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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


Optimisation of cryopreservation technologies to cellular therapeutics

A recent paper sets out the options for cryopreservation of cell therapies now and gives pointers to future research. Prof Barry Fuller et al summarise (Cell and Gene, Therapy Insights, June '17)  the applications of cryopreservation technologies to cellular therapeutics and their delivery. The ability to hold products in readiness for logistical, regulatory or potency reasons is highlighted and it usually involves cryopreservation. However, cryopreservation itself poses biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. 

The review sets out the history of applied cryopreservation, current understanding of the various processes involved in storage at cryogenic temperatures, and the challenges for the reliable uses of cryopreservation within cell therapy. The authors include observations on slow freezing: "Successful cryopreservation was also found to be influenced by the kinetics of the cooling process itself. As often practiced today, slow cooling (where slow as a relative term applies to cooling rates of between about -0.3 Deg C min-1 and -2 Deg C min-1) were found by empirical observation to relate to good success." And in another section:  " other cell therapies have entered the arena within the broad area of hematopoietic stem cell replacement, slow cooling cryopreservation has maintained its beneficial role, but with some addition to detail in terms of protocol management."

To read the full article

Procurement, processing and storage approaches for umbilical cord blood and tissue

A new article in Cell and Gene Therapy Insights neatly sets out the advances in procurement and storage in the umbilical cord blood and tissue bio processing field. The paper by Dr Qasim Rafiq of London's UCL, Kate Sneddon and members of Biovault Technical Ltd, a leading tissue bank in the UK, looks at regenerative medicine related to both autologous and allogeneic therapeutic applications and describes the current challenges and how these may be met to augment translation and use of therapeutics.

Cord blood and tissue banking is increasingly popular because of the stem cells they contain and the clinical research associated with such material is advancing. Umbilical cord blood (UCB) is a source of hematopoietic stem cells (HSCs) and is clinically applied for the reconstitution of hematopoiesis. Umbilical cord tissue (UCT) contains mesenchymal stem cells (MSCs) that have been widely investigated for applications in tissue restoration and repair, and the treatment of immune mediated disorders.

UCB is defined as the blood that remains in the umbilical cord and placenta following neonatal delivery, and UCT as the cord itself. Both tissues are procured immediately after birth in a process not deleterious to mother or child. In fact, banking of these tissues may confer future health benefits to the corresponding child or immediate family as they potentially comprise an exact or partial human leukocyte antigen match, reducing complications such as graft-versus-host disease upon transplantation. Additionally, both tissues may be applied in allogeneic models; UCT MSCs are immune evasive and donor-recipient mismatches can be well tolerated in transplantation.

The potential applications of UCB HSCs and UCT MSCs in both autologous and allogeneic therapeutics has encouraged the coexistence, sometimes within the same organization, of two tissue storage models. Parents may choose to publicly bank their UCB for use for any patient, or store privately for the event of disease in the immediate family.

Over 750,000 UCB units are thought to be banked publicly worldwide, with over 4 million banked in private family storage arrangements. There are a considerable number of clinical trials concerning the application of UCT MSCs, creating demand for UCT processing strategies that maintain the viability, efficacy and safety of derived therapeutics.

The authors conclude that to elucidate strategies that are optimal with respect to cell functionality, patient outcomes and cost to be developed, UCB volume reduction, antibiotic-free bio processes, low DMSO in cryopreservation media, and improved antimicrobial strategies may aid the readiness of UCB and UCT banking.

For further information
Citation: Cell Gene Therapy Insights 2017;3(6), 489-508. Published: Aug 4 2017

You can read the whole article by registering (free) at Cell and Gene Therapy Insights online 

Planer freezers at Biovault



Launch of ShipsLog3™. Monitor the temperature history of your vapour shipper throughout its transit.

Planer is pleased to announce the launch of the ShipsLog3™ temperature data logger. The new ShipsLog3™ is the latest edition of the well-established ShipsLog™ product range which has been providing protection to samples inside vapour and dry shippers for over 15 years. The ShipsLog3™ temperature data logger provides an accurate and downloadable temperature history of your vapour shipper throughout its transit.

