Cell Transplantation, Vol. 22, pp.

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Increasing Follicular and Stromal Cell Proliferation in Cryopreserved

Human Ovarian Tissue After Long-Term Precooling Prior to Freezing:
In Vitro Versus Chorioallantoic Membrane (CAM) Xenotransplantation
Vladimir Isachenko, Evgenia Isachenko, Peter Mallmann, and Gohar Rahimi

Research Group for Reproductive Medicine and IVF-Laboratory, CAM-Xenotransplantation Group,

Department of Obstetrics and Gynecology, Cologne University, Cologne, Germany

A positive effect on the future development of cells, which have been cooled to low suprazero temperatures
and then thawed, has been observed before and is not new. The aim of this study was to test the effectiveness
of postthawing culture of human ovarian tissue, which was either frozen just after operative removal or cooled
after removal to 5°C for 24 h before cryopreservation. Ovarian fragments from six patients were divided into
small pieces in the form of cortex with medulla and randomly divided into the following four groups: Group
1 consisted of pieces that just after removal had been cultured in vitro for 8 days in a big volume of medium
with mechanical agitation; Group 2 included pieces cooled after operation to 5°C for 24 h and then cultured
in vitro for 8 days; Group 3 was comprised of pieces frozen–thawed just after operation and then cultured for
5 days in the chorioallantoic membrane (CAM) culture system; and the pieces in Group 4 were cooled after
operation to 5°C for 24 h, frozen–thawed, and then cultured in the CAM system for 5 days. The effectiveness
of the tissue culture was evaluated by the development of follicles and by the intensiveness of proliferation
in the tissue (by expression of cytokeratin and Ki-67). For Groups 1, 2, 3, and 4, the mean densities of fol-
licles per 1 mm3 was 12.9, 12.2, 12.4, and 16.1, respectively (p1–2 > 0.1; p3–4 < 0.05). For these groups, 87%,
95%, 71%, and 84% of the preantral follicles were morphologically normal (p1–2, 3–4 < 0.05). The immunohis-
tochemical analysis showed increased proliferation after cooling of fresh and cryopreserved tissue. Long-term
(24 h) cooling of ovarian tissue to 5°C before cryopreservation increases the viability of the cells in the tissue
after thawing. Additionally, the efficacy of the CAM system for the culture of thawed human ovarian tissue
was demonstrated.

Key words: Human ovarian tissue; Freezing; Proliferation; Cytokeratin; Ki-67

INTRODUCTION Part of the ovarian tissue obtained before oncological

Owing to the increasing effectiveness of cancer treat-
ments and positive long-term prognosis for young women,
the problem of postcancer infertility is playing an increas-
ingly significant role because chemotherapy can be
gonadotoxic and lead to the functional death of ovaries.
Cryo­preservation of ovarian tissue before cancer therapy
with reimplantation after convalescence is the potential
key solution to this problem (6,33,34).
Several cases reporting restoration of ovarian function
after reimplantation of ovarian cortex in patients with pre-
mature ovarian failure after cancer treatment have been
published since 1998. Now, pregnancies and babies born
after retransplantation of frozen ovarian tissue have been
reported (31).
treatment is used for routine histological observation, a
mandatory procedure for monitoring and minimizing the
risks associated with any future implantation of tissues
that could be affected by metastases. After cryopreserva-
tion and storage, some ovarian tissue can be thawed and
cultured in vitro in order to screen for the presence of
metastases and to monitor follicles. The quality of fol-
licles in the cultured tissue will indicate whether it is
possible to restore a woman’s reproductive function. At
present, there are three ways to effectively determine the
quality of the cryopreservation procedure using ovar-
ian tissue from a given patient before the reimplantation
treatment: (i) evaluation of follicles after postthawing
xenotransplantation to severe combined immunodeficient

