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AVA Clinic, Latvia, chooses Planer benchtop incubator

Riga, in Latvia, is one of the most beautiful cities in Europe and is the third biggest city in the Baltic region, right after St. Petersburg and Stockholm. The AVA Clinic based there, is part of a European network of infertility treatment clinics founded in 1993 and this one opened in 2005.

The experienced Riga team get high infertility treatment results, and they adhere strictly to ISO 9001:2008 which is why they chose the Planer Origio BT37 benchtop incubator. The BT37, a worldwide success story, is a high accuracy bench-top incubator for growing and maintaining gametes and embryos. The incubator will keep cells at optimal temperature, humidity and gas content by maintaining a constant and clean environment and it has extremely accurate temperature control, systems for pH stability and regulated humidity. All the technology goes to making sure that an embryo suffers little or no exposure to temperature or pH level changes.



Improve post-thaw function in cryopreserved Mesenchymal Stromal Cells

Results from a multi disciplinary study into improving post thaw function in MSCs has just been published in the journal Stem Cells and Development. Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. The investigation, carried out by Pollock, Samsonraj et al with Professor Allison Hubel, (pictured here), a Mayo Clinic and University of Minnesota collaboration, developed a novel approach to preserving MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). As a part of the protocol the team used a Planer controlled rate freezer with a six part programme starting at -20oC  with differing rates cooling down to -100oC before transferring to liquid nitrogen. The authors point out that rapid cooling and warming steps in their freezing profile correspond to a temperature spike which is included in the freezing ramps to reliably induce extracellular ice formation in the samples before they undercool substantially - which can result in undesirable intracellular ice formation when nucleation does finally occur.

Osteogenic and chondrogenic differentiation assays showed that cells retained their mesenchymal lineage properties and genomic analysis indicated that the different freezing media evaluated had  different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrated that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. The studies validate the concept that DMSO-free solutions can improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

For further information:-
Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions

Allison Hubel, PhD Department of Mechanical Engineering University of Minnesota 
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Andre J. van Wijnen, PhD Department of Orthopedic Surgery Mayo Clinic,  
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Planer controlled rate freezer - kryo 560

40 years of Planer Incubators

Planer incubator 1980It's the tenth anniversary of our first concept for the BT37 benchtop incubator, which is now known around the world.  But it is not the only incubator we make. Or have made. While we are known for our controlled rate freezers, few people remember our first incubator - way back in 1976!

Looking through the archives we found a photo of one we used to sell 40 years ago. We collaborated 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 machine - shown here - was built around 1980 and was tested by a team led by a Dr Berry from the Department of Microbiology at St. Thomas's Hospital Medical School London. It was distributed for a while by Gallenkamp who became part of Fisons Scientific plc.

With several thousand sold, the precision benchtop BT37, pictured here, has proved a boon to both the IVF industry and researchers in other fields. It was introduced in 2009 and is now found in labs and clinics in each continent of the world. With or without humidity control its market leading temperature control is also making it increasingly useful in transgenic and animal ART as well as the huge success it is in the human fertility market.

However we have not stopped innovating or developing new products. With bench space in labs at a premium we will be showcasing a new, unique incubator at ESHRE 2017 in Geneva. It is designed for the modern laboratory and has the capability of market leading volume of dish spaces in a unique small footprint.

A unique modular approach (patents pending) allows users to add units as space and throughput require and units will fit in most laboratory hoods helping to improve work flow whilst reducing the risk of mishap during transfer for inspection. And all this whilst maintaining the legendary temperature control accuracy throughout the chamber environment. Details of this innovative incubator will be available on our website, from July once the product is launched.

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Round-up of interesting ISBER 2017 abstracts

The International Society for Biological and Environmental Repositories (ISBER) is an international forum that addresses the technical, legal, ethical, and managerial issues relevant to repositories of biological and environmental specimens. This year's Annual Meeting took place in Toronto Canada - here is a round of abstracts that we felt might be of interest:-

The Effect of Liquid Nitrogen Storage on ctDNA Extraction from Plasma
ISBER abstract reference BRS-8 
Kenney1, J. Sosa-Baez2, E. Lin1, E. Hernandez1, P. McNeil1, C. Mariano1, L. Villafania2, J. Padilla2, A. Samoila2, E. Peerschke2, M. H. Roehrl1
1Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, United States,
2Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, United States

