News 2015

Variables in “Passive” Cryopreservative Methods: Standardizing Cell-Based Assays By Reducing Cryopreservation-Induced Variability

Many studies have focused on the effects of cryopreservation on cells, looking at cryoprotectants, freezing rates on cell viability, sub-cellular and biochemical modifications etc. However, generally, what happens in the freezing process is treated as a given. The actual freezing profile - the variation of the freezing rate over time - has not often been precisely investigated; to do so is technically challenging as cryovials are normally 1 mL in volume and do not easily accommodate temperature probes.

Now two authors Drs Barradez and Lekishvili have measured temperature profiles by inserting a thin thermocouple temperature probe through a hole drilled in the cap of the cryovials and recorded the temperature in a passive alcohol freezer in a -80. They compared results with those from a Planer controlled rate freezer. Looking at Recovery and Toxicity they tested effects of freezing profiles on cell functions and demonstrated the strong differences in temperature profiles experienced by cell samples between the two freezing devices. Data suggest that the freezing rates before and after the freezing point, as well as the variability in freezing profiles experiences by the vials, all combine to affect cell recovery - viability and potency. 

Given the stakes in this cell based industry, simple and passive cryopreservation techniques seem not always to provide an adequate enough level of control for optimal cell performances. Temperature profiles experienced by cells during passive cryopreservation in a passive freezing box (e.g., alcohol filled container in a -80) ) can be highly variable from vial to vial as well as batch to batch and may not be optimised. Such variability may well have significant consequences on cell behaviour upon thawing. 

Read the study in the latest issue of BioProcess magazine
http://www.bioprocessintl.com/manufacturing/cell-therapies/variables-in-passive-cryopreservative-methods-standardizing-cell-based-assays-by-reducing-cryopreservation-induced-variability/?utm_source=Planer&utm_medium=Post&utm_campaign=promo

EATB 2015

The European Association of Tissue Bank’s 24th Congress is taking place this year in the beautiful city of Split. The Congress offers professionals involved in tissue, cell banking and transplantation from around the world the opportunity to share knowledge and experience.

This year’s programme will provide ideas, scientific data and guidance for future work as well as standardise the quality and safety of tissue and cell transplantation internationally. There will also be a half-day EATB-WUTBA joint Symposium on Ethics in donation and human application of MPHO.

The Planer sales team will be joining forces with our distributor Mediva. Why not come and see us on Booth 11 and find out more about our DATAcentre (http://www.planer.com/products/alarms-a-monitoring/datacentre.html) monitoring equipment or sample tracking software, Pro-Curo (http://www.planer.com/products/pro-curo.html).

Tendon decellularization

Decellularized tissues represent the ideal natural scaffold for many research applications in tissue science. They also hold great promise as possible transplants for medical applications. Decellularization of tissues can be performed using physical, chemical and enzymatic methods but to achieve good results on the removal of cells and the conservation of the extracellular matrix, a combination of techniques needs to be explored for the different tissue types to find the optimal.

For the dense tissue of tendons, researchers at the University of Leipzig, Germany, recently demonstrated that the application of freeze-thaw cycles prior to treatment with detergents enhances the effectiveness of the decelluralization procedure (Burk et al., 2014).

Dr. Janina Burk’s research group at the Translational Centre for Regenerative Medicine in Leipzig is now working on further improvements of this technique. Using our Planer controlled-rate freezer Kryo 360, they are developing freeze-thaw protocols for standardized decellularization of tendon samples. Preliminary results are promising, showing that the freeze-thaw cycles performed in the controlled-rate freezer are at least as effective as the difficult to control manually performed freezing and thawing. These first results are being published in the degree thesis of biotechnologist Sina Glauche who worked with the group with the Leipzig labs during her studies.

Translational Centre for Regenerative Medicine,
04103 Leipzig http://www.trm.uni-leipzig.de/

Cryopreservation of human cartilage allografts

A new article by Dr Alasdair Kay et al in Cryobiology has just been published. The paper gives the results of Dr Kay's investigations taking a liquidus tracking approach to the cryopreservation of human cartilage allografts. He used our special slow vitrifier - Liquidus Tracker - controlling both the temperature and the concentration of cryoprotectant so that the solution composition "tracked" the liquidus (melting point) line for that system. Ice crystal formation was prevented and the tissue was not exposed to CPA concentrations exceeding those experienced by cells during conventional cryopreservation.

