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Freezing Responses in DMSO Based Cryopreservation of Human iPS

A study on these freezing responses, relating to aggregates versus single cells, has just been published in Tissue Engineering (on-line February 2018) by Rui Li, Guanglin Yu (pictured here) and others working with Prof Allison Hubel's team at the Department of Mechanical Engineering University of Minnesota. Human induced pluripotent stem cells (hiPSCs) are multicellular aggregates attracting much interest in tissue engineering, disease modelling and personalised medicine. They can be frozen either as aggregates or single cells depending upon the application. For both clinical and scientific purposes, effective cryopreservation of hiPSCs is required for transportation, storage of frozen hiPSCs and other downstream uses. However, cryopreserved hiPSCs are vulnerable to the loss of viability, function or pluripotency.

It is known that inadequate preservation methods of hiPSCs have impeded efficient re-establishment of cell culture after their freeze-thaw. In the study the roles of cooling rate, seeding temperature and the difference between cell aggregates and single cells in controlled rate freezing were examined using, inter alia, Raman spectroscopy, as a tool for understanding cell responses to the freezing environment. The Raman spectroscopy was used to observe both hiPSC single cells and aggregates frozen at three cooling rates and two seeding temperatures; it suggested higher sensitivity of aggregates to supercooling than previously thought. The work will deepen understanding of behaviours of single cells and aggregates frozen at various conditions and help promote the development of improved cryopreservation protocols for human induced pluripotent stem cells.

For single cells, slow cooling rates allowed significantly better preservation of membrane integrity than higher cooling rate (10˚C/min) regardless of the seeding temperature. For aggregates, however, slow cooling rates (1, 3˚C/min) combined with high seeding temperature (4˚C) had little effect on the membrane integrity but resulted in significantly better cell attachment than higher cooling rate (10˚C/min) or low seeding temperature (8˚C). The authors say there are advantages of using a seeding temperature of 4˚C compared to 8˚C suggesting that the range of seeding temperatures of 7˚C to 12˚C quoted in much literature may be sub-optimal, and that seeding temperature should be considered as a critical parameter when designing cryopreservation protocol for hiPSCs. Guanglin Yu, see photo above, carried out a lot of the experimentation. He said "...we used manual seeding for nucleation of the sample. We sprayed the sample with a narrow stream of liquid nitrogen. We saw different cell responses of HiPSC with seeding temperature of -4˚C and -8˚C using Raman microscopy. This made it clear that we wanted to control the temperature at which ice formed in the extracellular space for controlled rate freezing experiments."

The paper indicates that hiPSCs respond to freezing in very complex fashion, and successful establishment of post thaw culture depends on various critical factors. Further studies will need to not only continue exploring additional factors to optimise the freezing protocol for hiPSCs but investigate the biological pathways connecting the factors and the observed cryopreservation outcomes to provide targets for future development of cryoprotectants.

For further information
Contact Prof Hubel's Lab: This email address is being protected from spambots. You need JavaScript enabled to view it.
Tissue Engineering:
Planer Controlled Rate Freezer:

The first frozen embryo baby

On the 34th anniversary of the birth of the first baby from a frozen embryo, the BBC World Service broadcast an interview with Alan Trounson the pioneer doctor who undertook the procedure.

Zoe Leyland was born in Melbourne, Australia on 28th March 1984, helped on her way by Drs Trounson and Wood who made medical history. The decision to try 'test tube' fertilisation and embryo freezing was taken by Zoe's parents – her mother a 33 year old New Zealander and father a 38 year old British born Australian resident. Her mother had hormonal stimulation and produced eleven eggs which were frozen using a then new type of controlled rate freezer made by Planer plc. One of those frozen embryos became Zoe - who weighed in at about 5 lbs or 2.5 kilos.

The world's first 'fresh’ test tube baby was Louise Brown born in England in 1978, but Zoe came from an embryo frozen for a time before being thawed and implanted. To allow cells to survive liquid nitrogen temperatures (-196°C) the embryos had to be treated with cryo-protectant, then slowly frozen down in the Planer freezer with extreme precision using different temperature ramps, before being stored in liquid nitrogen. This controlled rate freezing procedure is now used in cell laboratories worldwide. The controlled rate freezing technique, originally suggested some fifty years ago by British Scientist Professor David Pegg, enabled Planer plc to pioneer the equipment and today many thousands of units are in constant use all over the world in stem cell labs, IVF, hospitals and research institutions. This controlled rate freezing is often needed before storing certain types of cells in liquid nitrogen – in areas such as cord blood banking, bone marrow transplants, botanical matter, semen, oocytes, botanical seeds, skin, ovarian tissue, heart valves and blood vessels.

