Neuronal stem cells are cryopreserved with CoolCell freezing container

May 21, 2013

Independent publication cites use of the CoolCell freezing container for cryopreservation of human neuronal progenitor and stem cells[1].

27 stem cell neural 300x196 Neuronal stem cells are cryopreserved with CoolCell freezing container

A new protocol for directed differentiation of stem cells leads to the formation of all human cortical neural cell types. (Red, mature neuron derived from human iPSCs).

Despite a centuries-old fascination with the brain, we have a relatively thin grasp on how the brain works.  President Obama recently announced a new initiative to try to tackle the problem, which the NIH is calling the Brain Activity Map, to be funded by $3 billion over the next ten years.  The idea is that a map of the connections in the human brain might help us better understand diseases like Alzheimer’s, schizophrenia, and Parkinson’s. As people consider how best to study the brain, one avenue that scientists have been chipping away at is to use stem cells to model the brain’s connections, in vitro.

This can be accomplished by differentiating human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) into neurons through directed differentiation.  Developing the technology to do this in vitro has broad utility: differentiated stem cells could be used to model disease in a dish, or be developed for neuronal transplantation.  Also, in vitro human neuronal stem cell models might someday trump the mouse models of human disease currently used in the lab because they are more relevant to the human condition.

Until recently, scientists have had a hard time driving stem cells down a path that completely recapitulates that of normal neuronal development.  The cultures failed to produce all cortical cell types.  A recently published protocol describes a method for differentiating both embryonic stem cells and iPSCs into all types of human neuronal progenitor cells and cerebral cortex neurons[1].

Differentiating stem cells into neurons is time and labor intensive.  It takes three weeks to prepare stem cells for induction, two weeks to induce the cells, and a further three weeks to expand and differentiate neural stem cells[1].  Once differentiated, cultures can be maintained for up to 3 months for study in the lab.  However, they cannot be further passaged, meaning that cultures have an “expiration date” for study.

Fortunately, it is possible to cryopreserve established cortical neuronal stem cells before they terminally differentiate.  The published protocol endorses the use of the CoolCell freezing container to do so[1].  Rosettes of cells undergoing neurogenesis can be dissociated into single cell suspensions, frozen in cryovials using the CoolCell, and stored in liquid nitrogen.  They can be thawed and shared as needed, furthering both the reproducibility of science and our understanding of the human brain.

Reference:

1. Y. Shi et al.  Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.  Nature Protocols.  2012 Oct;7(10):1836-46. doi: 10.1038/nprot.2012.116.

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Nature’s Best Survivor: The Water Bear (a.k.a Tardigrade)

May 15, 2013

A few years ago we blogged about the toughest animal on the planet – the water bears a.k.a Tardigrades. Tardigrades are microscopically small, water-dwelling, segmented animals with eight legs from the phylum Tardigrada, superphylum Ecdysozoa, with fossils dating from 530 million years ago.

Last month NPR had a short segment about these fascinating creatures, which can withstand boiling, freezing, and the vacuum of space. They can survive these extreme conditions in part by undergoing a natural cryopreservation process, and replacing the water in their cells with sugar trehalose when they encounter below-freezing temperatures to prevent cell damage.

Professor Bob Goldstein, of University of North Carolina, Chapel Hill, recently updated NPR on his continuing studies involving these millimeter-long creatures and his efforts in trying to understand how organisms like this develop. Among other work, Goldstein’s lab is in the process of sequencing the Tardigrade genome with the goal to find out which genes make Tardigrades the ultimate survivor.

Watch the original NPR video about these fascinating little creatures:

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RT-PCR and qPCR: new sampling cooling method

May 14, 2013

If you are using ice for your PCR assays, you might like to know about a safer and more reproducible method, which was recently endorsed by TATAA, the European genomics biocenter.

PCR method 1024x641 RT PCR and qPCR: new sampling cooling method

A new sampling cooling method for RT-PCR and qPCR

For RT-PCR (reverse transcriptase PCR), here are some important take home messages:

  • It is crucial to note that mRNA is significantly less thermostable than DNA, and that when working with mRNA, extreme care should be taken in handling the reactions.
  • Unless you are using a DNA polymerase designed for Hot-Start PCR, the work MUST be performed at 4°C or less to reduce nuclease activity and prevent degradation. Even when using Hot-Start PCR, if low yield, no amplification or non-specific amplification is observed then this can often be eliminated by adopting a pseudo-hot start protocol, i.e Hot-Start set-up conducted at 4°C.

