- Email: [email protected]
An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography
Accepted Manuscript An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography Ariadna Corral, Marcin Balcerzyk, Miguel Gallardo, Christiani A. Amorim, Ángel Parrado-Gallego, Ramón Risco PII:
S0093-691X(18)30428-X
DOI:
10.1016/j.theriogenology.2018.06.031
Reference:
THE 14613
To appear in:
Theriogenology
Received Date: 13 April 2018 Revised Date:
27 June 2018
Accepted Date: 29 June 2018
Please cite this article as: Corral A, Balcerzyk M, Gallardo M, Amorim CA, Parrado-Gallego Á, Risco Ramó, An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography, Theriogenology (2018), doi: 10.1016/ j.theriogenology.2018.06.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT Title page Information
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An optimized controlled rate slow cooling protocol for bovine ovarian tissue
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cryopreservation by means of X-ray computed tomography
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Ariadna Corral1, Marcin Balcerzyk1, Miguel Gallardo2, Christiani A. Amorim3, Ángel Parrado-
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Gallego1, Ramón Risco1,4
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Affiliations:
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Centro Nacional de Aceleradores (Universidad de Sevilla-CSIC-Junta de Andalucía), Calle
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Thomas Alva Edison 7, 41092, Sevilla, Spain
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Ginemed Clínicas Sevilla, Calle Farmaceutico Murillo Herrera 3, 41010, Sevilla, Spain
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Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique,
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Université Catholique de Louvain, Avenue Mounier 52, Bte. B1.52.02, 1200, Brussels,
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Belgium
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Sevilla, Camino Descubrimientos S/N, Isla Cartuja, 41092, Sevilla, Spain
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Departamento de Física Aplicada III, Escuela Técnica Superior de Ingeniería, Universidad de
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Corresponding author:
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Prof Ramon Risco
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Departamento de Física Aplicada III, Escuela Técnica Superior de Ingeniería, Universidad de
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Sevilla, Camino Descubrimientos s/n, Isla Cartuja, 41092, Sevilla, Spain
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Phone Number: +34 954.48.61.85, +34 636.21.19.91
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FAX number: +34 95.448.60.03
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Email
address:
[email protected]
ACCEPTED MANUSCRIPT Abstract
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Cryopreservation and subsequent transplantation of ovarian tissue is the only option to
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preserve fertility in certain patients facing gonadotoxic treatment. So far, cryopreservation
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of ovarian tissue has been carried out mostly by a controlled rate slow cooling process,
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typically known as slow freezing. Even though there are still some concerns about the
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iatrogenic damage on the follicle population, this technique has been used in the more than
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100 live births reported to date. It is well known that the control of the cryoprotectant
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loading in the tissue is crucial to in a cryopreservation procedure. We have used the
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technology of X-ray computed tomography to assess the concentration and distribution of
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dimethyl sulfoxide (one of the cryoprotectants most used in fertility preservation) inside
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pieces of bovine ovarian tissue after its cryopreservation. The low voltage used in our device
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(75 kV) and the high electronic density of this cryoprotectant makes the X-ray attenuation
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proportional to its concentration. By assessing and comparing the permeation and
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homogeneity of the cryoprotectant inside ovarian tissue fragments subjected to a controlled
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rate slow cooling process, we have characterized the effect of variations in the main
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parameters involved in the process, with the goal of achieving an optimized protocol with
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higher permeation of the cryoprotectant in the tissue. The most promissory results were
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obtained by increasing the initial concentration of dimethyl sulfoxide in the vehicle solution
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from 10 to 20 % / .
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Keywords
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CT imaging, Fertility preservation, Cryoprotectant visualization, Tissue permeation
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ACCEPTED MANUSCRIPT Introduction
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Cryopreservation of ovarian tissue is the only option of fertility preservation for certain
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cancer patients, such as pre-pubertal girls, for whom being subjected to a hormonal
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stimulation cycle for oocyte preservation is not possible [1]. Advantages of ovarian tissue
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cryopreservation are the high tolerance of the follicle population to freezing and thawing,
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and the possibility for immediate surgical collection of the tissue, done at any time of the
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women’s cycle [1].
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The most widely employed and successful method of ovarian tissue cryopreservation for
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fertility preservation is a controlled rate cooling slow cooling, known as slow freezing [1-5],
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with more than 100 cases of live births after transplantation reported [6-7]. Despite this
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promising data, current slow freezing protocols are empirical adaptations from the ones
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used for oocyte and embryo cryopreservation [5]. As a result, when this cooling process is
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employed in ovarian tissue, a more complex and dense biological system, it can damage the
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follicle population and the ovarian stroma. This iatrogenic damage can affect the viability of
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the tissue after transplantation [8-9].
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The aim of this study was to optimize a conventional controlled rate slow cooling protocol
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for human ovarian tissue, focusing on achieving a more homogeneous distribution of the
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cryoprotectant (CPA) inside the tissue fragments during the cryopreservation procedure. For
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that purpose, we carried out a series of experiments with bovine ovarian tissue varying the
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following parameters of the controlled rate slow cooling protocol: the volume of the initial
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solution, the cooling rate (CR) from the seeding point to -40 oC, the initial Me2SO
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control of the cryoprotectants penetration in tissues is crucial for avoiding both ice
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formation and toxicity damage in tissues [10]. Several methods of measurements of
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cryoprotectant permeation in tissues haven been attempted so far: differences on density
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[11-12], electric impedance [13-14] or refractive index [15]. Imaging methods (mostly
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nuclear magnetic resonance) have also been applied to monitor the cryoprotectants
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concentration inside tissues [16-19]. Herein we used an imaging technique based on X-ray
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computed tomography in order to assess the equilibration of the CPA inside the tissue,
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previously developed by our group [20]. The low energies used in our device (75 keV) makes
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it an excellent way to visualize and quantify the concentration of dimethyl sulfoxide
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(Me2SO), one of the most used CPAs in the field of fertility preservation, thanks to the high
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electronic density of this molecule due to its sulfur atom [20].
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1. Materials and Methods
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2.1. Experimental design
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To optimize the freezing protocol for human ovarian tissue, we used bovine ovaries, due to
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their similarities with human ovaries in follicle size and growth pattern and stroma
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composition [21]. A total of 6 ovaries were cut into strips, cryopreserved using different
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freezing protocols and then analyzed by X-ray computed tomography to calculate the
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cryoprotectant concentrations in the fragments.
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2.2. Collection of ovarian tissue
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Ovaries were obtained from one-year-old heifers (n=6), sacrificed in a local slaughterhouse 4
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(Matadero del Sur, Sevilla, Spain). They were transported to our laboratory in a saline
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solution at 37 oC and processed within the following three hours. The ovaries were cut in a
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half and the medulla was removed. The cortex was then cut in 5x5x2-3 mm3 strips
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(supplementary material, Table S1).
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2.3. Freezing-thawing protocol
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2.3.1. Control protocol
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Freezing of bovine ovarian tissue samples was performed using a variation of Dolmans et al.
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protocol [22]. Briefly, one fragment was introduced in a cryovial (Nalgene, USA) containing
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1.8 mL of the freezing solution (minimal essential medium + GlutamaxTM (MEM; Gibco,
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Invitrogen, Belgium) supplemented with 10 % /
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cryovial was then placed in the freezing machine (Bio-Cool IV, model BCIV40D; FTS Systems,
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USA) at 0 oC and held for 15 min. The sample was subsequently cooled to -5.5 oC at 2 oC/min
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and held for another 15 min. The ice seeding was performed by touching the side of the
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cryovials at the height of the vehicle solution with some previously cooled forceps, until
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growth of ice crystals was observed. Ice was allowed to grow for another 15 min inside the
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ethanol bath. Afterwards, the cryovial was cooled down to -40 oC at 0.3 oC/min. The fast
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cooling (20 oC/min) until -150 oC was performed by exposing the cryovial to liquid nitrogen
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vapors, at 2 cm of height. The sample was finally quenched and stored in the liquid nitrogen
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tank. This protocol was considered as the control protocol for the optimization process.