For further information:
BBC World Service: The first frozen embryo baby
Forty years on ...Planer freezers

AVA Clinic, Latvia, chooses Planer low oxygen alarm system

Planer O2NE+ low oxygen alarm systemThe AVA Clinic in Latvia was opened in 2005. The experienced team get high infertility treatment results, and as they adhere strictly to ISO 9001:2008 , the same rigour must apply to safety in their cryo store - which is why they chose our O2NE+ low oxygen alarm system.

In the unlikely event that there were to be a leak in the cryo store causing a build-up of nitrogen gas, which is colourless and odourless, the depletion of oxygen levels might pose a serious danger. The O2NE+ system provides two audio visual alarms which are usually pre-set at 19.5% & 18% to warn personnel of a potential hazard should the oxygen levels deplete.  The main senor unit is wall mounted and repeater units can be sited at other entrances.  The system has a long life O2 sensor, allowing simple calibration with low maintenance.

Andris Grunskis AVA clinic LatviaAndris Grunskis, pictured here, embryologist at AVA, is also the Quality Systems Manager who specified the O2NE. He graduated from the University of Latvia with a Master’s degree in Biology and regularly participates in international conferences and hands-on workshops and is a member of European Society of Human Reproduction and Embryology and the Nordic Fertility Society. "  ... your engineers were great!" he commented after installation.



For further information:-
Ava clinic 
Planer O2NE+ low oxygen alarm system


Aspire 2018

The 8th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2018) will be held at Taipei International Convention Center, Taipei, Taiwan from 12 - 15 April 2018. Over the three days, the conference will consider the latest developments and their impact on the future of fertility preservation, embryology, andrology, clinical trials, PCOS, PGS, IVM, and related fields.

Planer will be exhibiting at Aspire 2018. If you are planning to visit the conference, don’t forget to come and see us on stand C53. We will be displaying the full range of Planer IVF products, including the CT37stax™ multi chamber incubator, the DATAssure™ wireless alarm and monitoring system as well as Shipslog3™ and PetriSense®.


Upper Egypt Assisted Reproduction Summit 2018

Mohamed Fawzy IVF Lab Director of Ibnsina and Banon IVF Centers Egypt
The Upper Egypt Assisted Reproduction Summit (UEARS) held in Cairo last February (21-23) saw the participation of 40 international speakers and 90 national ones for three days of scientific discussion with close to 1600 attendees from Egypt and overseas. With seven pre-congress workshops and a Consensus meeting, we looked into the micro environments, that can support the embryo in vivo and in vitro starting from how the human life began, navigating through the current and future techniques, including gene editing, with the current standards of embryo culture in between. New papers from 14 researches were presented for the first time during UEARS from young researchers.

Discussed in some detail was how the current standards of embryo culture have failed yet to make paradigm-shifting changes during the last 20 years, although some improvement has occurred. One of the elegant topics presented in the conference was from Catherine Racowsky of Harvard University who discussed the advantages of blastocyst transfer concluding that there remains a need for a robust evidence to support the superiority of blastocyst transfer compared with the cleavage-stage transfer. The new culture platforms that can improve the IVF outcomes were also discussed in a complete session.

In collaboration with The Egyptian Knowledge Bank, two research methodology workshops were conducted, navigating how reliable research should be robustly conducted and how a well conducted meta-analysis has to be done. These two research methodology workshops were arranged by Professor Ali Mahran from Asyut University and Dr Ola Laurence from the Egyptian Knowledge Bank, and delivered by Dr Léa Gagnon, PhD of Nature Research.

The conference had three parallel arms of infertility running throughout the three days. The gynaecology program arm, which was built by Drs Hazem Abdelghafar, Mohamed Sabry, Mohamed Yahya of Sohag University and Ahmed Nabil of Asyut University. The andrology program arm that was developed by Dr Ali Mahran. The embryology and genetics program arm, which was built by Mohamed Fawzy of Ibnsina and Banon IVF Centres, Egypt. The embryology arm had two parallel halls of lectures during the three days with a pre-congress four workshops. In the main program, Mohamed Fawzy delivered a talk navigating through his latest three publications discussing humidity, oxygen tension and temperature roles in human embryo culture. For the first time in RCT design, it has been shown that omitting humidity from some incubators was done with no robust evidence but with possible harm to the developing embryos and the downstream events. 

The UEARS 2018 lectures were generally considered to be of a very high scientific calibre. All UEARS Activities were well organized by the professional company Pure Spot Events Management; the closing remarks were focused on the concept of "no harm must be the goal in all IVF procedures and interventions". There is an open invitation to join UEARS in 2019 (Feb 20-22) which it is hoped will be of even higher quality and design.