Received May 22, 2012; final acceptance September 28, 2012. Online prepub date: November 1, 2012.
Address correspondence to Vladimir Isachenko, University Maternal Hospital, Department of Obstetrics and Gynecology, Cologne University,
Kerpener Str. 34, 50931 Cologne, Germany. Tel: +49-221-4783975; Fax: +49-221-47886201; E-mail: v.isachenko@yahoo.com
2053
2054 isachenko ET AL.
(SCID) mice (9,19,24,25,27–29), (ii) in vitro culture in a
large volume of culture medium and under constant agita-
tion (10–12), and (iii) culture on embryonic chorioallan-
toic membrane (CAM) within a hen’s egg (13,21).
The CAM system is an intermediate stage between
in vitro culture and animal experiments, and it could be
considered as an interface between in vitro and in vivo
models (including xenotransplantation). Importantly, it
does not raise ethical or legal questions, nor does it violate
animal protection laws.
At present, the suprazero temperature (usually from
0°C to 7°C) storage method is a widely used technol-
ogy in organ preservation. Low temperature retards
cellular metabolism and thus reduces cellular oxygen
demand and consumption. This furnished an approach
to reduce tissue autolysis without the need for vascular
perfusion.
The absence of a negative effect of hypothermic stor-
age on domestic cat (35) and rat (36) ovaries has been
noted. The positive effect of cooling on rat hypothalamus
was reported (7).
Comparative data about the effect of long-term cool-
ing of human ovarian tissues before cryopreservation on
their viability after thawing are limited.
We chose two culture methodologies for the current
investigation: the in vitro and the CAM system because the
chorioallantoic membrane system is a borderline model,
an intermediate stage between in vitro culture and animal
experiments.
The aim of this study was to compare the effectiveness
of postthawing culture of human ovarian tissue, which
was either frozen just after removal (surgical operation)
or cooled to 5°C for 24 h just after the operation and later,
after this 24-h cooling, was frozen.
MATERIALS AND METHODS
Full details of the study described in this article were
approved by the Ethics Boards of Universities Cologne
(permission 276-03 from 07.07.2003) and Ulm (permis-
sion 102/10 from 19.07.2010).
Written informed consents were obtained from all par-
ticipants aged 18 and over involved in our study. On behalf
of the patients under the age of 18, written consents were
obtained from the next of kin.
Except where otherwise stated, all chemicals were
obtained from Sigma (Sigma Chemical Co., St. Louis,
MO, USA).
Tissue Collection, Dissection, and Distribution
Into Groups
Informed consent was obtained from six female patients
aged between 13 and 32 (22.1 ± 5.0) years. According to
approved protocol, 10% of ovarian tissue was used for
patient-oriented research.
The basal medium used for manipulation of tissues
(transport and dissection) was Leibovitz L-15 with 5%
dextran serum substitute (Irvine Scientific, Santa Ana, CA,
USA), referred to below as “basal medium.”
Fresh ovarian tissue fragments were transported from
the surgical room to the laboratory within 20 min with tem-
perature maintained at 32–34°C. Using tweezers (Bochem
Instrumente GmbH, Weilburg, Germany) and scalpel
No. 22 (B Braun Melsungen AG, Melsungen, Germany),
ovarian fragments were dissected and divided into small
pieces (1.5–2.0 × 1.0–1.2 × 1.0–1.2 mm) and cryopreserved
as described below. The pieces were randomly divided
into four groups:
Group 1: Pieces were cultured in vitro for 8 days after
surgical operation (removal of these pieces).
Group 2: Pieces were cooled after operation to 5°C for
24 h and then cultured in vitro for 8 days.
Group 3: Pieces were frozen just after operation with-
out cooling and then thawed and cultured for 5 days in
a chorioallantoic membrane (CAM) culture system.
Group 4: Pieces were cooled after operation to 5°C for
24 h, then frozen, thawed, and cultured in the CAM
system for 5 days.
Eight pieces in one experimental group were used for
respective treatment (freezing and culture or only culture
with or without cooling) to determine the quality of fol-
licles and the degree of proliferation. After thawing, one
half of the experimental pieces (n = 16) were dissected on
32 fragments (~1.0 × 1.0 × 1.0 mm) and in vitro cultured.
The second half of the pieces were dissected on 48 frag-
ments (0.5–1.0 × 0.5–1.0 × 1.0–1.2 mm) and CAM cul-
tured. The volume of pieces for CAM culture is smaller
than pieces for in vitro culture. After respective treatment
and culture, 79 particles of ovarian cortex with medulla
(32 after in vitro culture and 47 after CAM culture) were
analyzed.
Cryopreservation (Freezing and Thawing)
Pieces of ovarian tissue were placed at room temper-
ature in 20 ml of freezing medium composed of basal
medium supplemented with 6% dimethyl sulfoxide, 6%
ethylene glycol, and 0.15 M sucrose. Then pieces were put
into a standard 1-ml cryostraw (MTG GmbH, Bruck­
berg, Germany) previously filled by freezing medium
and frozen in a CTE 2104 freezer (Cryo-Technik-
Erlangen, Hoechstadt, Germany). The freezing program
consisted of the following five stages: (1) start tempera­
ture 22°C, (2) cooling from 22°C to 4°C at a rate of −5°C/
min, (3) cooling from 4°C to −7°C at a rate of −1°C/min,
(4) cooling from −7°C to −34°C at a rate of −0.3°C/min,
(5) plunging into liquid nitrogen. It was noted that at
−10°C there was a formation of crystals in the place of
INCREASING OF CRYOPRESERVED OVARIAN CELL VIABILITY AFTER LONG-TERM PRECOOLING 2055