  • The ShipsLog3™ data logger stores more than 32,000 temperature readings from the thermocouple fitted to the inside of the shipper lid providing early alerts of potential warming issues.
  • The ShipsLog3™ is easily set up using free downloadable EasyLog software*, allowing the user to set alarm thresholds, logging rates and immediate, delayed or ‘push to start’ operation.
  • The same software can be used to download and view data from the data logger at the end of transit which can be graphed, printed and exported to provide a permanent audit trail.
  • Programming and downloading of data can be achieved by plugging the USB data logger into the USB port on the PC or using the ShipsLog3™ Data transfer unit+.
  • The ShipsLog3™ is fitted with a replaceable lithium metal battery which will typically give two years logging life.

There are two versions of ShipsLog3™ available;

  • The standard ShipsLog3™ comes complete with a high contrast LCD display showing a variety of information at the touch of a button.
  • Alternatively if you require the extra security then a 21CFR compliant version of the ShipsLog3™ is also available.

 Both versions of the ShipsLog3™ are fitted with red and green status LEDs for immediate recognition of the logger status.

Find out more about ShipsLog3™

*EasyLog software or EasyLog CFR software required dependent on model of ShipsLog3™
+Sold separately (not compatible with 21CFR version)

International Society for Fertility Preservation Meeting 2017

The 5th Biennial World Congress of the International Society for Fertility Preservation is being held in Vienna, Austria from November 16-18, 2017

The aims of this congress are to present and discuss important topics in the field such as advances in ovarian cortex transplantation, assessment of ovarian reserve, endometriosis, in vitro follicle growth maturation and artificial ovaries, medical protecting from chemotherapy, oocyte and embryo vitrification, reimplantation of ovarian tissue and many more cutting edge topics. There will be an opportunity for all to share protocols and clinical knowledge; to find areas for future multi-disciplinary scientific research and clinical collaboration; to spread education and to facilitate fertility preservation awareness. Fertility preservation is a substantial quality of life issue for young cancer survivors.

During the meeting there will also be practical workshops on cryopreservation of ovarian tissue with hands-on lab and clinics, tissue collection, lab preparation and tissue freezing, methods and transplantation techniques.

Planer equipment has been at the forefront of the development of the techniques used in fertility preservation with Planer controlled rate freezers being used in the cryopreservation of ovarian tissue in the majority of patients that have had successful births. We will therefore be attending this meeting to demonstrate our latest cryogenic equipment to this important and growing customer group.

You may have seen these earlier articles:-
Cryopreservation and transplantation course - human ovarian tissue
First British woman gives birth after an ovarian tissue transplant

For further information about the ISFP meeting please visit:
The 5th World Congress of the International Society for Fertility Preservation




Cryo Preserved Vascular Grafts - new work from the Hradec Králové Hospital , Czech Republic

A recent study on cryo preserved grafts has been undertaken by Dr Pavel Mericka et al who performed a detailed evaluation of the factors around the discard of cryopreserved vascular allografts. The study looked at those manufactured in 2014 and 2015 at the Tissue Bank department of the University Hospital.  This tissue bank, operational since 1952, have been cryopreserving specimens since the 1980's using, inter alia, Planer freezers. In the study, all vascular grafts transported to the unit for processing and cryopreservation were included. This amounted to 78 vessels collected from 28 donors. In 2014, 57 vessels were collected, and in 2015, 30.

All the tissue vessels were collected aseptically during multi-organ harvests from deceased donors, aseptically processed in a clean room, cryopreserved in medium containing 10% DMSO and stored in the vapour phase of liquid nitrogen.

The researchers were looking at factors causing discard which included: contra indications of tissue post collection; deviation from the SOPs, problems in processing and storage and microbial contamination and in addition any severe adverse reactions by the graft recipient. The results from the total of 30 grafts not released for clinical use showed that microbial contamination occurred in 22 cases and was the largest cause. In their conclusions the authors point to these results confirming previous experiences - that the most frequent cause of tissue discard was microbial contamination. They do emphasise though the possibility of occurrence of other causes such as contraindication of tissue harvest found at autopsy or unexplained adverse reaction in the recipient of an organ originating from the same donor which should not be neglected.