Pictured here: Mohamed Fawzy, Scientific organiser of the embryology arm of UEARS, IVF Lab Director of Ibnsina and Banon IVF Centers Egypt

For further information on UEARS, please visit their website:



Managing risks in cryogenic storage

Over the last year or so there have been reports in the press on problems with samples stored in liquid nitrogen. Cryo storage is such a reliable method that it is often taken for granted - equipment is generally very robust and supply of liquid nitrogen, LN2, is typically very reliable. Patients as well as new entrants to the field are often surprised to learn that both the freezing of a sample, and its long-term storage, can be more complicated than they seem.

Many people, even those working in ART laboratories, remain unaware that specimens in cryogenic storage will suffer damage due to ice recrystallization when their temperature rises above -132°C (the ice transition point of water). And, the smaller the specimen the greater risk it is at, because its low thermal mass will allow its temperature to rise more quickly, and hence follow more closely, the rising external temperature, that of the storage environment. So the risk of ice recrystallization damage increases when comparing cryotubes to straws to vitrified embryo carrier devices.

Quality and Risk Management in the IVF Laboratory

In a definitive textbook on the subject on quality and risk management in the IVF lab, Drs Sharon and David Mortimer (pictured here) provide extensive background on the whole topic including explaining general principles applicable to managing risk in an Assisted Reproduction lab’s cryobank.    

In Chapter 8 “Managing Risk” there is a section on “Monitoring storage cryotanks” where they recount a personal experience of dealing with a degrading dewar and, based on that experience, provide a series of recommendations for routine good practice. And as an extension to the book, Dr David Mortimer has provided the following practical advice.

When using liquid storage in small cryotanks (e.g. up to 50 litres):

  • Top up the tanks regularly at quite strict weekly intervals – but before doing so measure and record in a logbook the level to which the LN2 has fallen. Performing weekly re-fillings, rather than more frequent top-ups, is not unreasonable when a typical tank (e.g. Taylor Wharton HC35) has a static working time of more than 11 weeks. Then use these measurements to create a control chart – a Shewhart chart - so that not only can unusually low levels be readily identified, but degrading performance of the tank will be seen as a series of decreasing levels of remaining LN2 prior to top-up.

  • In addition, tanks should be monitored for temperature and LN2 level with sensors connected to an automatic dial-out alarm system. Since the temperature of LN2 will always be -196°C the temperature sensor should be placed somewhere just below the typical level to which the LN2 will have fallen after a week of normal operational use and set to alarm at, say, -160°C. The LN2 level sensor should be located a short distance above where the uppermost specimen is held in storage.

When using liquid storage in large cryotanks of say 150 litres and more:

  • These tanks must be connected to a large LN2 supply so they can operate on auto-fill.
    Note: Ideally the cryotank controller should be “smart” and able to communicate with external monitoring software to allow logging of the amount of LN2 used; these data can then be used to generate an alarm should usage increase beyond a certain limit. An example of this would be the MVE TEC 3000 controller from Chart Industries combined with Planer’s DATAssure software.

  • Temperature and LN2 level sensors should be installed using the same principles as with small tanks and connected to an automatic dial-out system.


When using vapour storage in smaller dewars manual monitoring of the LN2 level is impractical, and a combination of LN2 level and temperature sensors should be used:

Locate the temperature sensor a good distance above the uppermost specimen in storage and have it alarm when the measured temperature falls below -140°C (which is considered to be the limit for safe vapour storage).

  • Locate the LN2 level sensor at the manufacturer’s recommended minimum LN2 level for safe operation of the cryotank.
  • When using vapour storage in large dewars the same principles for positioning the temperature and LN2 level sensors as in small dewars apply. However, because these tanks operate on auto-fill their regular consumption of LN2 should be monitored as described for large liquid storage dewars.

At Planer we are keen to promote safe cryo storage. As well as controlled rate freezers we sell vessels, alarm systems, monitoring systems and cryo transport loggers through our distributors available all over the world.

For further information
For Planer Monitoring:
For ‘Quality and Risk Management in the IVF Laboratory’:










45 years - still pioneers

45 years logoPlaner plc celebrates 45 years
In 2018 Planer plc, formerly known as Planer Products, will be celebrating 45 years of designing, manufacturing, selling and supporting products for a variety of technical uses throughout the world. The company sprung from GV Planer Ltd., founded in the 1950s, primarily a design and research consultancy making one off products for research institutions. It was some of these earlier research products that were the foundations of the equipment that Planer plc went on to develop into commercially viable machines. These continued to evolve over the 45 years into the products we manufacture and sell today.