For q-PCR (quantitative or real-time PCR), here are some important take home messages:

  • Retain all samples, master mix, and reaction tubes/plate on the appropriate CoolBox 2XT PCR workstation to ensure consistent controlled temperature or < 4°C and uniform conditions for all samples. This eliminates problems due to evaporation, degradation of reaction components, non-specific binding, and amplification during set up.
  • Even Hot-Start Polymerase can potentially exhibit a low level of amplification at room temperature. In fact, setting up a Hot-Start reaction on ice is referred to as ‘pseudo Hot-Start’ protocol and is often used to trouble shoot qPCR problems. Pseudo hot start is frequently not used as a front line technique due to the potential for contamination, mess, and additional work that is involved working with ice. The Coolbox 2XT workstation however eliminate these problems.
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Cryopreservation: The Cause of Stem Cell Therapy Trials Failure?

May 9, 2013
Mesenchymal Stem cells Cryopreservation: The Cause of Stem Cell Therapy Trials Failure?

Mesenchymal stem cells after cryopreservation

We recently stumbled upon a fantastic stem cell site called Stem Cell Assays.com. One of the writers, Alexey Bersenev dissects a publication from earlier this year about a stem cell trial that failed. In his words, this publication is “one of the best examples of how the analysis of cell therapy trial failure should be done!” [1]

The analysis was published in Cryotherapy in January this year by Dr Jacques Galipeau and his team and is based on discrepancy between mesenchymal stem cells (MSCs) preparation for US industry-sponsored trial versus European academic trials. [2] The publication reflects on four distinct MSC product variables that may apply to the apparent conundrum at hand: donor variance, epigenetic reprogramming, immunogenicity and cryopreservation. We highlight below a detailed excerpt from the cryopreservation section:

 

  • Human clinical trials examining the use of MSCs almost universally use stored, cryopreserved products that are thawed and transfused within no more than a few hours. This logistical approach is convenient and logical and rests on the premise that similar handling of cryopreserved leukapheresis products used in autologous peripheral blood transplantation has good outcomes.
  • However, all pre-clinical studies of MSCs in mouse models of human disease universally involve transfusion, transplantation or implantation of live, log phase of growth MSCs.
  • Nearly no published pre-clinical report delivers cryopreserved MSCs immediately after thawing to experimental animals.
  • “Is cryopreservation a truly innocuous transient state without bearing on MSC function at thaw?”
  • This question has not been tested in a rigorous fashion in pre-clinical models. It is recognized that 20–30% of thawed MSCs are irreversibly compromised as ascertained by the trypan blue viability assay routinely used as a release parameter in most studies; this leaves roughly two thirds of a cell dose as “live” trypan-excluding cells at the time of transfusion.
  • “However, viability is not a synonym for functionality”

In a previous publication the same research group demonstrated that cryopreserved MSCs, upon thawing displayed impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing[3].  They also found that in vivo persistence of thawed human MSCs in transfused mice is markedly shortened compared with non-cryopreserved counterparts but that the deleterious effects of thawing are self-limited and reverse within 24 hours in vitro[2].

These results highlight a possible cause for the inefficacy of MSC-based immunotherapy clinical trials using cryopreserved MSC thawed immediately prior to infusion. It also raises the question whether it would be more beneficial for MSCs to undergo a post-thaw “rescue” prior to infusion to potentially improve performance and potency[2].

REFERENCES

[1] http://stemcellassays.com/2013/01/failure-mesenchymal-stem-cells-gvhd-devil-cell-prep/

[2] The mesenchymal stromal cells dilemma—does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road?  Jacques Galipeau. Cytotherapy 2013; 15:2-8

[3] Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing. Francois M et al., Cytotherapy. 2012;14:147–152

 

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Fieldwork Sample Handling is Improved by CoolCell Freezing Container

May 7, 2013

Independent publication cites the use of the CoolCell freezing container to cryopreserve mussel hemocytes in the field, resulting in better sample handling from field to lab [1]

26 sample handling mussel 300x243 Fieldwork Sample Handling is Improved by CoolCell Freezing Container

Blue mussels are bio-indicators of coastal pollution. Moving tissue from field to lab for analysis has proven problematic.

To monitor human impact on the marine environment, estuaries and coastal waters are a great place to go. With 22 of the world’s largest cities located on estuaries and more than 60% of the world’s population living along estuaries and the coast, the water in estuaries represents a snapshot of how people affect ocean water quality.

Estuaries are formed where the river meets the sea.  In this unique position, they accumulate pollutants from both the immediate vicinity and from further afield.  To analyze the degree of pollution in a marine environment, mussels are often used as bio-indicators.  These sedentary creatures have a high capacity for filtering water and tend to bioaccumulate environmental contaminants.  Exposure to toxic agents in the water causes DNA strand breaks in the mussels’ cells, including blood cells (hemocytes), which can be measured using a gel electrophoresis comet assay.