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Me2SO (Sigma Aldrich, Spain)). The
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2.3.2. Experimental variations Table 1 shows the six different protocols we created by varying the four parameters that can 5
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solution, the cooling rate, the initial cryoprotectant concentration, and the temperature and
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time of the tissue incubation. The total volume of the vehicle Me2SO solution was studied
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and two volumes compared (1.8 mL and 0.8 mL). We also varied the cooling rate from the
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temperature of the seeding until -40 oC. This cooling rate (CR) controls the process of the
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controlled rate slow cooling: the rate of the extracellular ice formation and the CPA
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permeation and dehydration of the tissue. The control group uses a CR of 0.3 oC/min;
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variations tested were 0.1 oC/min and 1 oC/min. In addition, the initial CPA concentration
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used in the solution is also a limiting factor for the concentration achieved in the tissue at
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the end of the controlled rate slow cooling process. The initial concentration of the control
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group was 10 % / Me2SO. We compared the effect on the tissue permeation of a lower
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concentration of 5 % /
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important to highlight that the seeding temperature must vary according to the initial
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Me2SO concentration (Table 1): the higher the concentration, the lower the seeding
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temperature (Ts). The temperature of the seeding for each concentration was calculated
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experimentally as the highest temperature which permitted the formation of ice at the top
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of the vial. Finally, the influence of the incubation temperature and time was studied. The
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equilibration of the tissue samples in the freezing solution should last enough time to
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ensure the CPA penetration throughout the tissue without inducing cell damage caused by
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the CPA toxicity. The incubation of the tissue in the freezing vehicle solution is done at the
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first step of the cooling process and consists of 15 min at 0 oC in the control group. This
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control incubation process compared to exposure at room temperature for 10 min and then
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5 more min at 0 oC.
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Me2SO, and a higher concentration of 20 % / Me2SO. It is
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Table 1. Variation of the different parameters for the optimization of the controlled rate slow
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cooling protocol. Control
Volume 0.8 mL
CR o 0.1 C/min
CR o 1 C/min
Volume
1.8 mL
0.8 mL
1.8 mL
1.8 mL
Cooling rate (*Ts to -40 o C)
0.3 C/min
0.3 C/min
0.1 C/min
1 C/min
0.3 C/min
0.3 C/min
0.3 C/min
[Me2SO] (% / ) *Ts
10 % / o -5.5 C
10 % / o -5.5 C
10 % / o -5.5 C
10 % / o -5.5 C
5 % / o -3 C
20 % / o -9 C
10 % / o -5.5 C
o
0 C 15 min
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0 C 15 min
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**Incubation
0 C 15 min
0 C 15 min
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20 % / Me2SO 1.8 mL
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Incubation RT 10 min
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Parameters
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0 C 15 min
1.8 mL
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RT 10 min o 0 C 5min
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*Ts: temperature of the manual ice seeding.
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**Incubation refers to the first stage of the controlled rate slow cooling protocol, before the ice seeding.
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Similarly to our control group, in the six treatments, one ovarian tissue fragment was put in
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a cryovial containing the freezing solution, which was then loaded in the Bio-Cool. After the
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cryovial was cooled down to -40 oC, the fast cooling (20 oC/min) until -150 oC was performed
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by exposing the cryovial to liquid nitrogen vapors. The samples were quenched and stored
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in the liquid nitrogen tank.
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2.4. CT Imaging
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The assessment of the CPA concentration inside the frozen tissue samples was determined
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with the NanoCT device (Bioscan NanoCT®, USA; currently Mediso, Hungary), located at the
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Centro Nacional de Aceleradores (CNA, Spain). The X-ray attenuation was proved to be
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proportional to the Me2SO concentration, as it was published by our group [20]. All images 7
ACCEPTED MANUSCRIPT were acquired under the following parameters: a current of 106 mA for a voltage of 75 kV,
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500 ms of exposition time per projection and 360 projections per rotation. Each image
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required a total acquisition time of 3 min. The images were reconstructed using a cone-
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beam filtered-back projection algorithm to a spatial resolution of 0.2 mm. In order to avoid
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the rewarming of the samples, the CT imaging of the cryopreserved tissues was performed
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below the glass transition temperature (~ - 140 oC), with a specific cooling equipment
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described elsewhere [20]. In brief, it consists of nitrogen gas pre-cooled with liquid nitrogen
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which cools the insulating container in which the cryovial is placed during the measurement.
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The temperature is monitored by a thermocouple located out of the CT area to scan.
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Three different software programs were used during the image processing: Nucline
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software was used for acquisition (Mediso, Hungary); IVS Image Processing software for
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reconstruction (Invicro, USA); and PMOD 3.8 software for analysis (PMOD Technologies LLC,
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Switzerland).
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2.5. Statistical analysis
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Student’s t-test was used to compare Me2SO concentrations in tissue samples calculated
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from the CT images. Values of at least p<0.05 were considered statistically significant.
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3. Results
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In order to evaluate the CPA concentration and distribution, the frozen tissue samples,
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always kept at -140 oC, were analyzed by X-ray computed tomography. The PMOD software 8
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permits to obtain the average X-ray attenuation within a certain VOI (volume of interest),
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which dimensions (3x3x1 mm3 in our case) are chosen by the user. The X-ray attenuation
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values obtained are expressed in
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ray absorption coefficients of the samples [23]. Since there is a linear relation between the
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X-ray attenuation and the Me2SO concentration [20], before analyzing the tissue, we
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previously obtained a calibration curve by imaging different concentrations of Me2SO and
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performing a linear regression analysis. In this way, the coefficients
, which are arbitrary units proportional to the X-
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∗ Att +
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in:
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and
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were obtained, where [Me SO] is the Me2SO concentration (in % / ) and Att is the
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average X-ray attenuation (in
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y-intercept of the curve were:
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% / ), with a regression coefficient " = 0.9993.
). The experimental values for the slope
= 0.423 ± 0.016 (in
% /
!
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and the
= - 0.54 ± 0.03 (in
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Figure 1 shows the CT images of bovine ovarian tissues cryopreserved with the control
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procedure and the different variations of the protocol. The spatial resolution of all images is
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0.2 mm. We used a color scale from PMOD software (cold scale) to visualize the images, in
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which the lowest attenuation corresponds to a dark blue color and the highest attenuation
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to an intense red color, passing through green, yellow and orange colors for intermediate
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attenuation values. The typical structure of extracellular ice formed during the controlled
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rate slow cooling protocol was observed in all the cryovials of each group, showing a low
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attenuation (dark blue color). This extracellular ice, which appears in a striped shape as
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dendritic crystals, brings about a solute rejection. As a result, small islands with very high
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Me2SO concentration are formed, especially in the inner part of the vials, showing an
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intense red color.
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characteristic of tissues used in each variation of the control group are described in Table S1
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at the supplementary material.
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A 3x3x1 mm3 pink VOI is located in the tissue. The number and
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Figure 1. CT images of bovine ovarian tissues: optimization of a conventional controlled rate slow
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cooling protocol. Spatial resolution of 0.2 mm. Pink 3x3x1 mm3 volume of interest (VOI) is located in
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the tissue. All images are at -140 oC. The parameters of the controlled rate slow cooling protocol
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varied were: (a) volume (1.8 mL for the control group and 0.8 mL), (b) cooling rate (0.3 oC/min for
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the control group, 0.1 oC/min and 1 oC/min), (c) initial Me2SO concentration (10 % /
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control group, 5 % / and 20 % / ) and (d) temperature of incubation (15 min at 0 oC for the
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control group and 10 min at room temperature followed of 5 min at 0 oC).
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The quantitative study of the average Me2SO concentration for bovine ovarian tissue is
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shown in Table 2. The average Me2SO concentration obtained in the bovine tissue for the
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control group is 7.50 ± 0.14 % / . Obviously, the distribution of the Me2SO is not uniform
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inside the tissue. Therefore, the minimum and maximum Me2SO within the VOI located
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inside the tissue region is also given in the table. Moreover, the value of the minimum
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concentration gives us an idea of the possibility of forming ice in the less concentrated parts
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of the tissue. The results obtained with the different experimental variations are analyzed
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following.
