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Evaluation of different protocols for the cryopreservation of mouse ovaries; T. Kolbe et al

Poster from the ISFP congress in Vienna November 2017
Institute of Laboratory Animal Science, University of Veterinary Medicine, Vienna

Evaluation of different protocols for the cryopreservation of mouse ovaries

Cryopreservation of gametes and embryos is a routine method for the long-term preservation of genetic material. The cryopreservation of ovarian tissue offers a valuable addition to these current approaches. The freezing of ovaries with subsequent thawing and surgical engraftment can restore fertility in young women undergoing cancer treatment with chemo- and/or radiotherapy and preserve important lines, strains or races of experimental, domestic and wild animals. During the last decade, several different protocols for the cryopreservation of mouse ovaries were published. In this study we compared four different protocols, which were mostly based on older protocols with some modifications (Liu et al., 2008; Migishima et al., 2003; Wang et al., 2009).

Summary
Ovarian cryopreservation is an easy but invaluable additional tool to archive mouse strains. In summary, all tested protocols used in this study resulted in good pregnancy rates, which did not significantly differ from the pregnancy rate of recipients transplanted with fresh ovaries. Ovarian tissue survived the aforementioned preservation techniques well and resumed follicle development and ovarian cycle after transplantation. Controlled freezing in straws with DMSO as cryoprotectant (protocol Gr. III) resulted in the best performance based on the fast resumption of reproduction and a generally high litter size.

 

Ovarian tissue cryopreservation slow freezing versus vitrification, Nikiforov et al

Dmitry Nikiforov At the world congress of the International Society for Fertility Preservation in Vienna in November important topics such as advances in ovarian cortex transplantation, assessment of ovarian reserve, in vitro follicle growth, medical protection from chemotherapy, reimplantation of ovarian tissue were on the agenda. Slow freezing of cryopreserved ovarian tissue (for fertility preservation in cancer patients) was a hot topic and there were a number of workshops and posters. One such - see below - was from Dmitry Nikiforov at the University of Teramo, Italy and also the Rigshospitalet in Copenhagen.

Dmitry is also an ESHRE/ReproUnion fellow in the Laboratory of Reproductive Biology. He has three years of clinical experience in human embryology and now is working on a doctoral project about ovarian tissue cryopreservation and in-vitro oocyte production from the tissue. At the Laboratory of Reproductive Biology in Copenhagen (under Prof Claus Yding Andersen) he is focusing on isolation of follicles from medulla tissue discarded during cryopreservation of ovarian cortex for further in-vitro culture. Another point of interest is to evaluate an effect of additional substances to the cryopreservation medium, which may improve survival rate of the tissue as well as improve surviving after implantation of the tissue back to the patient.

 

 

 

Ovarian tissue freezing

The fifth World Congress of the International Society for Fertility Preservation (ISFP) took place in November 2017 in Vienna, Austria. Attended by many delegates from around the world topics included Cryopreservation and Reimplantation of Ovarian Tissue, Oocyte and Embryo Freezing, the search for Cancer cells in the Ovaries. And of special interest were the workshops and particularly those given by Professor Christiani Amorim on the cryopreservation of Ovarian Tissue. These workshops included hands on lab and clinical aspects, tissue collection, lab preparation and tissue freezing, storing, thawing and transplantation techniques.

Cancer treatment can save a life but it can also bring infertility; chemo and radio therapy destroy cancer cells but they also destroy eggs and the gonadal toxicity results in early menopause and ovarian failure. UCL Louvain pioneered post-cancer female fertility restoration by freezing ovarian tissue prior to treatment and subsequently reimplanting it. Cryogenically slow freezing a patient’s own ovarian tissue prior to storage, treatment and then reimplanting it afterward has been successful with some 130 children born worldwide, including the world's first at UCL’s Saint-Luc University Hospital in 2004.

Professor Amorim was an Associate Professor at the Brasília University, in Brazil before moving to Belgium. In recent years, she has focused her attention on ovarian tissue transplantation for cancer patients. Her pioneering studies have served as the basis for establishing the field of ovarian tissue engineering and she organised the first study group on reproductive tissue engineering. In her Vienna workshop - which was oversubscribed - she went through and demonstrated the techniques of cryopreservation of ovarian tissue and the slow freezing method a process very much pioneered by UCL Louvain.

In the presentation, Professor Amorim summarised that there have been more than 130 live births using the slow freezing protocols in a controlled rate freezer. This contrasts with only 2 or 3 live births using the vitrification technique. UCL Louvain has itself had 14 live births since she and Professor Donnez first tried the technique.

Workshop on the cryopreservation of Ovarian Tissue
The workshop ran through the established Louvain procedure.

 

Ovarian tissue preparation
An ovarian biopsy is taken, tissue being retrieved from the operating theatre and then prepared in sterile conditions. It is treated with cryopreservatives before slow freezing and cryo storage. Analyses are carried out prior, to check suitability, testing with patient serum, microbiological analysis and via the anatomopathology lab

 

Preparing the cryovial
The treated tissue is placed in vials, marked and sealed and then frozen in a specific protocol in a controlled rate freezer - in this case a Planer Kryo 360.

 

 

Loading the vial in the controlled rate freezer
As can be seen from the protocol below, the vial holder is removed at a critical temperature and is seeded by touching with cold forceps. In this case it was undertaken at -8 Deg C. The computer controlled freezer cools the sample at a pre determined series of temperature rates and gives printable result for medical records and audit.

