0009
Structural integrity and bioenergetic activity of human
ovarian egg precursor cell-derived mitochondria are
maintained by a novel cryopreservation approach
Michael S. Bonkowski,1,2 Yuanyuan Wu,1 David A. Sinclair,2 James D. Luterman,1 Arthur O. Tzianabos,1 Bo Zhang1
OvaScience Inc., Waltham, MA 02451, USA; 2Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
1
I. Limited time window for maintaining activity of isolated
mitochondria at 4°C
• When isolated mitochondria were kept at 4°C, the ATP production
activity was maintained at full capacity up to 4 and 8 hours in human
and mouse egg precursor cell-derived mitochondria, respectively.
• However, substantial loss of activity was observed after 12 hours, and
at the 24-hour time point, activity loss reached approximately 50% for
both human and mouse; at 12 and 24 hours P<0.0001 (Figure 1).
B
A
Mito
Mito
Nucleus
20
0
0
0h
4h
8h
12 h*
16 h*
24 h*
40
20
0
10
20
30
Time (×30 seconds)
0
10
20
30
Time (×30 seconds)
Figure 1: ATP production over time in egg precursor cell-derived mitochondria
stored at 4°C. ATP production over a 24-hour period in mitochondria
isolated from cultured human (A) and mouse (B) egg precursor cells while
stored at 4°C. Experiments were replicated at least twice in two different
cell lines. Data expressed as mean±SEM. Significance was determined
using a repeated measures ANOVA for each time point compared to
0 hours. #P=0.0151, *P<0.0001.
II. Identification of optimal cryopreservation medium and process
for egg precursor cell-derived mitochondria
• To explore the optimal cryopreservation medium and process
for freezing and storing mitochondria, we first tested mouse
egg precursor cell-derived mitochondria preservation in four
cryopreservation media (labelled A–D) using two different freeze/thaw
processes (slow and fast).
• The cytochrome C assay indicated that medium C was the least effective
in protecting mitochondria, followed by medium D. Medium B was the
most effective in preserving mitochondrial structural integrity (Figure 2).
• Regardless of cryopreservation medium used, the fast freeze/thaw
process was more protective than the slow method (Figure 2).
500
IV. Human egg precursor cell-derived mitochondria maintained
activity after long-term cryopreservation
• OxPhos activity of human egg precursor cell-derived mitochondria
was maintained after long-term freezing (Figure 6; Table 1).
100,000
0
• To further develop flexibility for long-term storage of human
egg precursor cell-derived mitochondria, we sought to identify a
cryopreservation medium and corresponding freeze/thaw process in
order to retain their viability, including energy potential and structural
integrity, after freezing and storing in liquid nitrogen.
• To evaluate the functionality of egg precursor cell-derived mitochondria
after long-term storage.
Methods
Isolation of mitochondria
Mitochondria were isolated from cultured human and mouse egg
precursor cells using a syringe pump. Briefly, cells were suspended in
200 μl ice-cold isolation buffer and passed through a 24G needle 40
times using an automatic syringe pump (Harvard Apparatus, Holliston,
MA, USA). Cell lysates were then spun at 800 g for 10 minutes to remove
nuclei and unbroken cells. The mitochondrial fraction was obtained after
centrifugation at 7000 g for 30 minutes and suspended in indicated
media.
ATP assay
ATP production from isolated mitochondria, either fresh or post-freeze/thaw,
was determined using ATP Bioluminescence Assay Kit CLSII (Roche
Molecular Biochemicals, Mannheim, Germany). Succinate (S; 10 μM)
was used as the substrate in excess for the mitochondria electron
transport chain (Sigma-Aldrich Corp., St. Louis, MO, USA). Rotenone
(R; 3 μM) was used to block excess electron transfer activity from complex I.
S+R clamps mitochondrial respiration while addition of complex VI
inhibitor oligomycin (O; Sigma-Aldrich Corp.) inhibits mitochondrialspecific ATP production. This allows measurement of mitochondrial vs.
cytosolic ATP production from ADP. All experiments were performed
in the presence of excess ADP and measured every 30 seconds for 15
minutes. Oxidative phosphorylation (OxPhos) activity was expressed as
percentage of activity in the presence of O (10 μM) over the total ATP
activity in the presence of ADP (400 μM), S (10 μM) and R (3 μM).