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Table 2. Average % / Me2SO data of each sample calculated from a 3x3x1 mm3 cube within the
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tissue area. Data presented as average (± standard error) and percentage range (min – max Me2SO
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concentration found within the VOI). The Me2SO concentration is calculated from a calibration curve
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X-rays attenuation versus % / Me2SO.
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232 Samples
[Me2SO] (% $/$)
Min – Max [Me2SO] (% $/$) 11
7.50 (0.14)
0.32 – 32.44
Volume 0.8 mL (n=2)
7.8 (0.4)
1.2 – 16.3
CR 0.1 oC/min (n=2)
9.8 (0.8)
0.5 – 45.5
CR 1 oC/min (n=2)
8.8 (0.8)
2.0 – 18.4
5 % / Me2SO (n=2)
*4.7 (0.3)
20 % / Me2SO (n=2)
*19.8 (1.0)
Incubation RT 10 min (n=2)
*9.4 (0.2)
*p<0.05 when compared with the control group.
10.7 – 38.0 2.6 – 24.6
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0.0 – 19.2
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Control (n=6)
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3.1.Volume
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Figure 1.a shows the CT images obtained when we varied the volume: 1.8 mL (control
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group), and 0.8 mL, volume of UCL protocol. No apparent differences in the concentration
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were found between both images. Moreover, according to data of Table 2, when decreasing
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the volume of the solution to 0.8 mL, the average Me2SO concentration obtained was 7.8 ±
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0.4 % / , very similar to that of the control group (7.50 ± 0.14 % / ).
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3.2. Cooling rate
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The CT images for the parameter of the cooling rate are shown in Figure 1.b. It is not
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possible to see any difference from the images, although in the image of the fastest CR (1
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quantitative result for each cooling rate is compared in Table 2. There seems to be an
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increase of the average concentration when slowing down the cooling process to 0.1
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C/min), a less number of ‘islands’ of high Me2SO concentration is observed. The
C/min, with a concentration of 9.8 ± 0.8 % /
Me2SO (p = 0.3). An increase in the 12
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the much longer period of time necessary for cooling down to -40 oC may increase the
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likelihood of intracellular ice, which could explain the high variability expressed in the range,
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from 0.5 – 45.5 % / Me2SO. When increasing the cooling rate to 1 oC/min, the average
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concentration was 8.8 ± 0.8 % / Me2SO, lower than that observed at 0.1 oC/min. In this
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case, the ice formation is too fast and tissue does not have time enough to be permeated by
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the cryoprotectant. We also observed a decrease on the average size of the ice crystals as a
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consequence of this higher CR, with a thickness of 0.29 ± 0.04 mm versus 0.33 ± 0.05 mm in
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the case of the control group. However, the size of the dendritic crystals was maintained the
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same in the case of the cooling rate of 0.1 oC/min, with an average thickness of 0.33 ± 0.04
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mm.
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3.3. Initial Me2SO concentration
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Figure 1.c shows the CT images studying the influence of the initial Me2SO concentration.
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Evident differences between the three images are observed, showing a darker blue color for
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the 5 % / Me2SO concentration, indicating a lower concentration, while a green color
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appears along the vial in the case of the 20 % / Me2SO concentration, and therefore a
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higher concentration. This could imply a less likely of ice formation in tissues cryopreserved
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with the protocol using a higher Me2SO concentration. If we analyze the concentration
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achieved in the tissues using different initial concentrations of Me2SO, both results differ
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with the average concentration obtained for the control group (p<0.05). In the case of the
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lowest concentration used, 5 % / Me2SO, the average concentration obtained was close
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to the initial value: 4.7 ± 0.3 % / Me2SO. When using 20 % / Me2SO, there is also an
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final Me2SO concentration achieved in the tissue is determined by the initial concentration
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of the solution. It must be highlighted that the range of tissue CPA concentrations in this
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protocol was very homogeneous, with a minimum concentration of 10.7 % / Me2SO,
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indicating that every part of the tissue was likely permeated with a sufficient CPA
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concentration to avoid the formation of ice.
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3.4. Incubation temperature and time
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Figure 1.d shows the impact of the tissue incubation for Me2SO equilibration. No apparent
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differences were observed when compared the CT images between these two protocols.
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Nevertheless, the increase in the temperature of the incubation (room temperature instead
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of 0 oC) resulted in an increase of the average concentration to 9.4 ± 0.2 % / Me2SO
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(p<0.05).
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4. Discussion
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Even though there are more than 100 live births from transplanted ovarian tissue
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cryopreserved by controlled rate cooling slow, there is evidence of the damage of the
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follicle population associated with this cryopreservation procedure [1, 5]. To better
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understand the source of this cryodamage, we used bovine ovarian tissue and a CT scan to
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assess the final Me2SO concentration achieved in the tissue fragments.
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Initially, we tried to reproduce the freezing protocol applied by Dolmans et al. [22], but since
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we use the Bio-Cool to freeze our samples, the CT images showed that ice formed very fast 14
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necessary. In such adapted protocol, we observed a lower Me2SO concentration of 7.5
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% / Me2SO compared to 11 % / Me2SO found in human ovarian tissue samples with
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similar size and frozen with Dolmans et al. [22] protocol (Gallardo et al., unpublished data).
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This could be due to the different characteristics of the tissues and especially to the
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different cooling device used (ethanol bath instead of liquid nitrogen based CRF).
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Nevertheless, in the tissue samples from both species, the CPA distribution in the tissue was
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clearly not homogeneous and it is true that regions with low concentrations of Me2SO
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would be more prone to crystallization.
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We then set to determine the influence of four different parameters in the outcome of the
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controlled rate slow cooling. No statistically significant differences were found when using a
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lower volume of the solution (0.8 mL vs 1.8 mL). The total volume of the solution influences
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the time it takes the ice to reach the tissue from the top of the vial, where the seeding is
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always performed, to the bottom, where the tissue is always located. The dehydration of
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the tissue and its CPA loading critically depends on this fact. However, this variation in
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volume does not seem sufficient to observe any difference in the concentration or
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distribution of Me2SO within the tissues. The variations of the cooling rate (0.1 oC/min and 1
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lower thickness on the dendritic crystals obtained when the cooling rate was increased to 1
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followed of a second step at 0 oC for 5 more min, resulted in a significant increase in the
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C/min vs 0.3 oC/min) did not show significant differences either, although it was observed a
C/min. The increase of the incubation temperature at room temperature for 10 min
15
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Nevertheless, the concentration range found in the tissues pointed out to likely ice
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formation in some parts of it. Furthermore, it is well known that the toxicity of the CPA is
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increased at higher temperatures [24], which could affect the follicle population and the
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viability of the tissue after transplantation.
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The parameter that mostly influenced permeation seemed to be the initial Me2SO
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concentration in the freezing solution, to which the tissue was exposed for 15 minutes at
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0°C prior to cooling: a significant difference in the final Me2SO concentration was observed
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when comparing initial concentrations of 5, 10 and 20 % / Me2SO. An average of 19.8
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% / Me2SO was observed in the tissue when using 20 % / Me2SO. Also, the range of
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CPA concentrations within the tissue, when using 20 % / Me2SO, was very homogeneous,
329
with a minimum of more than 10 % / : the grade and homogeneity of the equilibration of
330
the cryoprotectant could avoid ice nucleation thoroughly in the tissue fragment, and may
331
result in improved cryoprotection and a decrease in the damage of the follicle population. A
332
concern of this protocol would again be toxicity resulting of this higher CPA concentration,
333
since it has been reported that Me2SO concentrations above 10 % / could have negative
334
effects on some kind of cells [25]. Nevertheless, any toxic effects might be reduced,
335
considering that the initial incubation time is short and performed at low temperature (0
336
o
337
exponential correlation between CPA toxicity and temperature [24]. For instance, it has
338
been reported that the number of morphologically normal follicles in pieces of zebu ovarian
339
tissue increased when the Me2SO concentration of the freezing solution was increased to
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C). Then, once cooling has started, the risk of toxicity is furtherly minimized, due to the
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3M (~ 21 % / ) [26]. Another concern about using high Me2SO concentrations might be
341
the cellular damage produced by the osmotic disruption. Morphological and viability
342
analysis were studied on human ovarian tissue after being cryopreserved by this protocol
343
(20 % /
344
cryopreserved by the conventional slow cooling protocols (10 % / Me2SO). This work has
345
been presented on a conference and a manuscript is under preparation [27].