This approach could be particularly appropriate for articular cartilage because chondrocytes in situ are susceptible to damage by the crystallisation of ice. This project aimed to investigate possibilities for a suitable process for tissue to be used in the surgical repair of damaged human knee joints. In this experiment, human articular cartilage was obtained from deceased donors and dimethyl sulphoxide (DMSO) was used as the CPA, with cooling at 0.14°C/min and warming at 0.42°C/min.  A program of increasing DMSO concentration was developed for cooling and this gave satisfactory tissue concentrations. Reduction of DMSO concentration during warming proved inadequate and firm conclusions were restricted by two factors: the broad spread of replicate data and the possibility that there might be significant non-solvent water interfering with DMSO concentration measurements. Empirical trials to optimise the system were inconclusive; the next steps indicated would be to resolve the problems of possible non-osmotic water in human cartilage and the provision of consistent quality tissue. A look at further optimisation of the rewarming phase would also be beneficial. Dr Kay worked for the R&D Tissue Services, NHS Blood and Transplant at Speke, Liverpool. His co authors were Messrs Hoyland, Rooney, Kearney and Pegg . See full details in "Cryobiology"

http://www.ncbi.nlm.nih.gov/pubmed/26012701

News in Ovarian Tissue Freezing

The news that a 27 year old woman in Belgium was the first ever to give birth to a baby using her own pre-adult frozen ovarian tissue, has caused interest around the world. The woman in Belgium was the first ever to give birth to a baby using her own pre-adult frozen ovarian tissue and the procedure may become increasingly important given the increasing number of long-term survivors of haematological diseases diagnosed in childhood. In the delicate process, fragments of ovarian tissue are very carefully frozen down before long term storage in liquid nitrogen. When needed they are thawed and transplanted.  

Planer plc have provided their controlled rate freezers to scientists around the world and they were used in 1984 for the world’s first human frozen birth. The Belgian team involved in this procedure used Planer freezers as did their colleagues in Louvain where the world's first ovarian tissue transplant was undertaken in 2004 – also with a Planer freezer.  

The procedure for freezing the tissue was described by Dr Isabella Demeestere in Human Reproduction journal March 2006 (http://m.humrep.oxfordjournals.org/content/21/8/2010.full). In this latest case, her team removed the ovary when the girl was 13 years old, just before she had begun treatment for cancer. As the chemotherapy needed can destroy ovarian function, they removed the ovary and froze tissue fragments in a Planer Kryo freezer. Tiny strips of ovarian tissue, one to two millimetres thick, were taken from the most productive part of the ovary and cut into sections and treated with cryo-protectants before very careful freezing.  

Scientists around the world choose controlled rate freezing and Planer plc are the world leaders in the area and have helped in many breakthroughs, including in 2014, the world’s first successful freezing and re-implantation of an adult mammal organ – an ovary. In 2004, in the world's pioneering procedure, a team led by Professor Donnez, whose labs have three Planer freezers, treated Ms. Ourda Touirat, a thirty two year old cancer patient. She had a daughter in September 2004 after undergoing the transplant; her tissue was removed in 1999, before her radiotherapy treatment. In 2013 a team from Melbourne IVF in Australia reported the world’s first pregnancy from ovarian tissue grafted onto a patient's anterior abdominal wall. Again her ovarian tissue was frozen using a Planer freezer and seven years after the frozen tissue was thawed and grafted back to help her successfully conceive with her own eggs.  

Further details
http://planer.com/company/news/news-2013/273-2013-2/609-a-world-first-pregnancy-from-frozenovarian-tissue-grafted-outside-a-womans-pelvis.html

Background on Planer freezers
http://planer.com/products/cryo-freezers/small-crf/kryo-360.html
http://planer.com/products/cryo-freezers/medium-crf/kryo-560.html  

What is virtual pH?

Petri pHpH remains an unforgiving biological parameter which is difficult and expensive to measure in established culture systems. The new Petri dish pH sensor provide a flexible and cost effective option to determining both CO2 and pH – find out how it works.

A new way to measure pH/CO2 anywhere, and in anything.

The new Petri dish pH sensor from Planer allows users to measure both CO2 and predict the pH of established culture systems.

The small sensor size (equivalent to a 35mm petri dish) combined with an ultra thin ribbon cable allows measurement of CO2 levels in benchtop incubators, environmental chambers and large incubators.

Using the established relationship between CO2 and pH, combines with characteristics of both the culture media and growth conditions, accurate pH values can be predicted without the expense or inconvenience of traditional methods.

Available as a fixed point sensor or portable validation device, the Petri dish pH sensor provide flexibility and innovation to ensure optimal growth conditions.

 

Dr Stephen Troupe presents “ISO 15189 – A survivors guide” at recent ABA conference

Dr Stephen Troup, Scientific Director of the Hewitt Fertility Centres, gave an interesting presentation at the recent Association of Biomedical Andrologists' conference in Leeds - “ISO 15189 – A survivors guide”. In it he illustrated his first hand experiences of the difficulties - and subsequent benefits - of attaining this high level of accreditation for his labs.