Slow freezing pioneers
The first birth from a frozen bovine embryo was reported in 1972 - using a very early Planer machine. Professors Rawson and Wilmut pioneering the animal husbandry field. With its successful development and the use of products for controlled rate freezing, the company received the Queen's Award for Technology and also awards from the British DTI for Innovation and Good Practice in Micro-electronics and achieved approval and for the demanding standards of medical device manufacture: ISO13485:2012 & Medical Devices Directive, Annex 11 93/42/EEC: LRQA. ISO 9001:2008.

The use of controlled rate freezers widens
IVF labs were the initial market but later hospitals and researchers found they needed programmable freezing for many types of cells – in areas such as cord blood banking, bone marrow transplants, botanicals, semen, oocytes, skin, ovarian tissue, heart valves and blood vessels. Planer Controlled Rate Freezers continue to be at the forefront of many of today’s scientific breakthroughs using cryopreserved cells.

Forty two years ago in 1976 we conceived our very first incubator whilst collaborating with the then UK Department of Health and Social Services in the production of a new form of anaerobic incubator as an alternative to the prevailing jar system for the isolation of anaerobes from clinical material. The prototype machine was built around 1980 and was tested by a team led by Dr Berry from the Department of Microbiology at St Thomas's Hospital Medical School London, SE1 in 1981. The Planer range of CO2 incubators has since been developed further with the introduction of the very successful BT37 in 2009 and the new CT37stax multi chamber incubator in 2017.

Throughout 2018, we plan to run a series of articles and events to celebrate our 45 years of working with and supporting scientists and healthcare professionals who use our equipment to preserve, protect and nurture all types of cells in many important applications throughout the world.

Pictured here: Professor David Pegg with a Planer freezer in the 1970s.






Preservation of Cells: A Practical Manual by Allison Hubel

Professor Allison HubelA new book has just been published by Professor Allison Hubel of the University of Minnesota. It aims to provide readers interested in cryo bio banking with the tools needed to develop or debug a preservation protocol for cells. The core structure and content of the text grew from a professional short course that she has offered at the Biopreservation Core Resource over the last 10 years. The text describes, step by step, the individual elements of a protocol, including the relevant scientific principles for each phase. It can be used by anyone who is involved in cell preservation - including those not expert in the freezing of cells; it does provide the scientific basis for those that want to understand the basis for the protocol.

The book covers pre-freeze Processing and Characterization; Formulation and Introduction of Cryopreservation Solutions; Freezing Protocols; Storage and Shipping of Frozen Cells; Thawing and Post Thaw Processing; Post-thaw Assessment; and Algorithm driven Protocol Optimisation. Professor Hubel explains the reasons behind every step in the development of a preservation protocol and the scientific principles behind them and provides alternative modes of preservation for when conventional methods of cryopreservation are not appropriate for a given cell type or application.

Preservation of Cells A Practical Manual by Allison HubelMammalian cells have become modern workhorses capable of a variety of applications, such as in production of therapeutic proteins, viral vaccines, antibodies and therapeutic agents. Cells that are to be used therapeutically must be properly stored to meet the safety and quality control testing prior to release of the cells. There are a variety of methods that can be used to preserve cells depending upon downstream application.  Specific practices are intended to improve both the outcome and the reproducibility of preservation protocols. The preservation toolkit is used to develop fit-for-purpose preservation protocols. Each element of a protocol is based on scientific principles and those principles are explained and can be used to rationally design that particular step to achieve the desired downstream use of the cell. The influence of processing procedures on the health of cells prior to cryopreservation and strategies to mitigate stress on cells is explained along with the formulation and introduction of cryopreservation solutions.

There is a specific chapter on controlled rate freezing with all the steps of a controlled rate freezing protocol and guiding principles for design of that step are given. Also described is a process for transferring units from a controlled rate freezer to a storage unit and there are examples showing good and bad freezing curves. Prof Hubel also covers storage and shipping of frozen cells, processing, assessment, protocol optimisation and the cryopreservation of endothelial cells in suspension, peripheral blood mono nuclear cells from whole blood, human adipose stem cells, red blood cells, oocytes and more.

Preservation of Cells: A Practical Manual is an important book for researchers, laboratory technicians and students in cell biology, stem cell biology, tissue engineering, and regenerative medicine. It is also useful to cell bankers, regenerative medicine, biomarker discovery or precision medicine companies, and cell therapy labs, blood bankers, biobankers, and biotechnology companies.

For further information:
Prof Allison Hubel
Preservation of Cells: A Practical Manual (John Wiley & Sons, Inc., Print ISBN: 9781118989845)




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