The Havel Králové Tissue Bank is an institutional member of the European Association of Tissue Banks and annually delivers around 1000 grafts that are used in University and county hospitals as well as in surgeons' private practices.

For more information

Study on fetal birth weights: vitrified group versus slow-frozen

Does the embryo freezing method have an impact on the birth weight of newborns from thawed embryo transfer cycles?  Prof Zhou and others from the Sun Yat-sen University Hospital in Guangzhou, China presented their finding as a poster (G17-1069) at ESHRE 2017 in Geneva. Their conclusion from a Large Scale Retrospective Study in Two Centers was that vitrification of cleavage stage embryos resulted in lower fetal birth weight compared to a slow-freezing method.

Prof Canquan Zhou (pictured here), Dr Fang Gu and others from the centres for reproductive medicines at the First Affiliated Hospital and the Jiangmen Central Hospital at Sun Yat-sen University in Guangzhou, China studied whether fetal birth weights were lower in a vitrified group than those in a slow-freezing group. A retrospective, two-center study was performed for 1797 singleton neonates born alive over 28 weeks of gestation after cleavage thawed embryo transfer was carried out in two large reproductive medical centers in South China, using the period January 2012 to December 2016. While vitrification was assumed superior to slow-freezing techniques regarding embryo survival rate and live birth rate the question of whether the vitrification cryopreservation method would affect fetal growth is still controversial.

In the study neonatal outcomes including gestational age, gender and birth weight were analysed with 1407 singletons born from vitrified group and 390 from slow-freezing embryo transfer. Although absolute birth weight was comparable between two groups, the adjusted birth weight controlled for gestational age and gender in the vitrified group was lower than in the slow-freezing group;  the rate of macrosomia was significantly decreased in babies from vitrified embryo transfer. The gestational age and the rate of preterm birth were comparable between the two groups. The sex ratio, rate of small for gestational age and large for gestational age , as well as the rate of low birth weight were all comparable between the two cryopreservation methods. Linear regression analysis demonstrated that maternal weight, gestational age, embryo freezing method and infant gender were significantly related to neonatal birth weight.

This was a Retrospective study so the authors point out that research on long-term effects on the health of children are needed. Nevertheless they suggest vitrified embryo transfer seems to give rise to a decrease in neonatal birth weight, suggesting the potential epigenetic changes resulted from the high concentration of cryoprotectants should be carefully considered.

Prof Zhou uses a Planer cryogenic slow rate controllable freezer and the work was funded by the Chinese Ministry of Health through their public welfare scientific research special fund.

Poster (G17-1069) from ESHRE 2017 


Cryopreservation and re-culture of a 2.3 litre biomass

In an article published in Plos One (August 25 2017) UCL researchers gave the first example of a large volume biomass being successfully cryopreserved in a single cassette and re-cultured. It demonstrates that a bioartificial liver device can be cryopreserved, and has wider applications to scale-up large volume cryopreservation.

The study optimised parameters such as excess media concentration and warming rates and used the findings to enable the successful cryopreservation of 2.3 litres of alginate encapsulated liver cell spheroids. The study of large volume cryopreservation is also essential to improve transplant results. Currently organs can only be preserved in a chilled state (typically between 0–4°C) for a maximum of between 4–24 h, depending on the organ. This greatly inhibits successful transplant numbers and outcomes.

The focus in cryopreservation of cells has largely been on small volumes, typically up to 1 ml samples. Exceptions are the cryopreservation of adult stem cells and T-cell therapies, typically in volumes 50-500ml, and ELS and other cell types in cryobags. There have been reports of successful cryopreservation of sheep ovaries and the vitrification of a rabbit kidney but no record of biomasses larger than 500ml cryobags (for cell suspensions), or volumes larger than a few hundred ml for more complex biomasses being cryopreserved in the frozen state.

For further information
Find full article at Plos One

See also

News Stories - 2017