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Cryopreservation of hepatocyte microbeads for clinical transplantation

Researchers at Kings College London have used Planer freezers for many years. Now Doctors Sharon Lehec and Ragai Mitry are among the authors of a new paper aiming to develop an optimised protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions in their Kryo 10.

Transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of these hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. So the establishment of banks for such cryopreserved microbeads would be an important step in emergency use. The aim of the study was to develop an optimised protocol for this cryopreservation for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated using human and rat hepatocytes. Cryopreservation was performed using a standard King’s College Hospital freezing protocol in a Planer controlled-rate freezer, the Kryo10.

The two freezing solutions which gave better results were studied with human and rat hepatocyte microbeads. Similar effects on cryopreserved microbeads morphology, viability and hepatocyte-functions post thawing were observed over seven days in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; ZVAD), an antioxidant (desferoxamine; DFO), and buffering and mechanical protection (human serum albumin; HSA) on RMBs. ZVAD (60µM) had beneficial effect on cell viability, greater than with DFO (1mM), HSA (2%) and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes, and a lower degree of cell apoptosis were observed with all three cytoprotectants, ZVAD tending to provide the greatest effect.

They concluded that cryopreservation of hepatocyte microbeads using UW solution containing 10% DMSO, 5% glucose and a cytoprotectant such as ZVAD had beneficial effects regarding cellular and physical damage leading to maintenance of cell viability and function post thawing. This initial optimised protocol for cryopreservation of hepatocyte microbeads supports the hypothesis that the development of freezing solutions containing cryoprotective agents and compounds targeting apoptosis during the cryopreservation process could improve the outcome of cryopreservation.

The hope is that, eventually, large amounts of hepatocyte microbeads may be cryopreserved and banked for immediate emergency clinical treatment in conditions such as acute liver failure. The work in the Dhawan lab at Kings College continues and a new PhD starts in April using clinical/GMP grade materials for future banking of these cryopreserved microbeads.

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Cryopreservation protocol for 100 strains of microalgae

The Culture Collection of Freshwater Microalgae, the Coleção de Culturas de Microalgas de Água-Doce, plays an important role in Brazilian micro algal research. It is based at the Universidade Federal de São Carlos ( and was founded in 1977. The collection provides biological materials, substrates and education for a large proportion of past, and indeed current, projects throughout the Brazilian territory. Microalgae are found in most aquatic and many terrestrial ecosystems; their diversity of lifestyles has resulted in a large group of organisms with differing metabolic and functional characteristics; as a result they possess considerable biotechnological potential, particularly in the bioenergy, cosmeceutical and nutraceutical markets.

The establishment of a cryopreserved biobank linked to the culture collection was recently the focus of a PhD project, aiming to reduce the costs associated with the maintenance regime of cultures and the secure maintenance of the collection catalogue. The optimised cryopreservation protocol developed as part of the project has been tested with around 100 strains of microalgae from CCMA-UFSCar, including exemplar taxa across the different taxonomic groups in the collection catalogue, with elevated levels of success, particularly for the smaller taxa, such as the small green algae.

Letícia Piton Tessarolli (pictured above) recently successfully defended this PhD in algal cryobiology under the supervision of Prof Armando Vieira (Universidade Federal de São Carlos UFSCar) and Prof John Day (Scottish Association for Marine Science, University of the Highlands and Islands). Her work was mostly undertaken at the Coleção de Culturas de Microalgas de Água-Doce. Letícia used one of our freezers for some of the work: cryopreservation is regarded as the preferred method for the long-term, genetically and phenotypically stable, storage of stock cultures. The Planer controlled rate freezer used provides adjustable protocols which can be optimised to ensure maximum post-thaw viability

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BT37 Benchtop Incubators at Primary IVF

If Lucy Ing, Lab Manager at Primary IVF’s first clinic in Sydney Australia had to pick her favourite piece of equipment, it would have to be the ‘mini bench top incubators'. Why?

“Because they are home to the little embryos that grow in the lab. Being placed in the centre of the lab means that we walk past these incubators hundreds of times a day, keeping a close watchful eye on the embryos. They provide a safe home for the embryos and work to make an environment similar to how it would be in the female uterus. These incubators constantly glow a warm soothing green (what I like to always see) to indicate that all conditions are ideal and optimal for embryo growth. It also has in-built alarms to quickly alert us if something is slightly out of range, which is an important quality control measure.”