Cytochrome C assay
Cytochrome C release was used as an indicator for mitochondrial
structural integrity. Mitochondria in different cryopreservation media
were spun at 7000 g for 10 minutes and the supernatant was loaded
onto a microplate coated with anti-cytochrome C antibody. Cytochrome
C level was evaluated using Quantikine Cytochrome C Immunoassay
(R&D Systems, Minneapolis, MN, USA).
Electron microscopy analysis
Mitochondrial isolates were pelleted and fixed in freshly-made 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer. Samples then underwent osmication and uranyl acetate staining,
dehydration in alcohols, and were embedded in Taab 812 Resin (Marivac
Ltd., Nova Scotia, Canada). Subsequent blocks were cut and sectioned
with a Leica Ultracut microtome (Leica, Buffalo Grove, IL, USA), picked
up on 100 mesh formvar/carbon coated Cu grids, stained with 0.2% lead
citrate, and imaged using a Tecnai Spirit BioTWIN electron microscope
(FEI, Dawson Creek, OR, USA).
0
CTL
A
4°C
B
C
D
A
Slow
freeze/thaw
B
C
D
Fast
freeze/thaw
Figure 2: Comparison of different cryopreservation media and freeze/thaw
methods using mouse egg precursor cell-derived mitochondria.
Cytochrome C released into the supernatant is an indication of
compromised integrity of isolated mitochondria. Medium A held at 4°C
(not frozen) is shown as a control (CTL). Four cryopreservation media
A–D were tested using fast and or slow freeze/thaw methods. Two
replicates were performed and data expressed as mean±SD. Significance
was determined using a two-way ANOVA for treatment*medium with a
Dunn’s multiple comparison post-hoc analysis. *P<0.05.
III. Human egg precursor cell-derived mitochondria retained
functionality and structural integrity post-cryopreservation
• Similar to mouse egg precursor cell-derived mitochondria (Figure 2),
freezing and thawing human egg precursor cell-derived mitochondria
using the selected cryopreservation medium (B) resulted in only
minimal cytochrome C release (1.9±0.1 ng/ml) when compared
with the freshly isolated mitochondria kept on ice for 30 minutes
(1.3±0.1 ng/ml) (Figure 3).
• In addition, the ATP assay showed that cryopreserved mitochondria
post-freeze/thaw demonstrated similar levels of total ATP production
and OxPhos activity to freshly prepared mitochondria (Figure 4).
• Electron microscopy showed that similar to mitochondria in oocytes,
egg precursor cell-derived mitochondria are less differentiated, with
rounded appearance, few cristae and high electron density (Figure 5A).
The isolated mitochondria maintained structural integrity (Figure 5B).
In particular, morphology was maintained after freeze/thaw using the
selected cryopreservation medium and process (Figure 5C).
Cytochrome C release
20
10
3
2
1.3±0.1
1.9±0.1
1
Total
Freeze/thaw
4°C
Figure 3: Structural integrity of human egg precursor cell-derived
mitochondria was maintained after rapid freeze/thaw. To confirm
that cryopreservation medium B was optimal, we tested cytochrome C
retention when using medium B and a fast freeze/thaw technique. Two
replicates were performed and data expressed as mean±SD.
4°C
A
15
Mito only
S+R
S+R+O
10
B
9.87 pM
OxPhos
5
0
0
0
Freeze/thaw sample
1 month
OxPhos
30.1%
100,000
10
20
30
Time (×30 seconds)
Freeze/thaw sample
2 months
250,000
S+R
S+R+O
150,000
0
200,000
S+R
S+R+O
OxPhos
29.6%
150,000
100,000
50,000
0
0
10
20
30
Time (×30 seconds)
Freeze/thaw sample
3 months
OxPhos
31.5%
200,000
150,000
100,000
100,000
50,000
50,000
0
10
20
30
Time (×30 seconds)
Freeze/thaw sample
14 months
250,000
S+R
S+R+O
150,000
0
0
10
20
30
Time (×30 seconds)
0
S+R
S+R+O
OxPhos
23.2%
10
20
30
Time (×30 seconds)
Figure 6: OxPhos activity of human egg precursor cell-derived mitochondria
was maintained after long-term freezing. Freshly isolated human egg
precursor cell-derived mitochondria were used as a baseline (Day 0).