The identification of a biomarker for cancer that is procured in a safe, non-invasive, and efficient manner for the detection of disease has been made possible in recent years via the discovery of cell-free circulating tumor DNA (ctDNA). The full scope of potential for the uses of ctDNA have not yet been realized; therefore, there is a call to preserve valuable samples in the hope that these resources can be utilized with forthcoming technologies. Traditionally, either due to cost or ease of use, tissue samples are stored at −80°C where they can be kept for many years, whereas in LN2, they can potentially be preserved indefinitely without degradation of the sample for future genetic and proteomic applications. Currently, plasma samples taken for ctDNA extraction are routinely stored at −80°C for extended periods of time. The trend toward banking samples in LN2, however, presents the question of whether the quality ctDNA in plasma samples stored in LN2 will be suitable for assays in the future. It has been shown that LN2 preserves many elements of the plasma, such as antibodies/proteins; however, the ability to preserve ctDNA has not yet been tested.

Methods and Results 
ctDNA from standard plasma samples stored at room temperature, −4°C, −80°C, and in vapor phase LN2 (−180°C) over various amounts of time (days to months) was extracted in a Hamilton easyBlood robot and ctDNA yields were compared.

Storing quality control plasma standards at −80°C produces a mean concentration of ctDNA of 0.95 ng/μl. We stored aliquots of a parallel standard in LN2 for 2 weeks and were able to extract a ctDNA concentration of 1.019 ng/μl. Additional data will be presented at the meeting. Initial results indicate that plasma storage in LN2 is at least not inferior, if not superior, to storage at −80°C. Storage at temperatures higher than −80°C is not recommended and further data will be acquired to study how time-sensitive cfDNA is with respect to “needle-to-freezer” speed and handling.

 We aim to provide evidence that ctDNA extracted from plasma stored in LN2 for various amounts of time will have equal to or better yields of ctDNA than plasma stored at other temperatures. Encouraged by the results, we plan to test more samples at varying temperatures, optimal and suboptimal, for extended periods of time in order to discover the ideal long term storage conditions for plasma used for ctDNA extraction.

Impact of Room Temperature on Tissue Quality as Assessed by RNA Integrity Number (RIN)
ISBER abstract reference HSR-12 
Bhanot1, C. Lai1, M. Santin1, C. Mariano1, P. McNeil1, M. R. Weiser2, M. H. Roehrl1
1Pathology, MSKCC, New York, New York, United States
2Surgery, MSKCC, New York, New York, United States

The practice of oncology is being propelled by new emerging technologies in genomics, transcriptomics, and proteomics to help better understand molecular events that result in tumor initiation, development, and progression. However, the results of genomic, transcriptomic, and proteomic analysis can reflect true alterations only when biospecimens used are of very high quality. Therefore, the impact of pre-analytic variables on tissue quality needs to be studied, as this will help identify areas for improvement in biobanking protocols.

Due to high volume and engagement of biobank personnel in various operations in a busy cancer center, it is not always possible to transfer the tissue in to liquid nitrogen immediately upon receipt. We are studying the impact of transport/storage at room temperature on fresh tissue kept at room temperature (RT) for various time points.

Tissues aliquots from the same patient (tumor and normal) were divided and frozen immediately in vapor phase liquid nitrogen or kept at room temperature (20°C) for various amounts of time (0-24 hours) before freezing. Total RNA was extracted from tissues using RNeasy Mini Kit (Qiagen) and RNA analysis was performed on the Agilent Bioanalyzer.

Initial data show that the quality of tissue as determined by RNA integrity numbers (RIN) is best maintained when the tissue is transferred to liquid nitrogen immediately upon arrival. When the tissue is transferred to liquid nitrogen immediately on arrival, tissues yielded RIN values close to 10 (optimal). When kept at RT for short time intervals, i.e., up to 1 hour, the RIN values are relatively unchanged. However, when the transfer of tissues into liquid nitrogen was delayed and the tissue is kept at RT for longer than 2 hours, a decline in the RIN scores was observed. Interestingly, we noticed that RIN values are typically higher in tumor vs. normal tissues from the same patient.

Any modern biorepository needs to closely monitor its protocols and implement an ongoing internal as well as external quality improvement plan that will help identify areas for improvement to better maintain the integrity of banked tissues.