When setting up the Primary IVF labs the Scientific Director, Rosemarie Cullinan, thought carefully about the choice of incubators. “Having had experience with the Planer controlled rate freezers in the 1990s, a reliable workhorse, I had confidence that the BT37s would be built to the same high standard. The in-built battery was also a big plus for me. Along with the sleek design that suits our state-of-the-art laboratories!”

Primary IVF offers Australians IVF treatment through the Australian government Medicare system making IVF accessible and affordable for all Australians.

Since Primary IVF began operations in August 2014, an additional two labs have been established in Melbourne and Brisbane and over 600 babies have been born who started life in the BT37 incubators.

See a profile of Lucy Ing at

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UEARS 2017

At the second recent gathering of the Upper Egypt Assisted Reproduction Conferences in February 2107, 163 lectures were presented by 46 international and Egyptian ART scientists - and attendance was close to 1900 attendees. The main theme was “translational research in ART: basic science to improve clinical practice” and the conference included four workshops. The program covered, from a new perspective, laboratory procedures including for culture media, incubator management, oocytes and embryo manipulation, pH impact and adjustment, quality control, vitrification, embryo selection and other topics.

Participants also had a look at the emerging technologies that may have future applications in ART, including gene editing, stem cell researches, and mitochondrial transfer.  For the first time in this area, the meeting conducted two consensus meetings that focused on controversial issues in the field; the first was to give some recommendations on laboratory air quality and environment and the resulting work will could be published September.

The next UEARC 2018 conference will be held in Cairo on February 21-23, 2018. 


Live births from semen stored for 40 years

Back in 2008 we reported that a child had been born with semen stored for 22 years. We thought we did well - but our record seems to have been well and truly broken.

We wrote then that Mike Kuzminski, a rock musician from Canada with Hodgkin‘s Lymphoma, banked his sperm and forgot about it till 22 years later when he wanted to start a family and learned he was infertile. In 2003 he tracked down the clinic and got the news from them “We totally have you here -- and you owe us $2,000 for storage." And on the 1st of November 2007 a baby boy was born.  The sperm they used had been frozen down in a Planer controlled rate freezer, and kept at liquid nitrogen temperatures.

But Andreas Szell and others report two live births from semen around 40 years old! In 1971, a man aged about 52, banked semen for frozen for long-term storage. In 2012 a patient using the donor sperm became pregnant after a second FET and had two healthy girls delivered. The authors conclusions are that low temperature storage in liquid nitrogen should maintain the viability of cells almost indefinitely because reactions that require molecular motion and/or activation do not occur at −196 °C. The adverse effects of background radiation during storage appear to be negligible since exposure of mouse embryos to the equivalent to 2,000 years of background radiation did not have a detectable effect on their viability.

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A Gap Exists Inside In Vitro Fertilisation Laboratories

The Upper Egypt Assisted Reproduction Conference 2017, produced some interesting discussions but Dr Mohamed Fawzy of the IVF Laboratory, Egypt pinpointed one in particular: current culture media and incubator designs. He says "Culture media for in vitro human embryo development affects not only the embryo development and the subsequent clinical outcomes but also offspring health and gene expressions. Many brands of culture media are commercially available with encouragement from the manufacturers in usage, to get superior results for IVF outcomes. These brands include two paradigms (sequential media and single-step media). Sequential media paradigm formed of three sequences of media, one for fertilization, the second is to culture from zygote to the cleaved embryo until day 3, and the third is for culturing the embryo from day 3 to 5 or 6. This approach of culture is based on the assumption that each stage of embryo development needs different requirements in regard to nutrition, and others. The single-step approach has been built on the assumption that the embryo should be provided with all the nutrients and to let the embryo choose from them. Both paradigms are basically accepted and supportive to embryo development. The emerging evidence in two meta-analyses suggests that both approaches are equal and there is no preferred medium yet. A recent publication that has presented at UEARS 2017 inferred that adding insulin into single-step culture media is associated with better embryological and clinical outcomes. The culture media effect on gene expression is, to some extent, lacking. For that, a gap exists in the current design of culture media and the culture media need revisiting, however, from different perspectives."

Dr Fawzy goes on: "The incubator is a second crucial piece that can affect the human embryo development and the subsequent outcomes. The incubator is responsible for maintaining a non-stressful regulatory environment for the developing embryo. Inside the incubator, pH, gas exchange, recovery time and humidity should be optimal. According to Armstrong et al. in a Cochrane review, they inferred that there is no evidence to support time-lapse superiority over the conventional incubator. However, there is an anecdotal belief that the Benchtop incubators may offer faster recovery of temperature and gas and this, in turn, could improve the results. To date, there is no agreement on which model of Benchtop is superior. This could notify us that a gap exists in defining the best incubator and leaving us to the designer's wishes." He suggests culture media and incubators should be the first items to be validated through the concept of no harm to the embryo, clinical outcomes, and more importantly the offspring health.