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Me2SO). Preliminary tests show no difference when compared with samples
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In the light of these results, the following modifications to the control protocol could be
348
implemented to achieve higher Me2SO concentrations: a volume of 1.8 mL of 20 % /
349
Me2SO vehicle solution, cooling down to -5.5 oC at 2 oC/min with a cooling device based on
350
an alcohol bath (when using a device based on liquid nitrogen cool down to -8 oC), seeding
351
and holding for 15 min, slow cooling down to -40 oC at 0.3 oC/min and fast cooling to -150
352
o
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cryoprotection. Further morphological and viability studies, performing histology and
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immunological analysis of tissue samples after cryopreservation and transplantation are
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necessary.
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C/min. It is yet to be determined if said adaptations in the protocol will result in improved
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5. Conclusions
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The capability of X-ray Computed Tomography to 3D map the concentration of Me2SO, a
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widespread cryoprotectant, opens unforeseen possibilities in the field of cryopreservation.
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3D mapping process can be done in a wide range of temperatures (from room to
361
cryogenics), as a straightforward consequence, ice crystal and fractures can also be detected
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by the same strategy. These characteristics constitute an essential tool for the design and 17
ACCEPTED MANUSCRIPT optimization of any cryopreservation process, representing a step forward to the challenge
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of tissue and organ cryopreservation. This technology was used in this work to understand
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the influence of different parameters of the controlled rate slow cooling protocol for bovine
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ovarian tissue cryopreservation, giving as result an optimized protocol and a better
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understanding of the conventional cryopreservation protocol used for ovarian tissue.
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The two parameters that showed to have more influence on the final Me2SO concentration
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achieved on tissues were the initial concentration of the Me2SO solution and the
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temperature and time of the tissue incubation, both showing significant differences with the
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control groups. For the concrete case of increasing the Me2SO concentration up to
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20 % / , apart from achieving a higher average Me2SO concentration within the tissues,
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the minimum value of the Me2SO concentration found was also quite high comparing to the
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other groups, which might provide a better protection of tissues against the possibility of ice
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formation.
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Conflict of interest
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The author declares no conflict of interest.
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Acknowledgements
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J. De la Fuente for his assistance in the management of the bovine ovarian tissue. This work
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has been partially supported by the Junta de Andalucía, Proyectos de Investigación de
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Excelencia (P08-CTS-03965), Siemens Healthcare S.L.U. and Retos-Colaboración RTC-2016-
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4733-1 (MINECO, Spain). C.A. Amorim is Research Associate, FRS - FNRS. 18
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References
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2 Amorim CA, Dolmans MM, David A, Jaeger J, Vanacker J, Camboni A, Van
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2013; 99(6): 1503-1513. 9 Schubert B, Canis M, Darcha C, Artonne C, Smitz J, Grizard G. Follicular growth and
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21 Amorim CA, Rondina D, Rodrigues APR, Gonçalves PBD, de Figueiredo JR, Giorgetti A.
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Cryopreservation of isolated ovine primordial follicles with propylene glycol and
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glycerol. Fertil Steril 2004; 81(1): 735-740.
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22 Dolmans MM, Jadoul P, Gilliaux S, Amorim CA, Luyckx V, Squifflet J, Donnez J, Van
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Langendonckt A. A review of 15 years of ovarian tissue bank activities. J Assist
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23 Adams JE. Quantitative computed tomography. European journal of radiology 2009; 71(3): 415-424.
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24 Elmoazzen HY, Poovadan A, Law GK, Elliott JA, McGann LE, Jomha NM. Dimethyl
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sulfoxide toxicity kinetics in intact articular cartilage. Cell Tissue Bank 2007; 8(2):
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125.
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25 Best BP. Cryoprotectant toxicity: facts, issues, and questions. Rejuv Res 2015; 18(5): 21
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422-436.
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26 Lucci CM, Kacinskis MA, Lopes LHR, Rumpf R, Báo SN. Effect of different
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cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos
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indicus) ovarian tissue. Theriogenology 2004; 61(6): 1101-1114. 27 Gallardo M, Paulini F, Corral A, Balcerzyk M, Lucci CM, Merola M, Gallego ALP,
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Dolmans MM, Risco R, Amorim CA. Utilization of a new freezing protocol containing
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a higher DMSO concentration to cryopreserve human ovarian tissue. Society for Low
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Temperature Biology (SLTB) Science Meeting 2017, Cambridge, United Kingdom.
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Manuscript in preparation.
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Figures and tables captions
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Figure 1.CT images of bovine ovarian tissues: optimization of a conventional controlled rate
468
slow cooling protocol. Spatial resolution of 0.2 mm. Pink 3x3x1 mm3 volume of interest
469
(VOI) is located in the tissue. All images are at -140 oC. The parameters of the controlled rate
470
slow cooling protocol varied were: (a) volume (1.8 mL for the control group and 0.8 mL), (b)
471
cooling rate (0.3 oC/min for the control group, 0.1 oC/min and 1 oC/min), (c) initial Me2SO
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concentration (10 % /
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temperature of incubation (15 min at 0 oC for the control group and 10 min at room
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temperature followed of 5 min at 0 oC).
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for the control group, 5 % /
and 20 % / ) and (d)
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Table 1. Variation of the different parameters for the optimization of the controlled rate
477
slow cooling protocol. 22
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Table 2. Average % / Me2SO data of each sample calculated from a 3x3x1 mm3 cube
480
within the tissue area. Data presented as average (± standard error) and percentage range
481
(min – max Me2SO concentration found within the VOI). The Me2SO concentration is
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calculated from a calibration curve X-rays attenuation versus % / Me2SO.
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Supplementary material
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Table S1. Characterization and experiments of the bovine ovarian tissue samples used for
487
the different cooling protocols. Dimensions (mm3)
Cooling protocol
2235
5x5x2.4
Optimization CRSC, Control
2236
5x5x2.6
Optimization CRSC, Control
2237
5x5x2.2
Optimization CRSC, Cooling rate
2238
5x5x2.2
Optimization CRSC, Cooling rate
2239
5x5x2.0
Optimization CRSC, Cooling rate
2240
5x5x2.2
Optimization CRSC, Cooling rate
2241
5x5x2.5
Optimization CRSC, Control
2242
5x5x2.2
Optimization CRSC, Control
2243
5x5x2.3
Optimization CRSC, Volume
2244
5x5x2.2
Optimization CRSC, Volume
2245
5x5x2.4
Optimization CRSC, % / Me2SO
2246
5x5x2.5
Optimization CRSC, % / Me2SO
2247
5x5x2.0
Optimization CRSC, % / Me2SO
2248
5x5x2.1
Optimization CRSC, % / Me2SO
2249
5x5x2.8
Optimization CRSC, Control
5x5x2.6
Optimization CRSC, Control
5x5x2.6
Optimization CRSC, Incubation
5x5x2.4
Optimization CRSC, Incubation
2250 2251 2252
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* CRSC = controlled rate slow cooling
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ACCEPTED MANUSCRIPT Highlights:
Ovarian tissue cryopreservation as fertility preservation options for cancer patients. Monitoring of cryopreservation procedures by X-ray Computed Tomography.
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Understanding and optimization of controlled rate slow cooling protocols for tissue cryopreservation. Dimethyl sulfoxide concentration proportional to X ray attenuation at low voltage.
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Assessment of dimethyl sulfoxide concentration and 3D distribution in cryopreserved ovarian tissue by X-ray Computed Tomography.