Sample Handling from Field to Lab

Hemocytes can easily be collected in the field, but the follow up work needs to be done in a laboratory.  This has been a stumbling block for environmental toxicology studies because the variable of time can introduce new DNA damage to sensitive samples.  Appropriate sample handling techniques must be developed to accurately preserve samples from the field to the lab.

Hemocytes from mussels can be stored for a week at 4°C, but if the stored hemolymph contained a genotoxicant, the chemical continues to do damage to DNA during the storage period.  This sort of sample handling skews the results and causes variability between experiments.

In an effort to develop better sample handling techniques for mussel hemocytes collected in the field, one group recently found a way to adapt the CoolCell freezing container for use outside the lab[1].  They placed the CoolCell in the vapor phase of liquid nitrogen (LN2) to cryopreserve hemocytes for later analysis.  Liquid nitrogen is convenient to use in the field, and the samples can be stored in the vapor phase of LN2 for up a month. Compared to freshly isolated hemocytes, DNA damage was not significantly altered when the cells were cryopreserved in this way.  This new method of storing mussel hemocytes expands the utility of the comet assay, and gives scientists better tools to understand human impact on coastal environments.

Reference:

1. Kwok A. et al.  Cryopreservation and storage of mussel (Mytilus spp.)  haemocytes for latent analysis  by the  Comet  assay.  Mutation Research 750 (2013) 86-91.

 

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Sequester Impacts NIH funding

May 2, 2013
Sequester Impacts NIH funding

Rolf Ehrhardt and Michelle Nemits in Washington, DC, showing their support

Last month, BioCision’s CEO and VP of Marketing took a detour from the AACR meeting to show their support for the funding of scientific research at the NIH.

In a recent interview with Dr Francis Collins, Director of the NIH, he expressed his frustrations about the political environment and recent budgetary squeeze.

Traditionally, “biomedical research has had bi-partisan support.” says Dr Francis Collins. After all, “medical research is about all of us — our loved ones, ourselves — and it’s also a great way to stimulate the economy so what’s not to love?” But sequester brought $1.6 billion in cuts to the NIH.

We can only hope that the reduction in NIH funding is a temporary glitch.

To listen to the full interview, please see the podcast below.

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The CoolCell LX Container: a Lean, Green, Freezing Machine

April 30, 2013
ATCC CoolCell edit 1006x1024 The CoolCell LX Container: a Lean, Green, Freezing Machine

ATCC CoolCell Freezing Container

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The Need for Standardization in Biobanking

April 26, 2013

There are more than 700 biobanks in the United States, spread across 43 states plus the District of Columbia[1].  In a recent survey, nearly two-thirds of biobanks were found to be established in the last decade[1].  With this explosion of growth, the biobanking industry is battling growing pains.  The lack of standardization is an underlying problem: how samples are handled, how patients are consented, and bureaucratic rules and regulations vary from bank to bank.  This landscape confuses and frustrates doctors, patients, and scientists alike.

As a recently published article in GenomeWeb states “What works best for the biobanks may also make life hard for scientists, particularly if they are trying to use specimens from multiple banks…[2]”

29 biobanking 300x200 The Need for Standardization in Biobanking

Biobanking will be a more powerful resource with improved standardization.

Biobanks can’t always control sample handling prior to banking.  Banked specimens may be cast-offs from pathology departments, whose first priority will always be the patients, not the samples.  Or, specimens may come from patients who wish to donate, but who are being treated at a facility that is not equipped with the resources or the training to properly preserve samples.  The lack of industry standardization results in a hodge-podge of banked tissue.  According to Nicole Lockhart, a program director in the National Human Genome Research Institute’s Ethical, Legal, and Social Implications research program,

“There are a lot of differences, even in the way people freeze specimens. Are they snap-frozen in liquid nitrogen? Are they frozen in isopentane? Are they formalin-fixed and paraffin-embedded? There can be kind of a huge range in how specimens are processed”[2]

Advances in technology make it easier to use samples that were preserved before standardization was a focus.  For example, we can analyze the genomics of samples that have sat preserved in paraffin for 15 years.  However, this might be something of a double-edged sword.  Results from these more sensitive analytical technologies could be misleading if they amplify sample handling artifacts!

Besides the preservation of such variables, many of these older specimens lack proper consent for the genome-wide association studies that researchers want to do.  This essentially renders the samples unusable.  To avoid this problem in the future, the industry needs to educate doctors and nurses about how tissues will be used for research, and the importance of tracking medical information with donated tissues.  Healthcare providers, in turn, can educate their patients.  The broadest possible consent, which includes deriving stable cell lines, sharing tissue between researchers, use of donated material in animal studies, and genetic profiling will make banked tissues a better resource [2].