The next UEARC 2018 conference will be held in Cairo on February 21-23, 2018. 

Further information
Planer BT37 Benchtop incubator



Tendon decellularisation by freeze thawing

Decellularisation of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for the translation of graft based tendon restoration in clinics. Automation of the decellularisation steps such as freeze-thawing is crucial for the development of more standardised decellularisation protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. Decellularised tissues represent the ideal natural scaffold for many research applications in tissue science and hold great promise as possible transplants for medical applications. Decellularisation of tissues can be performed using physical, chemical and enzymatic methods but to achieve good results for the removal of cells with the conservation of the extracellular matrix, a combination of techniques needs to be explored for the different tissue types to find the optimal method.

Researchers at the Translational Centre for Regenerative Medicine at the University of Leipzig in Germany recently demonstrated that the application of freeze-thaw cycles prior to treatment with detergents enhanced the effectiveness of the decellularisation procedure.  A new paper published in BMC Biotechnology by Susanne Roth, Sina Glauche, Janina Burk and others shows that using a controlled rate freezer gives precision and repeatability to the process.

Automated freeze-thaw cycles performed by the Planer Kryo 360 liquid nitrogen based controlled rate freezer were effective for the freeze-thaw procedures for decellularisation of equine superficial digital flexor tendons. The automation of this key procedure in decellularisation of large tendon samples is an important step towards the processing of large sample quantities under standardised and GMP conditions with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine.

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Cryo safety for the embryologist

Following the recent death of a police officer investigating a leakage of liquid nitrogen (LN2) in Atlanta, Georgia, USA, cryo safety has become a topical issue. A survey of the risks associated with it by Mathew Tomlinson and David Morroll, reported in Human Fertility (, concluded that incidents involving liquid nitrogen were more frequent than expected, and that training and awareness of risks associated with cryo stores was generally lacking. Steve Fleming and Alex Varghese have edited a book with a useful chapter on ‘Cryobank Management’, (by John Ryan) called the Organization and Management of IVF Units, published by Springer Science.

Steve (pictured here) and Alex say that an important starting point in liquid nitrogen cryo safety is the actual design of the cryo store where most handling of liquid nitrogen will occur. Because liquid nitrogen expands rapidly as it boils at -196 ºC, the liquid:gas expansion ratio being approximately 1:700, it can dramatically reduce the concentration of oxygen in the atmosphere within an enclosed or poorly ventilated space, leading to asphyxiation without any warning symptoms. Therefore, it is essential to install an oxygen monitor within a cryo store to provide early visible and audible warning.

It is also well known that liquid nitrogen and its vapour can potentially inflict burns or frostbite, if mishandled. So staff working within a cryo store will need special gloves and safety glasses along with face shields, aprons, fully enclosed shoes and long sleeved clothing. It will be necessary to educate staff in the variety of hazards associated with handling and storing liquid nitrogen. Instructions should include written standard operating procedures (SOPs) and prominently displayed notices within the cryo store, including evacuation maps. In addition to burns and frostbite, the typical hazards that staff may be exposed to include eye injury and/or infection resulting from explosion of straws or cryovials due to rapidly expanding nitrogen upon warming of any liquid nitrogen that may have seeped into them during cryo storage.

In many countries, provision of a licence to provide a clinical embryology service is contingent upon having in place appropriate monitoring, alarms, safety equipment, policies and procedures necessary to deal with unexpected leaks or spills of liquid nitrogen from bulk liquid nitrogen supply tanks or storage dewars. From a European perspective, the European Society of Human Reproduction guidelines for good practice ( cover cryo safety in embryology laboratories in considerable detail. However, not all countries where cryo storage of gametes and embryos is performed have a similar regulatory framework for protection of staff. Under such circumstances, it is very important that cryo store working areas remain well ventilated when in use and that staff do not work alone or investigate any low oxygen alarms on their own.

In an ideal world, nobody would suffer any injuries from working with liquid nitrogen, but in the real world tragedies such as those that occurred recently can happen so we must insist on best practice when handling liquid nitrogen to minimise the risks to ourselves and our colleagues working within embryology laboratories.

Further information

Steven D. Fleming & Alex C. Varghese, Editors, Organization and Management of IVF Units
A Practical Guide for the Clinician  Springer Science  ISBN 978-3-319-29371-4

Cryosafety in the Embryology Laboratory: Download full text PDF

BCGA code on LN2 50 litre dewars