OxPhos activity was maintained during frozen storage in liquid nitrogen
at day 0 and for extended time periods of 1, 2, 3, and 14 months. ATP
production is shown in the presence of succinate + rotenone (S+R), which
clamps the mitochondrial respiration, while the addition of oligomycin
(O) inhibits mitochondrial-specific ATP production. Data expressed as
mean±SEM.
Table 1: Summary of maximum ATP production and OxPhos activity of
cryopreserved mitochondria after different time periods of storage
Fresh
Fast frozen/thawed
Day 0
Day 0
1 month 2 months 3 months 14 months
Maximum ATP
production±SD
(RLU)
198477
±1344
191558
±4435
192202
±1142
203917
±492
204127
±6360
204429
±12285
OxPhos activity
(% inhibition)
32.5
35.4
30.1
29.6
31.5
23.2
OxPhos, oxidative phosphorylation; RLU, relative light units.
Conclusions
• O
nly a limited time window is available for maintaining
activity of mitochondria isolated from human egg precursor
cells when stored at 4°C. This suggests development of
a cryopreservation medium and method for long-term
cryopreservation of isolated mitochondria to increase flexibility
for point of delivery.
•We have successfully determined an optimal cryopreservation
medium and method for freezing mitochondria isolated from
human egg precursor cells. After thawing, mitochondrial
functionality was maintained, as demonstrated by the
preservation of ATP production potential, prevention of
cytochrome C release, and conservation of structural integrity.
15
0
100,000
50,000
RLU
ng/ml
5
OxPhos
35.4%
150,000
10
20
30
Time (×30 seconds)
200,000
0
10
20
30
Time (×30 seconds)
ATP (picomoles)
Objectives
*
0
10
ng/ml
•
*
S+R
S+R+O
50,000
250,000
60
20
200,000
50,000
200,000
40
OxPhos
32.5%
150,000
Freeze/thaw sample
Day 0
250,000
S+R
S+R+O
200,000
80
*
Unfrozen sample
Day 0
250,000
250,000
15
ATP (picomoles)
•
nm
Figure 5: Transmission electron microscopy of human egg precursor cells preand post-freezing. (A) Egg precursor cells (3000X magnification) were
FACS sorted and fixed for imaging. Mitochondrial isolates were pelleted
and fixed for imaging (8000X magnification) before (B) and after (C)
freezing, utilizing our cryopreservation technique.
Cytochrome C release
*
100
nm
RLU
40
60
RLU
0h
4 h#
8 h*
12 h*
16 h*
24 h*
RLU
60
• In mammals, oocyte quality may be related to the number and
•
500
Mouse
B
RLU
Human
RLU
A
Background
•
nm
C
0
function of mitochondria present,1-3 and poor egg quality can be
improved by the transfer of cytoplasm containing mitochondria from
donor eggs.3,4
As mitochondria contain their own genome, there are potential safety
concerns associated with their transfer from donor tissue, including
mitochondrial heteroplasmy in offspring and possible increased risk of
heritable mitochondrial disease.3,4
Egg precursor cells (also called oogonial stem cells, female germline
stem cells, EggPCSM cells) are oocyte progenitors found in the ovarian
cortex,5 and represent an ideal source of autologous germline
mitochondria for transfer to oocytes.
AUGMENTSM (AUtologous Germline Mitochondrial ENergy Transfer)
treatment is a new fertility treatment involving transfer of a woman’s
mitochondria from her egg precursor cells into her own oocytes
during in vitro fertilization (IVF), designed to improve IVF success
rates by enhancing energy production in poor quality oocytes.