localization of ovarian pieces (this construction of freezer
implies the autoseeding).
The procedure of thawing was achieved by holding the
straws for 10 s at room temperature followed by immersion
in a 100°C (boiling) water bath for 20 s and expelling the
contents of the straw into the solution for the removal of
cryoprotectants. The exposure time in the boiling water was
visually controlled by the presence of ice in the medium; as
soon as the ice was 2 to 1 mm apex, the straw was removed
from the boiling water, at which point, the final temperature
of the medium was between 4°C and 10°C. Within 5–10 s
after thawing, the pieces from the straw were expelled
into 10 ml of thawing solution (basal medium containing
0.5 M sucrose) in a 100-ml specimen container (Sarstedt,
Nuembrecht, Germany). The stepwise dilution of cryopro-
tectants was achieved using the same principle as that used
for saturation by ethylene glycol by Isachenko et al. (12). The
container was placed on a shaker and continuously agitated
at 200 osc/min for 15 min at room temperature. Stepwise
rehydration of the tissue pieces for 30 min at room tempera-
ture was also performed using the same “dropping” meth-
odology: slow addition of basal medium (see above) to the
solution of sucrose with ovarian pieces. For “dropping,” we
used 50 ml of basal medium in a 50-ml tube (Greiner Bio-
One GmbH, Frickenhausen, Germany). The final sucrose
concentration was 0.083 M, resulting in almost isotonic con-
ditions. Finally, the pieces were washed thrice each in basal
medium for 10 min and transferred for CAM culture.
In Vitro Culture
Individual thawed pieces of two experimental groups
(Group 1: pieces immediately after operation and Group
2: pieces cooled after operation to 5°C for 24 h) were
placed in 200-ml dishes for suspension culture (Cellstar,
Greiner Bio-One GmbH) with 30 ml of AIM-V medium
(Gibco/Invitrogen, Gaithersburg, MD, USA). They were
incubated for 8 days at 37°C in 5% CO2 with 75 osc/min
agitation using a rotation shaker.
CAM Culture
Fertilized eggs (n = 48) of White Leghorn chickens,
purchased at a local hatchery and incubated at 37°C with
60% relative humidity, were prepared for implantation
on day 4 of incubation. Standard microbiology assess-
ment was performed to exclude subclinical infections.
Preparation of the CAMs was performed essentially as
previously described (3,11,13,16,17,21). Each egg was
washed with warm 70% ethanol, after which a hole was
drilled through the pointed pole of the shell (Fig. 1).
The following day, part of the CAM of the embryo was
exposed by peeling a 1.5- to 2.0-cm window in the shell.
This window was covered with tape and the incubation
continued. The CAM has two epithelial layers and, in its
intact form, represents a “dry” impermeable barrier for all
invasions, including ovarian fragments. For “connection”
of ovarian pieces with the egg system, the latter must be
open. For this, the upper peridermal part of the double
epithelial layer was removed in each egg, leaving the
basal layer intact. On day 10 of incubation, a silicone
ring 0.5  mm thick with a 5-mm inner diameter (Sudhof
Technik GmbH, Ulm, Germany) was placed on the mem-
brane (Fig. 1). An ovarian piece was placed into this sili-
cone ring using microsurgical forceps. Thereafter, the shell
window was covered again, and the egg was replaced in
the incubator. After 5 days of CAM culture, the viability
of ovarian pieces was evaluated.
Histology of Follicles
For histological investigation, the cultured tissue pieces
were fixed in Bouin’s solution, embedded in paraffin wax,
serially sectioned at 4 μm, stained with hematoxylin/eosin,
and analyzed under a microscope (400×, Olympus Co,
Tokyo, Japan).
The number of viable and damaged follicles was
counted. Before the counting of follicles, sections were
coded and scored “blind.” To avoid overcounting of the
same follicles, only the section with a visible oocyte nucleus
was taken into account. Morphology of the follicles was
evaluated on the basis of parameters previously described
(26). Two types of preantral follicles were evaluated:
(1) primordial follicles composed of an oocyte surrounded
by a layer of flattened follicular cells and (2) primary and
secondary follicles, which are similar to primordial fol-
licles, but in which the oocyte is surrounded by one to
two layers of spheroid granulosa cells.
The quality of follicles was graded on the scale from
1 to 3. A follicle of grade 1 is spherical in shape and
contains a spherical oocyte, which is surrounded by an
even distribution of granulosa cells and has a homog-
enous cytoplasm and slightly granulated nucleus, with
condensed chromatin in the form of a dense spherical
structure detectable in the center of the nucleus. A fol-
licle of grade 2 has similar characteristics, but the oocyte
is without condensed chromatin within the nucleus and
is often irregular in shape; the surrounding granulosa
cells can be flat and pulled away from the edge of the
follicle. A follicle of grade 3 contains a misshapen oocyte
with or without nuclear vacuolation; theca and granulosa
cells are separated from the edge of the follicle, and the
partly or fully disrupted granulosa have pyknotic nuclei.
Follicles of grades 1 and 2 were denoted as normal, and
those of grade 3 were denoted as degenerated. Examples
of the different follicular degenerations can be observed
elsewhere [e.g., see Isachenko et al. (11,13)].
Immunohistology for Proliferation
For these investigations, ovarian pieces were fixed in
4% paraformaldehyde, embedded in paraffin wax, and
2056 isachenko ET AL.