Assessing the Quality of RNA from Fresh Frozen Human Tumour Tissues Stored Long-Term at Cryogenic Temperatures
ISBER Abstract Reference RS-15 
Kelly1, M. de Ladurantaye2, M. Albert1, M. Moore3, S. Dokun4, J. Bartlett1,5
1Ontario Tumour Bank, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
2Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montreal, Quebec, Canada,
3Ontario Health Study, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
4Health Services Research, Ontario Institute for Cancer Research, Toronto, Ontario, Canada,
5Transformative Pathology, Ontario Institute for Cancer Research, Toronto, Ontario, Canada

It is widely recognized that the integrity of tissue specimens preserved at temperatures below the glass state is stable long-term. Scientific studies in literature, however, do not generally extend beyond a few years. With biobanks reaching a degree of maturity where specimens may be stored for over a decade, this assumption should be tested to provide the data to support extended long-term storage and demonstrate continued fit-for-purpose. Since its inception in 2004, the Ontario Tumour Bank (OTB) has had an ongoing commitment to quality and, in accordance with biobanking best practices, has embedded stringent quality control (QC) and quality assurance (QA) measures into its routine procedures. One such measure, triggered twice annually, includes the random selection of cryopreserved tissues to undergo external quality assessment (EXTQA) by a third party to measure the integrity of the tissue's DNA and RNA. Here, as an extension of OTB's routine EXTQA, we analyzed second aliquots of previously evaluated tissues collected between 2005 and 2014 to determine if RNA integrity is affected by extended long-term storage in liquid nitrogen vapor phase.

RNA was extracted from duplicate aliquots of 70 cryopreserved tissue samples across 11 disease sites previously analyzed in prior years during routine EXTQA and having recorded RNA integrity number (RIN) scores of 7.5 or greater. Extractions and quality determinations were performed at the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) according to CTRNet SOPs. RNA quality was determined by the RIN assigned by the Agilent Bioanalyzer.

The results of external quality assurance 1 (EXTQA1) and external quality assurance 2 (EXTQA2) for RNA extracted from fresh frozen tissues were compared; 94% of EXTQA2 samples were Acceptable to Very Good in quality. There was no significant correlation (r = 0.10, p = 0.80) between the quality of RNA extracted in EXTQA2 and storage time. There was also no significant correlation (r = 0.04, p = 0.92) between the change in RIN score between EXTQA1 and EXTQA2 and storage time.

These data suggest that extended long-term storage of tumor tissue samples in vapor phase does not negatively affect the quality of RNA derivatives. From this, we conclude that OTB samples banked since inception continue to be viable for downstream applications and are fit to be used in high-impact cancer research studies.

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ESHRE 2017

The European Society for Reproduction and Embryology (ESHRE) will be holding their annual conference in the beautiful city of Geneva, Switzerland this year from the 2nd to the 5th July.

On Sunday 2nd July, there will be fifteen special interest group pre-congress presentations, before the main programme itself starts on Monday 3rd July.

Planer will be exhibiting once again at ESHRE, with some exciting new products. Don't forget to come and see us on stand D16 to find out more.

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Planer monitoring system installed in AVA clinic, Latvia

The AVA Clinic in Latvia is part of a European network of infertility treatment clinics founded in 1993 and the Riga clinic, shown here, opened in 2005. The experienced Riga team get high success rates, and as they adhere strictly to ISO 9001:2008 the quality of the equipment they buy is very important. So we were honoured that they chose Planer equipment - incubators, monitoring and low oxygen alarms when they recently upgraded their lab. 

Shown here is the DATAcentre monitoring system on two incubators. This affordable system can monitor temperature, humidity, CO2, liquid nitrogen level, Oxygen (O2), door status and more.  It can connect to up to 120 transmitters, including a mixture up to 120 wireless sensors or 14 hard wired sensors.  Real time and historical logged data can be viewed in graphical or text format and any alarms can be easily managed. Optionally the system may be connected directly to a PC or to a LAN port in the building.

Andris Grunskis is both an embryologist with AVA and the Quality Systems Manager (for ISO9001:2008 & EUTCD) and has worked at AVA since 2009. After our engineers had finished installing the system, Andris commented " ... the engineers were great! They fixed all our issues and now the DATAcentre seems to be performing flawlessly".

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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.

For further information

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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