryos grow in microdroplets of fallopian tubal fluid and because such in vivo
conditions are simulated in vitro by using microdroplets of culture medium
containing the optimal metabolic requirements for the embryo, high-quality
blastocysts are generated (8).
3.3.2. Sequential Culture Media
Knowledge of how coculture systems work by positive and negative conditioning (9) led to the evolution of sequential culture media. Human embryos are
metabolically dynamic and their nutritional requirements change from day to day
and from stage to stage. For example, early cleavage embryos require minimal
glucose, whereas late blastocysts require plenty of glucose; for pyruvate, the
requirements are the other way around. The antioxidant taurine is beneficial for
blastocyst development, whereas hypoxanthine is detrimental. Late morula and
blastocyst stage embryos require essential and nonessential amino acids, because
transcription has commenced after genomic activation around d 3; these will be
the building blocks for further protein synthesis. As such, to obtain optimal blastocyst development, early cleavage-stage embryos (2PN to four-cell stage) are
grown in the first medium (G-1 version 3 and HSA), and from d 3 onward, when
the genome is being activated, the embryos are grown in a more complex second
medium (G-2 version 3 and HSA) that contain salts, energy substrates, proteins,
amino acids, vitamins, and hormones (10). It is crucial when transferring embryos
from new to fresh or from G-1 to G-2 version 3 and HSA medium that drawn-out
pipets are used to avoid the dilution of the second medium with the first.
3.3.3. Methods for Embryo Culture (Days 1–6)
Day 0: G1-version 3 and HSA dishes are made for thawed d 1 (2PN)
embryos. Microculture droplets (20 µL) of G1-version 3 and HSA are placed in
the wells of four-well or small Petri dishes and overlaid with sterile oil (Ovoil).
Two separate rinse drops of G1-version 3 and HSA are made in the center of the
same four-well dish or Petri dish. The dishes are incubated at 37°C in 5% of
CO2 in air.
Day 1: The thawed 2PN embryos are washed in the two rinse drops, before
transfer to the microculture droplet. Each 2PN embryo is cultured individually
in each microculture droplet and the dishes incubated at 37°C in 5% CO2 in air.
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Check the embryos once more at 24 to 25 h postinsemination to record the first
cleavage (two-cell stage). 2PN and first cleavage embryos are scored according
to the markers described earlier (see Subheading 1.1.).
Day 2: (1) Each embryo is assessed for cleavage on d 2 and scored further
according to the three grades (1, 2, and 3) based on fragmentation and regularity
of blastomeres. There is no change of medium and the embryos are grown in
the same G-1 version 3 and HSA medium. (2) G-2 version 3 and HSA dishes
are made up for the following day. Microculture droplets (20 µL) of G-2
version 3 and HSA are placed in each well of four-well dishes or in small Petri
dishes and overlaid with Ovoil. Two rinse drops of G-2 version 3 and HSA are
placed in the center of the same four-well or Petri dish. The dish is incubated
at 37°C in 5% CO2 in air.
Day 3: On the morning of d 3, embryos are scored and transferred from G-1
version 3 and HSA medium into the G-2 version 3 and HSA culture dish prepared the previous day. The embryos are rinsed in the two central drops of the
culture dish before transfer to the microculture droplet (see Note 6). The
embryos are incubated at 37°C in 5% CO2 in air.
Day 4: (1) On the morning of d 4, embryos are scored. There is no change of
medium and the embryos are incubated in the same G-2 version 3 and HSA at
37°C in 5% CO2 in air. (2) Day 6 growth dishes containing fresh 20 µL microculture droplets of G-2 version 3 and HSA medium are prepared and incubated
at 37°C in 5% CO2 in air.
Day 5: (1) On the morning of d 5, embryos are scored using the blastocyst
markers described earlier (see Subheading 1.5. and Tables 1 and 2). Goodquality expanded blastocysts with large ICMs generated from high 2PN scores
are given priority when selecting embryos for deriving hES cell lines (11,12).
(2) Remaining embryos that are slow in development are transferred to fresh
20 µL microculture droplets of G-2 version 3 and HSA medium made on d 4
and monitored until d 6 for further blastocyst development. Expanded blastocysts
with large ICMs generated on d 6 can also be used for hES cell line derivation.
4. Notes
1. During this time, handle the straws carefully and examine them for air bubbles,
cracks in the seal, and any leakage of LN2.
2. Note that embryos are osmotically stressed and have to be handled very carefully.
3. Handle the embryos very gently because their membranes will be very fragile.
4. Do not place in the CO2 incubator because the buffer capacity of Cryo-PBS is
inadequate for the 5% CO2 atmosphere.
5. Human embryos are usually not frozen at compacted and cavitating stages.
6. Care must be taken not to dilute the G-2 version 3 and HSA medium with G-1
version HSA medium.
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