Steve thought that progression from Clinical Pathology Accreditation to ISO 15189 was inevitable; the change though meant even more focus on technical and clinical aspects, comprehensive reporting processes, and more frequent assessments for the clinics. It brought the inevitable increased need for detailed Training records, upgrades in Equipment Maintenance standards, and more H & S emphasis. The quid pro quo for all this extra work for laboratory staff was the increase in real benefits, including enhanced customer and peer recognition, the adding of real day to day value from the deeper assessment reports plus more consistent quality performance. Additionally, there were knock on benefits for customers and users, which included the comfort of greater transparency and the assurance that every test within the laboratory's scope of accreditation is fit for purpose. However, one interesting conclusion was that for The Hewitt Fertility clinics - including their five ‘specialist’ Andrology Labs undertaking some 10,000 total semen analyses pa - the real additional cost of ISO 15189 was the equivalent of an additional £4 per test – an increase of some 10-15%.
 


Steve has worked in the field of clinical embryology for nearly 30 years and as well as being a practising clinical embryologist and Scientific Director has held the prestigious positions of Scientific Advisor to the Human Fertilisation and Embryology Authority is an Honorary Senior Fellow and Visiting Reader at Edge Hill University and an Honorary Senior Fellow at the University of Liverpool.

Some of the other speakers at the ABA annual meeting included Paul Hancock, Stephanie Brooks from Liverpool Women's Hospital and Louise Davidson of UKAS. Planer plc were also at the ABA, giving attendees the opportunity to find out more about Medical Device controlled rate freezers and incubators and, relevant to the conference talks, real time monitoring. Planer's flexible alarm and monitoring systems help users look after samples as well as look after the laboratory and even the people in the laboratory. The ReAssure package has the option for cloud based, compliant storage of monitoring data - which allows users to report alarm events and associated annotations at the touch of a button, reducing the regulatory burden. A new product on show was the Sperm Selection Kit, to help improve pregnancy rates helping to selectively remove apoptotic spermatozoa from sperm preparations.

Incubators in the Reproduction Clinic Osaka

The state of the aging population in Japan, and indeed in many parts of Europe, is well known. In 2011, a Japanese government report suggested that the Japanese population, which peaked in 2004, will decrease in the next 100 years to the same level it was as 100 years ago, going down by approximately 33 million to 95.2 million in 2050. Given such predictions, assisted reproduction techniques take front place in national thinking and a wide number of clinics there practice new techniques and promote new equipments for IVF. 



So, we were delighted when the Reproduction Clinic Osaka decided to install the Planer/Origio bench top incubator, BT37. An embryology lab, while not often seen by patients, is a key part of a centre and a great deal of planning goes into it. At the Reproduction Clinic Osaka they use the very latest scientific techniques and equipment for culturing blastocysts. Precise conditions facilitate the selection of good quality embryos and increase implantation rates while decreasing multiple gestation rates. As part of this it is important to alter the environment of the embryo as little as possible so as to diminish cellular stress. The benchtop incubator supplied by Origio Japan showed itself ideal for the cultivation of embryos helping to control and stabilize temperature, moisture and gas, attempting to best reproduce the conditions found in the uterus. 



The Reproduction Clinic Osaka offers a broad range of infertility treatments from induction of ovulation, artificial insemination, IVF, ICSI, semen analysis and most notably, TESE. Head of the laboratory there is Mr. Shimpei Mizuta supported by Dr. Hidehiko Matsubayashi, Medical Director, and Dr. Tomomoto Ishikawa, CEO. The Reproduction Clinic Osaka was opened in September 2013 and has treated already, in just a year and a half, over 1500 cycles and 500 TESE cases. The Clinic’s spokesperson said "The greatest distinction for our clinic to others is the visiting of couples with male infertility, especially azoospermic men. Actually, the percentage of TESE-ICSI cycle is very high, and it is typically 30% of all cycles in our clinic. In cases like TESE-ICSI, we need multiple opening of the incubator lids. Recovery of gas concentration in the BT37 is very fast, and we have a good blastocyst development".

Mr. Kazuhito Shinoda, sales manager at Origio in Yokohama City, oversaw the placing of the equipment and from initial trials. The Reproduction Clinic Osaka now has 18 incubators installed; Mr. Shinoda said "The Reproduction Clinic Osaka does not only offer top quality treatment for infertile couples who visit the clinic but also are willing to take an active role in communicating through various kinds of mass media to people in general. They’re very serious about the impact they have on the society. In Japan most people feel too conservative about the topic to discuss in public especially when it’s related to male infertility. Both Origio and Planer are so pleased to assist such a client who is so influential and proactive for improvement of overall awareness of IVF in the country".

More Information

http://planer.com/
http://www.origio.com/us/contact/Japan

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