Figure 1.  Ovarian pieces precooled to 5°C for 24 h. (a, b) Eight-day in vitro culture. (a) Piece after precooling to 5°C and before in
vitro culture. (b) The same piece after 8 days in vitro culture. (c, d, d1) Five-day chorioallantoic membrane (CAM) culture. (c) Piece after
24 h precooling to 5°C, freezing and thawing, before CAM culture. (d, d1) The same piece after 5-day CAM culture. Scale bar: 1 mm.

serially sectioned at 4 μm. After deparaffinization, slides
were processed for staining for human cytokeratin and
Ki-67 (antibodies and autostainer from Dako, Hamburg,
Germany) according to manufacturer’s protocol. FLEX
monoclonal mouse anti-human Ki-67 antigen, clone
MIB-1 and EnVision, FLEX Target Retrieval Solution,
low pH, Code K8005, as well as FLEX monoclonal mouse
anti-human cytokeratin 5/6, clone D5/16 B4, Code IS780
and EnVision, FLEX, high pH, Autostainer Plus, Code
K8010 were used for immunization and visualization.
Images were digitally scanned at 50× magnification with
an Axiophot microscope (Carl Zeiss, Jena, Germany) and
recorded.
Our scoring system was similar to that described by
Abir et al. (1,2). The intensity of immunoreactivity with
different monoclonal antibodies was subjectively assessed
as follows: lack of immunoreactivity, immunoreactivity
low, medium, and high.
Statistical Analysis
Integrity rate of tissue after treatment was evaluated
by ANOVA. Various characteristics were summarized by
INCREASING OF CRYOPRESERVED OVARIAN CELL VIABILITY AFTER LONG-TERM PRECOOLING 2057