   

'Seeding' the vial with cold forceps

  

 

Ovarian tissue freezing cooling curve

Once the sample has been controlled frozen it is carefully removed and placed in frozen quarantine at liquid nitrogen temperatures awaiting test results. If all is correct, it is then transferred to a cryo storage tank for longer term storage at liquid nitrogen temperatures; when needed it is retrieved and warmed quickly in a water bath prior to use. It can be many years before the patient is ready to use the sample; in 2015 a Dubai patient had tissue frozen in 2001 thawed giving a successful transplant and a baby girl.

More information:-
Kryo 360 controlled rate freezers:-  
https://planer.com/products/cryo-freezers/small-crf/kryo-360.html
C
ryopreservation of ovarian tissue for fertility preservation in young girls:-
https://planer.com/company/news/latest-news/149-latest-news/869-cryopreservation-of-ovarian-tissue-for-fertility-preservation-in-young-girls.html

For more information on the UCL courses at Louvain, email:
This email address is being protected from spambots. You need JavaScript enabled to view it.

 

 

Fertility 2018

Fertility 2018, the joint Conference of the Association of Clinical Embryologists, British Fertility Society and the Society for Reproduction & Fertility, returns to the Arena Convention Centre (ACC), Liverpool. The ACC is situated on the Kings Waterfront, close to the Grade 1 listed Albert Dock and the World Heritage site at Pier Head.

Now the largest of its kind in the UK, the conference will bring together over 700 fertility experts for a three day programme on the latest scientific and clinical developments and updates in fertility, sexual health and reproductive biology.

Planer will be showcasing our recently launched CT37stax™ Multi Chamber Benchtop Incubator, our DATAssure™ alarm and monitoring system and our Shiplogs3™ temperature data logger. To find out more, please come and see us on stand 13.

For further information

Fertility 2018 is the 11th Joint Conference of the following UK Fertility Societies. Further information about these societies can be found at:-

PIMS - FREE incubator management software released

Planer Incubator Management Software (PIMS) is an easy-to-use management software suite for Planer BT37, CT37stax™ and INC-A20 benchtop CO2 incubators. The software, which can be downloaded from the Planer website, is free for all Planer incubators users.

The intuitive and user friendly suite provides the convenience of controlling multiple Planer CT37stax™ units as well as monitoring key parameters of multiple INC-A20 and BT37 incubators connected to your existing IT network with access from any connected PC. 

Why use PIMS?

  • FREE to download
  • Provides management of all the Planer incubators at a single point
  • Compatible for use with Planer BT37 and INC-A20 incubators
  • Displays real time graphical performance information for each incubator
  • Data logging of all incubator parameters
  • Allows Administrators access to full user management and audit tools
  • Provides full alarm notification management
  • Is fully regulatory legislation (CFR 21 part 11, etc.) compliant

PIMS is free to download - all you need to do is visit any incubator page on the Planer website for details. Alternatively, please feel free to contact the sales team for more details.

 

 

World’s first DATAssure™ alarm and monitoring system installed

Planer recently installed the world’s first DATAssure™ alarm and monitoring system in an ART clinic in a private hospital just outside London. The system comprises of 40 sensor points monitoring their incubators' temperature and CO2 levels, their storage dewars’ temperature and liquid nitrogen levels and their cryo bank's oxygen levels.

The DATAssure™ is the next generation alarm and monitoring system to replace the original Planer DATAcentre system and was launched at ESHRE in July. Part numbers and prices for the European 433 MHz version of the DATAssure™ system will be released in the price lists shortly with the worldwide 2.4Ghz version to follow in early 2018.

The DATAssure™ system provides all of the features you would expect from a modern monitoring system, including a clear colour touchscreen, 12 hour battery back-up and up to 10 years plus data storage. Up to 150 wireless sensors monitoring a wide variety of different environmental and equipment parameters can be connected to the system providing truly independent monitoring throughout the laboratory.

For more details on the new DATAssure™ system, please contact the sales team at Planer.

Effect of Warming Rate On Post-Thaw Recovery

A bioartificial liver is used as an extra-corporeal organ which is designed to supplement the function of the liver in patients with acute liver failure. This is to allow time to find a suitable donor or for the liver to undergo self-repair. The University College London's bio artificial liver consists of liver cells encapsulated in alginate, which have formed spheroids grown in a fluidised bed bioreactor. For patients with acute liver failure an “off-the-shelf treatment needs to be available and to that end Mo Awan, Joana Mendonca-Silva, Eloy Erro and the team have endeavoured to develop a cryopreservation protocol to preserve a large biomass and recover it after thawing.

The aim of recent research, published at the Society for Low Temperature Biology meeting in Cambridge 2017, was to see whether the rate at which the biomass is warmed affects post thaw recovery. The results suggested slow thawing produces better post thaw recovery than when the same volume biomass was fast thawed. This would infer that slow thawing aids recovery of cryopreserved spheroids. The cooling rates for two samples was not the same though: during one cooling the cryo bag cooled faster than the cryo bottle and remained at -50°C for longer. This likely resulted from better heat transfer in bags, and allowed dissipation of latent heat of ice nucleation. This may potentially alter ice crystal formation and have a negative effect on the biomass. Slower warming may also allow less stressful water re-equilibration in the spheroids as the ice melts.

UCL's bio artificial liver team are at the Institute for Liver and Digestive Health, UCL Medical School, Royal Free Campus, London NW3 2PF in the UK

Further information
A PDF of the full poster is available here

 

News Stories - 2017