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S0093-691X(18)30428-X
DOI:
10.1016/j.theriogenology.2018.06.031
Reference:
THE 14613
To appear in:
Theriogenology
Received Date: 13 April 2018 Revised Date:
27 June 2018
Accepted Date: 29 June 2018
Please cite this article as: Corral A, Balcerzyk M, Gallardo M, Amorim CA, Parrado-Gallego Á, Risco Ramó, An optimized controlled rate slow cooling protocol for bovine ovarian tissue cryopreservation by means of X-ray computed tomography, Theriogenology (2018), doi: 10.1016/ j.theriogenology.2018.06.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT Title page Information
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An optimized controlled rate slow cooling protocol for bovine ovarian tissue
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cryopreservation by means of X-ray computed tomography
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Ariadna Corral1, Marcin Balcerzyk1, Miguel Gallardo2, Christiani A. Amorim3, Ángel Parrado-
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Gallego1, Ramón Risco1,4
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Affiliations:
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1
Centro Nacional de Aceleradores (Universidad de Sevilla-CSIC-Junta de Andalucía), Calle
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Thomas Alva Edison 7, 41092, Sevilla, Spain
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2
Ginemed Clínicas Sevilla, Calle Farmaceutico Murillo Herrera 3, 41010, Sevilla, Spain
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3
Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique,
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Université Catholique de Louvain, Avenue Mounier 52, Bte. B1.52.02, 1200, Brussels,
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Belgium
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Sevilla, Camino Descubrimientos S/N, Isla Cartuja, 41092, Sevilla, Spain
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Departamento de Física Aplicada III, Escuela Técnica Superior de Ingeniería, Universidad de
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Corresponding author:
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Prof Ramon Risco
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Departamento de Física Aplicada III, Escuela Técnica Superior de Ingeniería, Universidad de
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Sevilla, Camino Descubrimientos s/n, Isla Cartuja, 41092, Sevilla, Spain
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Phone Number: +34 954.48.61.85, +34 636.21.19.91
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FAX number: +34 95.448.60.03
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address:
[email protected]
ACCEPTED MANUSCRIPT Abstract
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Cryopreservation and subsequent transplantation of ovarian tissue is the only option to
27
preserve fertility in certain patients facing gonadotoxic treatment. So far, cryopreservation
28
of ovarian tissue has been carried out mostly by a controlled rate slow cooling process,
29
typically known as slow freezing. Even though there are still some concerns about the
30
iatrogenic damage on the follicle population, this technique has been used in the more than
31
100 live births reported to date. It is well known that the control of the cryoprotectant
32
loading in the tissue is crucial to in a cryopreservation procedure. We have used the
33
technology of X-ray computed tomography to assess the concentration and distribution of
34
dimethyl sulfoxide (one of the cryoprotectants most used in fertility preservation) inside
35
pieces of bovine ovarian tissue after its cryopreservation. The low voltage used in our device
36
(75 kV) and the high electronic density of this cryoprotectant makes the X-ray attenuation
37
proportional to its concentration. By assessing and comparing the permeation and
38
homogeneity of the cryoprotectant inside ovarian tissue fragments subjected to a controlled
39
rate slow cooling process, we have characterized the effect of variations in the main
40
parameters involved in the process, with the goal of achieving an optimized protocol with
41
higher permeation of the cryoprotectant in the tissue. The most promissory results were
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obtained by increasing the initial concentration of dimethyl sulfoxide in the vehicle solution
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from 10 to 20 % / .
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Keywords
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CT imaging, Fertility preservation, Cryoprotectant visualization, Tissue permeation
47 1
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ACCEPTED MANUSCRIPT Introduction
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Cryopreservation of ovarian tissue is the only option of fertility preservation for certain
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cancer patients, such as pre-pubertal girls, for whom being subjected to a hormonal
52
stimulation cycle for oocyte preservation is not possible [1]. Advantages of ovarian tissue
53
cryopreservation are the high tolerance of the follicle population to freezing and thawing,
54
and the possibility for immediate surgical collection of the tissue, done at any time of the
55
women’s cycle [1].
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The most widely employed and successful method of ovarian tissue cryopreservation for
58
fertility preservation is a controlled rate cooling slow cooling, known as slow freezing [1-5],
59
with more than 100 cases of live births after transplantation reported [6-7]. Despite this
60
promising data, current slow freezing protocols are empirical adaptations from the ones
61
used for oocyte and embryo cryopreservation [5]. As a result, when this cooling process is
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employed in ovarian tissue, a more complex and dense biological system, it can damage the
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follicle population and the ovarian stroma. This iatrogenic damage can affect the viability of
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the tissue after transplantation [8-9].
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The aim of this study was to optimize a conventional controlled rate slow cooling protocol
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for human ovarian tissue, focusing on achieving a more homogeneous distribution of the
68
cryoprotectant (CPA) inside the tissue fragments during the cryopreservation procedure. For
69
that purpose, we carried out a series of experiments with bovine ovarian tissue varying the
70
following parameters of the controlled rate slow cooling protocol: the volume of the initial
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solution, the cooling rate (CR) from the seeding point to -40 oC, the initial Me2SO
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ACCEPTED MANUSCRIPT concentration and the temperature and time of incubation of the initial solution. The
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control of the cryoprotectants penetration in tissues is crucial for avoiding both ice
74
formation and toxicity damage in tissues [10]. Several methods of measurements of
75
cryoprotectant permeation in tissues haven been attempted so far: differences on density
76
[11-12], electric impedance [13-14] or refractive index [15]. Imaging methods (mostly
77
nuclear magnetic resonance) have also been applied to monitor the cryoprotectants
78
concentration inside tissues [16-19]. Herein we used an imaging technique based on X-ray
79
computed tomography in order to assess the equilibration of the CPA inside the tissue,
80
previously developed by our group [20]. The low energies used in our device (75 keV) makes
81
it an excellent way to visualize and quantify the concentration of dimethyl sulfoxide
82
(Me2SO), one of the most used CPAs in the field of fertility preservation, thanks to the high
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electronic density of this molecule due to its sulfur atom [20].
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1. Materials and Methods
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2.1. Experimental design
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To optimize the freezing protocol for human ovarian tissue, we used bovine ovaries, due to
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their similarities with human ovaries in follicle size and growth pattern and stroma
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composition [21]. A total of 6 ovaries were cut into strips, cryopreserved using different
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freezing protocols and then analyzed by X-ray computed tomography to calculate the
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cryoprotectant concentrations in the fragments.
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2.2. Collection of ovarian tissue
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Ovaries were obtained from one-year-old heifers (n=6), sacrificed in a local slaughterhouse 4
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(Matadero del Sur, Sevilla, Spain). They were transported to our laboratory in a saline
96
solution at 37 oC and processed within the following three hours. The ovaries were cut in a
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half and the medulla was removed. The cortex was then cut in 5x5x2-3 mm3 strips
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(supplementary material, Table S1).
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2.3. Freezing-thawing protocol
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2.3.1. Control protocol
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Freezing of bovine ovarian tissue samples was performed using a variation of Dolmans et al.
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protocol [22]. Briefly, one fragment was introduced in a cryovial (Nalgene, USA) containing
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1.8 mL of the freezing solution (minimal essential medium + GlutamaxTM (MEM; Gibco,
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Invitrogen, Belgium) supplemented with 10 % /
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cryovial was then placed in the freezing machine (Bio-Cool IV, model BCIV40D; FTS Systems,
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USA) at 0 oC and held for 15 min. The sample was subsequently cooled to -5.5 oC at 2 oC/min
108
and held for another 15 min. The ice seeding was performed by touching the side of the
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cryovials at the height of the vehicle solution with some previously cooled forceps, until
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growth of ice crystals was observed. Ice was allowed to grow for another 15 min inside the
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ethanol bath. Afterwards, the cryovial was cooled down to -40 oC at 0.3 oC/min. The fast
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cooling (20 oC/min) until -150 oC was performed by exposing the cryovial to liquid nitrogen
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vapors, at 2 cm of height. The sample was finally quenched and stored in the liquid nitrogen
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tank. This protocol was considered as the control protocol for the optimization process.