Figure 2.  Histological micrographs of follicles from ovarian tissue precooled to 5°C for 24 h and cultured in vitro for 8 days or
cultured in the CAM system for 5 days. (a) Fresh (not cryopreserved) tissue after 24-h precooling to 5°C and 8 days in vitro culture.
Degenerated antral follicles after cooling are shown by a bold black arrow. (b, c) Tissue after cryopreservation and 5-day CAM cul-
ture. (b) Tissue cryopreserved immediately after operation, without precooling. (c) Tissue cryopreserved after 24-h precooling to 5°C.
(b1, b2) Degenerated follicles. (a1, b3, c1) Normal follicles. Scale bar: 25 µm.

mean and SD within groups. The level of statistical sig-
nificance was set at p < 0.05.
RESULTS
The survival rate of chick embryos was 97.9% (47
of 48).
After 8 days of in vitro culture, as well as 5 days after
transfer onto the extraembryonic vascular system of the
chorioallantoic membrane, the ovarian fragments were
observed to have developed a spherical shape (Fig. 1).
After completion of CAM culture (on the fifth day),
the propagation of ovarian pieces in the mesenchymal
layer of the membrane was noted (Fig. 1).
Histology of Follicles
Histological examination after 8 days of in vitro and
5 days of CAM culture of ovarian pieces of Group 2 (24-h
postoperation cooling to 5°C and following 8 days in
vitro culture), Group 3 (cryopreservation just after opera-
tion without cooling and following 5 days CAM culture)
and Group 4 (cryopreservation after 24-h cooling to 5°C
and following CAM culture) showed that only prean-
tral (primordial, primary, and secondary) follicles were
viable. All the antral follicles in these three groups were
degenerated (Fig. 2a) and, hence, were not counted.
The mean preantral follicle density per 1 mm3 was
12.9 ± 2.8, 12.2 ± 1.9, 12.4 ± 3.3, 16.1 ± 1.5 for Groups 1, 2,
3, and 4, respectively (p1–2 > 0.1; p3–4 < 0.05). It was detected
that 87.0 ± 1.6, 95.0 ± 3.1, 71.0 ± 3.9, and 84.0 ± 1.2% fol-
licles for Groups 1, 2, 3, and 4, respectively, were normal
(p1–2, 3–4 < 0.05) (Fig. 3).
Immunohistology for Proliferation
Immunoreactivity of cells cultured without cool-
ing varies from “lack of immunoreactivity” to “low,”
whereas cells cultured with cooling varied from “low” to
“high” (Figs. 4 and 5). The immunohistochemical analy-
sis showed increased proliferation after cooling of fresh
and cryopreserved tissue.
DISCUSSION
It is important to improve the posttransplantation sur-
vival of human ovarian tissue (1).
A positive effect of cooling of cells to low suprazero
temperatures on their future development after rewarm-
ing has been observed before and is not new.
2058 isachenko ET AL.

Figure 3.  Effect of cooling on the quality of follicles [expressed as quantity (a) and percentage (b) of normal follicles]. Different
superscripts indicate statistical differences between respective systems of culture, that is, in vitro versus in vitro (Group 1 vs. Group 2)
and CAM versus CAM (Group 3 vs. Group 4) (p < 0.05; n = 8).