Initial clinical experience with AUGMENT treatment using freshly
prepared mitochondria suggests improved clinical pregnancy rates
compared with historical outcomes in the population assessed.6
–– Clinical pregnancy rates per initiated cycle with AUGMENT
treatment vs. previous IVF were 35% vs. 11% (Canada) and 22% vs.
4% (United Arab Emirates).
500
Mito
ATP (picomoles)
Introduction: Studies in animals and humans demonstrate
that oocyte quality is related to the number and function of
mitochondria, and that poor egg quality can be restored by the
transfer of mitochondria from healthy donor eggs. Egg precursor
cells, the oogonial progenitor cells present in the ovarian cortex,
provide an ideal source for autologous, germline-derived
mitochondria. Since mitochondria are maternally inherited and
contain their own genome (mtDNA), transfer of egg precursor
cell-derived mitochondria preserves homoplasmy. AUGMENT
(AUtologous Germline Mitochondrial ENergy Transfer) is a new
fertility treatment using the transfer of egg precursor cell-derived
mitochondria, and is designed to improve oocyte quality and
success rates by enhancing energy production in poor-quality
oocytes in women who are undergoing in vitro fertilization (IVF).
Objective: Our goal was to develop a novel technique for the
cryopreservation of egg precursor cell-derived mitochondria,
through which the viability, energy potential, and structural
integrity of mitochondria can be successfully retained after
freezing and storing in liquid nitrogen.
Methodology: After isolation from egg precursor cells,
mitochondria were frozen in liquid nitrogen in the presence of
various cryopreservation media with different freezing settings.
In addition, mitochondria were frozen in the selected media for
different time periods to evaluate long-term storage stability.
After thawing, mitochondrial function was determined using
bioluminescent ATP and cytochrome C release assays. Structural
integrity was evaluated using transmission electron microscopy.
Results: One medium was found to be capable of maintaining
egg precursor cell mitochondrial viability following freezing,
as demonstrated by the preservation of both ATP activity and
structural integrity. Furthermore, a time course study showed
that mitochondrial function could be maintained after longterm storage for up to 14 months using this cryopreservation
approach.
Conclusions: These data support the feasibility of freezing and
storing mitochondria isolated from egg precursor cells prior to
performing AUGMENT.
Results
ATP (picomoles)
Abstract
15
Medium B - freeze/thaw
Mito only 10.03 pM
S+R
S+R+O
10
OxPhos
5
0
0
10
20
30
Time (×30 seconds)
•A time course study showed that with use of the selected
cryopreservation medium and method, the structural
integrity and functional activity of mitochondria isolated from
human egg precursor cells were maintained after long-term
cryopreservation.
• T
ogether, these data support the feasibility of freezing and
storing mitochondria isolated from human egg precursor cells,
which may be incorporated in the AUGMENT process.
Funding information
This work was supported by OvaScience Inc. DAS and MSB are
consultants to OvaScience Inc. DAS is an inventor on patents licensed
to OvaScience Inc.
WY, JDL, AOT and BZ are employees and stock holders of
OvaScience Inc.
Writing assistance was provided by Helen Varley, PhD, CMPP of Excel
Scientific Solutions, Horsham, UK and supported by OvaScience Inc.
References
1. Reynier P et al. Mol Hum Reprod 2001;7:425–9.
2. May-Panloup P et al. Hum Reprod 2005;20:593–7.
3. Bentov Y et al. J Assist Reprod Genet 2011;28:773–83.
4. Chappel S. Obstet Gynecol Int 2013;2013:183024.
5. White YA et al. Nat Med 2012;18:413–21.
6. Fakih MH et al. JFIV Reprod Med Genet 2015;3:154.
Figure 4: ATP production potential of human egg precursor cell-derived
mitochondria retained after rapid freeze/thaw. ATP production was
determined in fresh (A) and rapidly frozen/thawed (B) mitochondrial
isolations from fresh cultured human egg precursor cells. Two replicates
were performed and data expressed as mean±SEM.
Presented at the 5th Annual Scientific Meeting of the Obstetrical and Gynaecological Society of Hong Kong and the Ovarian Club VII Meeting, 21–22 May, 2016, Hong Kong