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Me2SO (Sigma Aldrich, Spain)). The
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2.3.2. Experimental variations Table 1 shows the six different protocols we created by varying the four parameters that can 5
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119
solution, the cooling rate, the initial cryoprotectant concentration, and the temperature and
120
time of the tissue incubation. The total volume of the vehicle Me2SO solution was studied
121
and two volumes compared (1.8 mL and 0.8 mL). We also varied the cooling rate from the
122
temperature of the seeding until -40 oC. This cooling rate (CR) controls the process of the
123
controlled rate slow cooling: the rate of the extracellular ice formation and the CPA
124
permeation and dehydration of the tissue. The control group uses a CR of 0.3 oC/min;
125
variations tested were 0.1 oC/min and 1 oC/min. In addition, the initial CPA concentration
126
used in the solution is also a limiting factor for the concentration achieved in the tissue at
127
the end of the controlled rate slow cooling process. The initial concentration of the control
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group was 10 % / Me2SO. We compared the effect on the tissue permeation of a lower
129
concentration of 5 % /
130
important to highlight that the seeding temperature must vary according to the initial
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Me2SO concentration (Table 1): the higher the concentration, the lower the seeding
132
temperature (Ts). The temperature of the seeding for each concentration was calculated
133
experimentally as the highest temperature which permitted the formation of ice at the top
134
of the vial. Finally, the influence of the incubation temperature and time was studied. The
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equilibration of the tissue samples in the freezing solution should last enough time to
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ensure the CPA penetration throughout the tissue without inducing cell damage caused by
137
the CPA toxicity. The incubation of the tissue in the freezing vehicle solution is done at the
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first step of the cooling process and consists of 15 min at 0 oC in the control group. This
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control incubation process compared to exposure at room temperature for 10 min and then
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5 more min at 0 oC.
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Me2SO, and a higher concentration of 20 % / Me2SO. It is
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Table 1. Variation of the different parameters for the optimization of the controlled rate slow
143
cooling protocol. Control
Volume 0.8 mL
CR o 0.1 C/min
CR o 1 C/min
Volume
1.8 mL
0.8 mL
1.8 mL
1.8 mL
Cooling rate (*Ts to -40 o C)
0.3 C/min
0.3 C/min
0.1 C/min
1 C/min
0.3 C/min
0.3 C/min
0.3 C/min
[Me2SO] (% / ) *Ts
10 % / o -5.5 C
10 % / o -5.5 C
10 % / o -5.5 C
10 % / o -5.5 C
5 % / o -3 C
20 % / o -9 C
10 % / o -5.5 C
o
0 C 15 min
o
0 C 15 min
o
**Incubation
0 C 15 min
0 C 15 min
o
o
o
o
20 % / Me2SO 1.8 mL
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0 C 15 min
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0 C 15 min
1.8 mL
o
RT 10 min o 0 C 5min
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*Ts: temperature of the manual ice seeding.
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**Incubation refers to the first stage of the controlled rate slow cooling protocol, before the ice seeding.
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Similarly to our control group, in the six treatments, one ovarian tissue fragment was put in
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a cryovial containing the freezing solution, which was then loaded in the Bio-Cool. After the
150
cryovial was cooled down to -40 oC, the fast cooling (20 oC/min) until -150 oC was performed
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by exposing the cryovial to liquid nitrogen vapors. The samples were quenched and stored
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in the liquid nitrogen tank.
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2.4. CT Imaging
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The assessment of the CPA concentration inside the frozen tissue samples was determined
156
with the NanoCT device (Bioscan NanoCT®, USA; currently Mediso, Hungary), located at the
157
Centro Nacional de Aceleradores (CNA, Spain). The X-ray attenuation was proved to be
158
proportional to the Me2SO concentration, as it was published by our group [20]. All images 7
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160
500 ms of exposition time per projection and 360 projections per rotation. Each image
161
required a total acquisition time of 3 min. The images were reconstructed using a cone-
162
beam filtered-back projection algorithm to a spatial resolution of 0.2 mm. In order to avoid
163
the rewarming of the samples, the CT imaging of the cryopreserved tissues was performed
164
below the glass transition temperature (~ - 140 oC), with a specific cooling equipment
165
described elsewhere [20]. In brief, it consists of nitrogen gas pre-cooled with liquid nitrogen
166
which cools the insulating container in which the cryovial is placed during the measurement.
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The temperature is monitored by a thermocouple located out of the CT area to scan.
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Three different software programs were used during the image processing: Nucline
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software was used for acquisition (Mediso, Hungary); IVS Image Processing software for
171
reconstruction (Invicro, USA); and PMOD 3.8 software for analysis (PMOD Technologies LLC,
172
Switzerland).
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2.5. Statistical analysis
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Student’s t-test was used to compare Me2SO concentrations in tissue samples calculated
176
from the CT images. Values of at least p<0.05 were considered statistically significant.
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3. Results
179 180
In order to evaluate the CPA concentration and distribution, the frozen tissue samples,
181
always kept at -140 oC, were analyzed by X-ray computed tomography. The PMOD software 8
ACCEPTED MANUSCRIPT 182
permits to obtain the average X-ray attenuation within a certain VOI (volume of interest),
183
which dimensions (3x3x1 mm3 in our case) are chosen by the user. The X-ray attenuation
184
values obtained are expressed in
185
ray absorption coefficients of the samples [23]. Since there is a linear relation between the
186
X-ray attenuation and the Me2SO concentration [20], before analyzing the tissue, we
187
previously obtained a calibration curve by imaging different concentrations of Me2SO and
188
performing a linear regression analysis. In this way, the coefficients
, which are arbitrary units proportional to the X-
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∗ Att +
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in:
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and
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were obtained, where [Me SO] is the Me2SO concentration (in % / ) and Att is the
192
average X-ray attenuation (in
193
y-intercept of the curve were:
194
% / ), with a regression coefficient " = 0.9993.
). The experimental values for the slope
= 0.423 ± 0.016 (in
% /
!
) and
and the
= - 0.54 ± 0.03 (in
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Figure 1 shows the CT images of bovine ovarian tissues cryopreserved with the control
197
procedure and the different variations of the protocol. The spatial resolution of all images is
198
0.2 mm. We used a color scale from PMOD software (cold scale) to visualize the images, in
199
which the lowest attenuation corresponds to a dark blue color and the highest attenuation
200
to an intense red color, passing through green, yellow and orange colors for intermediate
201
attenuation values. The typical structure of extracellular ice formed during the controlled
202
rate slow cooling protocol was observed in all the cryovials of each group, showing a low
203
attenuation (dark blue color). This extracellular ice, which appears in a striped shape as
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dendritic crystals, brings about a solute rejection. As a result, small islands with very high
205
Me2SO concentration are formed, especially in the inner part of the vials, showing an
206
intense red color.
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characteristic of tissues used in each variation of the control group are described in Table S1
208
at the supplementary material.
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A 3x3x1 mm3 pink VOI is located in the tissue. The number and
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210 10
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Figure 1. CT images of bovine ovarian tissues: optimization of a conventional controlled rate slow
212
cooling protocol. Spatial resolution of 0.2 mm. Pink 3x3x1 mm3 volume of interest (VOI) is located in
213
the tissue. All images are at -140 oC. The parameters of the controlled rate slow cooling protocol
214
varied were: (a) volume (1.8 mL for the control group and 0.8 mL), (b) cooling rate (0.3 oC/min for
215
the control group, 0.1 oC/min and 1 oC/min), (c) initial Me2SO concentration (10 % /
216
control group, 5 % / and 20 % / ) and (d) temperature of incubation (15 min at 0 oC for the
217
control group and 10 min at room temperature followed of 5 min at 0 oC).