It was shown that the acclimation of the warm-blooded
rat to cold stimulates mitosis indirectly in cells capable of
division because it directly stimulates the mitotic activ-
ity in mouse and human cells cultured and adapted to
the cold in vitro. In situ hybridization analysis of hypo-
thalamic tissue showed that cold exposure causes a two-
fold increase in the total number of neurons expressing
thyrotrophin-releasing hormone mRNA in the paraven-
tricular nucleus (7).
In addition, our previous results showed good survival
of bovine trophoblastic fragments that had been subjected
to chilling for 48 h at 4°C. The survival/formation of ves-
icles in these fragments was not different from that of the
untreated controls (both 98%) (14).
INCREASING OF CRYOPRESERVED OVARIAN CELL VIABILITY AFTER LONG-TERM PRECOOLING 2059

Figure 4.  Cytokeratin expression in the cryopreserved and 5-day CAM-cultured tissue. (a–c) Three examples of tissue cryopre-
served immediately after operation, without precooling. (d–f) Three examples of tissue cryopreserved after 24-h precooling to 5°C.
Cytokeratin-positive cells are shown in red. Scale bar: 25 µm.

Figure 5.  Ki-67 expression in the cryopreserved and 5-day CAM-cultured tissue. (a, a1) Tissue cryopreserved without precooling, imme-
diately after operation. (b, b1) Tissue cryopreserved after precooling. Ki-67-positive cells are shown in brown. Scale bar: 25 µm.
2060 isachenko ET AL.
The exposure of human ovarian tissue to low positive
temperatures of up to 26 h does not inhibit the devel-
opment of follicles during subsequent in vitro culture.
Compared with the untreated controls, the number of
primordial follicles in all treatment groups significantly
decreased owing to development to the advanced stages
(10). The results of an investigation by Yin et al. (36),
who found that, although ovaries showed fewer follicles,
hypothermic storage of rat ovary at 4°C for 24 h did not
disrupt ovarian function, does not support our results.
This could be explained by the species-specific sensitiv-
ity of ovarian tissue to hypothermia.
Interesting investigations were performed by Wood et
al. (35). The influence of long-term hypothermic storage
of whole domestic cat ovary for 48 h at 4°C on follicle-
oocyte atresia and temporal taphonomy was investigated.
It was found that the highest (but statistically insignifi-
cant) degeneration rate of follicles occurred at 48 h, with
inhibition of taphonomy. Our data support these results.
Our experimental design included the use of two cul-
ture methodologies for ovarian tissue, in vitro, and CAM
culture because animal testing has ethical and legal impli-
cations in Germany. Taking into account this fact as well
as published results (13,21), we established chorioallantois
membrane (CAM) of fertilized chicken eggs as a culture
system in the present study. The chorioallantoic membrane
system provides rapid results and may be used as a simple,
inexpensive method in any laboratory. The extraembryonic
vessel system of the chorioallantois membrane is similar to
that of the mammalian placenta, which also is not inner-
vated. This system is naturally immunodeficient. In addi-
tion, we used chick embryos in the first half of the usual
breeding time up to the stage when the embryonic neural
and immune systems are still underdeveloped. CAM can
be used for xenotransplantation of different types of cells.
CAM culture of bovine and mice ovarian tissue has
been reported by Cushman et al. (5) and Gigli et al. (8).
It is established that primordial follicles during the CAM
culture retained the ability to activate and grow. These
authors believe that extracellular matrix constitution of
CAM is similar to peritoneum, to which human ovarian
tissue is transplanted by intraperitoneal xenotransplanta-
tion and orthotopic autotransplantation (5,8), CAM forms
a bursa-like structure around ovarian pieces that is similar
with in vivo condition. Recently, the similar investiga-
tions were performed on human ovarian tissue (13,21).
The CAM system is used to study angiogenesis in tumor
(4) and endometriosis tissues (20,23). CAM also has been
used for culture of human skin (17), liver (15), and skel-
etal muscle tissue (22), for surgical retinal research (18),
for testing of different biomaterials in tissue engineering
(32), and also for investigations of a bright spectrum on
biological objects (30).
In conclusion, it appears that cooling of ovarian tis-
sue to 5°C for 24 h before cryopreservation has a posi-
tive effect on viability of this tissue after thawing.
Additionally, the efficacy of the CAM system for culture
of thawed human ovarian tissue was demonstrated.
ACKNOWLEDGMENTS: The authors are very grateful to Mr.
F. V. Braun from University Bonn for helpful discussion of our
results as well as to Ms. I. Orth and Ms. D. Peters for technical
assistance. The authors declare no conflicts of interest.
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