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Progress on a bio-artificial liver

A bio-artificial liver - a BAL - is used as an extra-corporeal organ designed to aid the function of the liver in patients with acute failure and University College London's team are working on one where liver cells are encapsulated in alginate. The challenge as a clinical product is related to immediate availability, because of the unpredictability of disease progression in acute liver failure.

The aim of a recent study, presented at the Society of Low Temperature Biology annual meeting by Eloy Erro, Joana da Silva (both pictured here) et al, was to achieve BAL biomass cryopreservation with different systems for post-thaw culture and recovery, aiming at an off-the-shelf product. HepG2 cells were encapsulated in 1% alginate solution, producing ~500µm beads which were encapsulated and cultured for 12-14 days in a fluidised bed bio reactor (FBB) to obtain sufficient biomass.

Cryopreservation of >1L of alginate cell beads was achieved in a cryo-bag using our controlled rate freezer Kryo 750 and DMSO was used as cryoprotectant with an ice nucleator.

The team concluded that successful cryopreservation of encapsulated liver cell spheroids on a large scale, at 16-48% of a total human liver size (the biomass of 30% liver cell numbers is the likely requirement for such a bio-artificial liver) represented the correct order of magnitude and provided sufficient post-thaw recovery. They also found the process could be easily scalable in freezing bags using the Kryo750 and the same fluidised-bed bioreactor for cell recovery. The use of micro-organoids within an alginate matrix proved an interesting choice for large-scale cryopreservation avoiding problems associated with large tissue freezing. However a more rapid recovery would be ideal, and the team is exploring improvements.

Further information:
Poster - The BAL Team : http://www.ucl.ac.uk/livergroup/theteam.html
Planer's controlled rate freezer: https://planer.com/products/cryo-freezers/medium-crf/kryo-750.html
To download PDF of poster, please click here.

bio artificial liver poster ucl

 

 

 

New controlled rate freezer at Cell Tech Pharmed

controlled rate freezers at CellTech Pharmed

Professor Nasser Agdami
Prof Nasser Aghdami, a user of Planer freezers when he was at Iran's Royan Institute, recently helped to set up a new company Cell Tech Pharmed to meet an increasing demand for stem cell therapy. The focus on several distinct areas - systemic diseases with an underlying inflammatory and immunologic etiology, cardiac and vascular diseases, orthopaedic diseases of the knee, ankle and pelvis where cell therapy products can be locally administered, improving outcomes of bone marrow transplantation and also skin augmentation where current methods are ineffective.

Embryonic stem cells or pluripotent stem cells can differentiate into any cell in the body. Because stem cells in the umbilical cord are less developed than adult stem cells, they don't have to be "matched" as stringently to a patient's human leukocyte antigen, or HLA, type. In the USA for example more than 80,000 unrelated bone marrow and umbilical cord blood transplants have been facilitated since 1987.

Embryonic stem cells or pluripotent stem cells can differentiate into any cell in the body. Because stem cells in the umbilical cord are less developed than adult stem cells, they don't have to be "matched" as stringently to a patient's human leukocyte antigen, or HLA, type. In the USA for example more than 80,000 unrelated bone marrow and umbilical cord blood transplants have been facilitated since 1987.

Planer Kryo 360 Controlled Rate Freezer at CellTech PharmedThe new company has the technical help and financial support of Dr Aghdami's old employer, the Royan Institute, along with the commercial backing of the Barakat Pharmed Company, a large pharmaceutical holding company.  In the pharma world, quality is of course paramount - the Barakat group complies with all common pharmaceutical standards such as the FDA, WHO, EMEA. Likewise in Cell Tech Pharmed who use one of our precision slow freezers, a Kryo 360, (pictured here) for their cell freezing.

 

Professor Brian Grout speaks at Stem Cell Users Group Meeting

We were pleased to be one of the commercial sponsors of this important meeting at Kings College Hospital, London, bringing together more than 60 stem cell practitioners to share knowledge and experiences and discuss current issues and difficulties. For this group, cryopreservation is a key enabling technology contributing to advances in both basic research and clinical practice. Consequently, a strong understanding of the principles underlying cryopreservation protocols is important to group members and we were happy to be able to support Professor Brian Grout*, a widely experienced cryobiologist and recent Chair of the Society for Low Temperature Biology, to contribute to the meeting.

Brian presented the audience with a cryobiologist’s view of the cryochain central to a stem cell user’s activities, moving from cell preparation to end use in the laboratory or on the clinical front-line. The intention was to present the pitfalls experienced by the audience in a cryobiological context and to point out where stronger biological understanding can be used to eliminate costly, and typically unintended, poor practice. Throughout, the concept was that all the cryopreservation team members need to be aware of the biological issues, especially where frozen material has to be recovered by separate groups e.g. clinicians, with the expectation of reproducible, high performance of the frozen product.

In particular, a good deal of attention was given to the benefits of controlled ice nucleation and the risks of damaging temperature rise to samples that are lifted up into the neck of storage vessels when searching for specific samples. Further, the potentially lethal effects of poorly controlled thawing protocols were examined together with some of the risks of microbial contamination of the cryogen in storage vessels and transit shippers.  The need for strong, supporting biological knowledge to underpin successful cryopreservation was underlined and also the continuing need for sustained research and developments in technology; this, to strengthen the level of control available to those using stem cell cryopreservation as a near-routine tool. Positive feedback from the meeting was strong and we are looking forward to continuing our links with this stem cell users group in the coming year.

*Emeritus Professor, Life Sciences, Copenhagen University

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:-

News Stories - 2019

News Stories - 2018

News Stories - 2017