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for the
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The quantitative study of the average Me2SO concentration for bovine ovarian tissue is
220
shown in Table 2. The average Me2SO concentration obtained in the bovine tissue for the
221
control group is 7.50 ± 0.14 % / . Obviously, the distribution of the Me2SO is not uniform
222
inside the tissue. Therefore, the minimum and maximum Me2SO within the VOI located
223
inside the tissue region is also given in the table. Moreover, the value of the minimum
224
concentration gives us an idea of the possibility of forming ice in the less concentrated parts
225
of the tissue. The results obtained with the different experimental variations are analyzed
226
following.
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Table 2. Average % / Me2SO data of each sample calculated from a 3x3x1 mm3 cube within the
229
tissue area. Data presented as average (± standard error) and percentage range (min – max Me2SO
230
concentration found within the VOI). The Me2SO concentration is calculated from a calibration curve
231
X-rays attenuation versus % / Me2SO.
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232 Samples
[Me2SO] (% $/$)
Min – Max [Me2SO] (% $/$) 11
7.50 (0.14)
0.32 – 32.44
Volume 0.8 mL (n=2)
7.8 (0.4)
1.2 – 16.3
CR 0.1 oC/min (n=2)
9.8 (0.8)
0.5 – 45.5
CR 1 oC/min (n=2)
8.8 (0.8)
2.0 – 18.4
5 % / Me2SO (n=2)
*4.7 (0.3)
20 % / Me2SO (n=2)
*19.8 (1.0)
Incubation RT 10 min (n=2)
*9.4 (0.2)
*p<0.05 when compared with the control group.
10.7 – 38.0 2.6 – 24.6
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0.0 – 19.2
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Control (n=6)
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3.1.Volume
236
Figure 1.a shows the CT images obtained when we varied the volume: 1.8 mL (control
237
group), and 0.8 mL, volume of UCL protocol. No apparent differences in the concentration
238
were found between both images. Moreover, according to data of Table 2, when decreasing
239
the volume of the solution to 0.8 mL, the average Me2SO concentration obtained was 7.8 ±
240
0.4 % / , very similar to that of the control group (7.50 ± 0.14 % / ).
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3.2. Cooling rate
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The CT images for the parameter of the cooling rate are shown in Figure 1.b. It is not
244
possible to see any difference from the images, although in the image of the fastest CR (1
245
o
246
quantitative result for each cooling rate is compared in Table 2. There seems to be an
247
increase of the average concentration when slowing down the cooling process to 0.1
248
o
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C/min), a less number of ‘islands’ of high Me2SO concentration is observed. The
C/min, with a concentration of 9.8 ± 0.8 % /
Me2SO (p = 0.3). An increase in the 12
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250
the much longer period of time necessary for cooling down to -40 oC may increase the
251
likelihood of intracellular ice, which could explain the high variability expressed in the range,
252
from 0.5 – 45.5 % / Me2SO. When increasing the cooling rate to 1 oC/min, the average
253
concentration was 8.8 ± 0.8 % / Me2SO, lower than that observed at 0.1 oC/min. In this
254
case, the ice formation is too fast and tissue does not have time enough to be permeated by
255
the cryoprotectant. We also observed a decrease on the average size of the ice crystals as a
256
consequence of this higher CR, with a thickness of 0.29 ± 0.04 mm versus 0.33 ± 0.05 mm in
257
the case of the control group. However, the size of the dendritic crystals was maintained the
258
same in the case of the cooling rate of 0.1 oC/min, with an average thickness of 0.33 ± 0.04
259
mm.
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3.3. Initial Me2SO concentration
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Figure 1.c shows the CT images studying the influence of the initial Me2SO concentration.
263
Evident differences between the three images are observed, showing a darker blue color for
264
the 5 % / Me2SO concentration, indicating a lower concentration, while a green color
265
appears along the vial in the case of the 20 % / Me2SO concentration, and therefore a
266
higher concentration. This could imply a less likely of ice formation in tissues cryopreserved
267
with the protocol using a higher Me2SO concentration. If we analyze the concentration
268
achieved in the tissues using different initial concentrations of Me2SO, both results differ
269
with the average concentration obtained for the control group (p<0.05). In the case of the
270
lowest concentration used, 5 % / Me2SO, the average concentration obtained was close
271
to the initial value: 4.7 ± 0.3 % / Me2SO. When using 20 % / Me2SO, there is also an
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13
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273
final Me2SO concentration achieved in the tissue is determined by the initial concentration
274
of the solution. It must be highlighted that the range of tissue CPA concentrations in this
275
protocol was very homogeneous, with a minimum concentration of 10.7 % / Me2SO,
276
indicating that every part of the tissue was likely permeated with a sufficient CPA
277
concentration to avoid the formation of ice.
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3.4. Incubation temperature and time
280
Figure 1.d shows the impact of the tissue incubation for Me2SO equilibration. No apparent
281
differences were observed when compared the CT images between these two protocols.
282
Nevertheless, the increase in the temperature of the incubation (room temperature instead
283
of 0 oC) resulted in an increase of the average concentration to 9.4 ± 0.2 % / Me2SO
284
(p<0.05).
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4. Discussion
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Even though there are more than 100 live births from transplanted ovarian tissue
288
cryopreserved by controlled rate cooling slow, there is evidence of the damage of the
289
follicle population associated with this cryopreservation procedure [1, 5]. To better
290
understand the source of this cryodamage, we used bovine ovarian tissue and a CT scan to
291
assess the final Me2SO concentration achieved in the tissue fragments.
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292 293
Initially, we tried to reproduce the freezing protocol applied by Dolmans et al. [22], but since
294
we use the Bio-Cool to freeze our samples, the CT images showed that ice formed very fast 14
ACCEPTED MANUSCRIPT over the whole cryovial (data not showed). Thus, an adaptation of the freezing protocol was
296
necessary. In such adapted protocol, we observed a lower Me2SO concentration of 7.5
297
% / Me2SO compared to 11 % / Me2SO found in human ovarian tissue samples with
298
similar size and frozen with Dolmans et al. [22] protocol (Gallardo et al., unpublished data).
299
This could be due to the different characteristics of the tissues and especially to the
300
different cooling device used (ethanol bath instead of liquid nitrogen based CRF).
301
Nevertheless, in the tissue samples from both species, the CPA distribution in the tissue was
302
clearly not homogeneous and it is true that regions with low concentrations of Me2SO
303
would be more prone to crystallization.
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We then set to determine the influence of four different parameters in the outcome of the
306
controlled rate slow cooling. No statistically significant differences were found when using a
307
lower volume of the solution (0.8 mL vs 1.8 mL). The total volume of the solution influences
308
the time it takes the ice to reach the tissue from the top of the vial, where the seeding is
309
always performed, to the bottom, where the tissue is always located. The dehydration of
310
the tissue and its CPA loading critically depends on this fact. However, this variation in
311
volume does not seem sufficient to observe any difference in the concentration or
312
distribution of Me2SO within the tissues. The variations of the cooling rate (0.1 oC/min and 1
313
o
314
lower thickness on the dendritic crystals obtained when the cooling rate was increased to 1
315
o
316
followed of a second step at 0 oC for 5 more min, resulted in a significant increase in the
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C/min vs 0.3 oC/min) did not show significant differences either, although it was observed a
C/min. The increase of the incubation temperature at room temperature for 10 min
15
ACCEPTED MANUSCRIPT Me2SO concentration when compared with the control protocol (15 min at 0 oC).
318
Nevertheless, the concentration range found in the tissues pointed out to likely ice
319
formation in some parts of it. Furthermore, it is well known that the toxicity of the CPA is
320
increased at higher temperatures [24], which could affect the follicle population and the
321
viability of the tissue after transplantation.
322
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The parameter that mostly influenced permeation seemed to be the initial Me2SO
324
concentration in the freezing solution, to which the tissue was exposed for 15 minutes at
325
0°C prior to cooling: a significant difference in the final Me2SO concentration was observed
326
when comparing initial concentrations of 5, 10 and 20 % / Me2SO. An average of 19.8
327
% / Me2SO was observed in the tissue when using 20 % / Me2SO. Also, the range of
328
CPA concentrations within the tissue, when using 20 % / Me2SO, was very homogeneous,
329
with a minimum of more than 10 % / : the grade and homogeneity of the equilibration of
330
the cryoprotectant could avoid ice nucleation thoroughly in the tissue fragment, and may
331
result in improved cryoprotection and a decrease in the damage of the follicle population. A
332
concern of this protocol would again be toxicity resulting of this higher CPA concentration,
333
since it has been reported that Me2SO concentrations above 10 % / could have negative
334
effects on some kind of cells [25]. Nevertheless, any toxic effects might be reduced,
335
considering that the initial incubation time is short and performed at low temperature (0
336
o
337
exponential correlation between CPA toxicity and temperature [24]. For instance, it has
338
been reported that the number of morphologically normal follicles in pieces of zebu ovarian
339
tissue increased when the Me2SO concentration of the freezing solution was increased to
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C). Then, once cooling has started, the risk of toxicity is furtherly minimized, due to the
16
ACCEPTED MANUSCRIPT 340
3M (~ 21 % / ) [26]. Another concern about using high Me2SO concentrations might be
341
the cellular damage produced by the osmotic disruption. Morphological and viability
342
analysis were studied on human ovarian tissue after being cryopreserved by this protocol
343
(20 % /
344
cryopreserved by the conventional slow cooling protocols (10 % / Me2SO). This work has
345
been presented on a conference and a manuscript is under preparation [27].
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Me2SO). Preliminary tests show no difference when compared with samples
346
In the light of these results, the following modifications to the control protocol could be
348
implemented to achieve higher Me2SO concentrations: a volume of 1.8 mL of 20 % /
349
Me2SO vehicle solution, cooling down to -5.5 oC at 2 oC/min with a cooling device based on
350
an alcohol bath (when using a device based on liquid nitrogen cool down to -8 oC), seeding
351
and holding for 15 min, slow cooling down to -40 oC at 0.3 oC/min and fast cooling to -150
352
o
353
cryoprotection. Further morphological and viability studies, performing histology and
354
immunological analysis of tissue samples after cryopreservation and transplantation are
355
necessary.
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C/min. It is yet to be determined if said adaptations in the protocol will result in improved
357
5. Conclusions
358
The capability of X-ray Computed Tomography to 3D map the concentration of Me2SO, a
359
widespread cryoprotectant, opens unforeseen possibilities in the field of cryopreservation.
360
3D mapping process can be done in a wide range of temperatures (from room to
361
cryogenics), as a straightforward consequence, ice crystal and fractures can also be detected
362
by the same strategy. These characteristics constitute an essential tool for the design and 17
ACCEPTED MANUSCRIPT optimization of any cryopreservation process, representing a step forward to the challenge
364
of tissue and organ cryopreservation. This technology was used in this work to understand
365
the influence of different parameters of the controlled rate slow cooling protocol for bovine
366
ovarian tissue cryopreservation, giving as result an optimized protocol and a better
367
understanding of the conventional cryopreservation protocol used for ovarian tissue.
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368
The two parameters that showed to have more influence on the final Me2SO concentration
370
achieved on tissues were the initial concentration of the Me2SO solution and the
371
temperature and time of the tissue incubation, both showing significant differences with the
372
control groups. For the concrete case of increasing the Me2SO concentration up to
373
20 % / , apart from achieving a higher average Me2SO concentration within the tissues,
374
the minimum value of the Me2SO concentration found was also quite high comparing to the
375
other groups, which might provide a better protection of tissues against the possibility of ice
376
formation.
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Conflict of interest
379
The author declares no conflict of interest.
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Acknowledgements
382
J. De la Fuente for his assistance in the management of the bovine ovarian tissue. This work
383
has been partially supported by the Junta de Andalucía, Proyectos de Investigación de
384
Excelencia (P08-CTS-03965), Siemens Healthcare S.L.U. and Retos-Colaboración RTC-2016-
385
4733-1 (MINECO, Spain). C.A. Amorim is Research Associate, FRS - FNRS. 18
ACCEPTED MANUSCRIPT 386 387
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25 Best BP. Cryoprotectant toxicity: facts, issues, and questions. Rejuv Res 2015; 18(5): 21
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26 Lucci CM, Kacinskis MA, Lopes LHR, Rumpf R, Báo SN. Effect of different
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indicus) ovarian tissue. Theriogenology 2004; 61(6): 1101-1114. 27 Gallardo M, Paulini F, Corral A, Balcerzyk M, Lucci CM, Merola M, Gallego ALP,
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Dolmans MM, Risco R, Amorim CA. Utilization of a new freezing protocol containing
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a higher DMSO concentration to cryopreserve human ovarian tissue. Society for Low
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Temperature Biology (SLTB) Science Meeting 2017, Cambridge, United Kingdom.
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Manuscript in preparation.
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Figures and tables captions
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Figure 1.CT images of bovine ovarian tissues: optimization of a conventional controlled rate
468
slow cooling protocol. Spatial resolution of 0.2 mm. Pink 3x3x1 mm3 volume of interest
469
(VOI) is located in the tissue. All images are at -140 oC. The parameters of the controlled rate
470
slow cooling protocol varied were: (a) volume (1.8 mL for the control group and 0.8 mL), (b)
471
cooling rate (0.3 oC/min for the control group, 0.1 oC/min and 1 oC/min), (c) initial Me2SO
472
concentration (10 % /
473
temperature of incubation (15 min at 0 oC for the control group and 10 min at room
474
temperature followed of 5 min at 0 oC).
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for the control group, 5 % /
and 20 % / ) and (d)
475 476
Table 1. Variation of the different parameters for the optimization of the controlled rate
477
slow cooling protocol. 22
ACCEPTED MANUSCRIPT 478
Table 2. Average % / Me2SO data of each sample calculated from a 3x3x1 mm3 cube
480
within the tissue area. Data presented as average (± standard error) and percentage range
481
(min – max Me2SO concentration found within the VOI). The Me2SO concentration is
482
calculated from a calibration curve X-rays attenuation versus % / Me2SO.
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Supplementary material
485
Table S1. Characterization and experiments of the bovine ovarian tissue samples used for
487
the different cooling protocols. Dimensions (mm3)
Cooling protocol
2235
5x5x2.4
Optimization CRSC, Control
2236
5x5x2.6
Optimization CRSC, Control
2237
5x5x2.2
Optimization CRSC, Cooling rate
2238
5x5x2.2
Optimization CRSC, Cooling rate
2239
5x5x2.0
Optimization CRSC, Cooling rate
2240
5x5x2.2
Optimization CRSC, Cooling rate
2241
5x5x2.5
Optimization CRSC, Control
2242
5x5x2.2
Optimization CRSC, Control
2243
5x5x2.3
Optimization CRSC, Volume
2244
5x5x2.2
Optimization CRSC, Volume
2245
5x5x2.4
Optimization CRSC, % / Me2SO
2246
5x5x2.5
Optimization CRSC, % / Me2SO
2247
5x5x2.0
Optimization CRSC, % / Me2SO
2248
5x5x2.1
Optimization CRSC, % / Me2SO
2249
5x5x2.8
Optimization CRSC, Control
5x5x2.6
Optimization CRSC, Control
5x5x2.6
Optimization CRSC, Incubation
5x5x2.4
Optimization CRSC, Incubation
2250 2251 2252
SC
M AN U
EP
* CRSC = controlled rate slow cooling
AC C
488
RI PT
ID Number
TE D
486
24
ACCEPTED MANUSCRIPT Highlights:
Ovarian tissue cryopreservation as fertility preservation options for cancer patients. Monitoring of cryopreservation procedures by X-ray Computed Tomography.
RI PT
Understanding and optimization of controlled rate slow cooling protocols for tissue cryopreservation. Dimethyl sulfoxide concentration proportional to X ray attenuation at low voltage.
AC C
EP
TE D
M AN U
SC
Assessment of dimethyl sulfoxide concentration and 3D distribution in cryopreserved ovarian tissue by X-ray Computed Tomography.
1