In an effort to improve, key players in biobanking are calling for more standardization.  The College of American Pathologists has established a Biorepository Accreditation Program.  It’s the first of its kind, and it aims to set standards for biobanking that would ensure more consistent sample handling and quality.  Similarly, the National Cancer Institute has established guidelines for NCI-supported biorepositories to address the lack of standardization which they’ve recognized as a “significant roadblock to the progress of cancer research.”[3]  Hopefully the field will learn from past mistakes and establish a solid foundation going forward.

References:

1. Henderson GE et al.  Characterizing biobank organizations in the US: results from a national survey.  Genome Medicine 2013, 5:3. doi:10.1186/gm407

2. M. Jones.  GenomeWeb Feature: Amid Array of Biobanking Obstacles, Researchers Work on Solutions.  GenomeWeb Daily News.  April 24, 2013.

3.  National Cancer Institute, Biorepositories and Biospecimen Branch.  NCI Best Practices.

 

 

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Sample Handling: Quality Assurance Could Improve Biomarker Search

April 25, 2013

A recent publication cites BioCision’s CoolCell® freezing container for cryopreserving biobanked blood in the development of a novel measure of sample quality[1]. The CoolCell ensured all blood samples started on equal footing before being subjected to variable sample handling.

28 biobank 199x300 Sample Handling: Quality Assurance Could Improve Biomarker Search

The integrity of biobanked samples is important to researchers using those tissues for biomarker studies.

Biomarkers are measurable characteristics, such as proteins or cytokines, which can be used to diagnose and track diseases. Developing good biomarkers is important for clinical research and disease management.  Because of their clinical value, researchers continue to look for new biomarkers.  Biobanks can help in that search by providing tissues, but the samples they offer are only useful if the researcher can be assured that the sample has not been significantly altered due to poor sample handling.

Some variables a biobank can’t control, like hormonal differences from one patient to the next.  However, there are a large number of variables, all related to sample handling, that biobanks can control.  For example, the length of time that samples sit at room temperature, the speed at which samples are centrifuged, or how they are cryopreserved are paradigms of controllable parameters.  With poor sample handling, proteins degrade.  This includes potential biomarkers.

Researchers looking for biomarkers could save time and money if the samples they obtain from biobanks underwent some form of quality control.  A recently published paper makes a step in that direction[1].  The authors identified proteins that can be used as markers of sample integrity. They translated their findings into a measurement called SPIN, or Sample-specific Protein Integrity Number.

Samples were collected and subjected to different sample handing techniques, causing changes to some proteins.  To do this, the researchers aliquoted and froze samples of blood, urine, and thyroid tissue from five different donors.  The blood was frozen using the BioCision CoolCell freezing container, while the other tissues were snap-frozen[1].  On thawing, each aliquot was treated differently: samples were left at room temperature for 30-120 minutes, or subjected to repeated freeze-thaw cycles.  Mass spectrometry (SELDI-TOF MS) identified peaks representing proteins that reliably degraded with poor sample handling (“dynamic proteins”).  They also identified peaks that represented proteins that did not degrade, which serve as internal controls. The SPIN index was calculated by dividing the peak intensity of a dynamic protein by the peak intensity of a stable protein.

We can’t know that every sample in a biobank was handled perfectly.  But tests like these provide after the fact quality control that could prevent researchers from using “bad” samples.  The authors hope that this study and others like it could be used to develop a dip-stick test to assess protein integrity that would make quality control cheap and ubiquitous across biobanks[1].

Reference:

1. TJ Geddes et al.  SPIN: Development of Sample-specific Protein Integrity Numbers as an Index of Biospecimen Quality.  Biopreservation and Biobanking.  2013 Vol 11(1):25-32. DOI: 10.1089/bio.2012.0039

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Cellular stress research is aided by CoolCell freezing container

April 23, 2013

Independent publication cites the use of the CoolCell® freezing container to test a protein’s ability to protect against cellular stress that’s caused by the environment[1]

25 cell stress brine 300x185 Cellular stress research is aided by CoolCell freezing container

Brine shrimp are protected from environmental stress by a protein that targets the mitochondria.

Dehydration and freezing kill, but they don’t have to.  Some plants and animals have adapted mechanisms to protect themselves against severe cellular stress.  Environmental changes, like the absence of water or freezing temperatures, can instead send cells into a state of virtual suspended animation.  The ability to survive cellular stress is a result of the proteins and sugars hardy plants and animals produce.  Researchers recently used the CoolCell freezing container to show that one of these proteins, a late embryogenesis abundant (LEA) protein, helps mitochondria survive freezing conditions1.

A common strategy that draught- and freezing-tolerant plants and animals employ to weather cellular stress is to express LEA proteins, along with the disaccharide trehalose. LEA proteins were first discovered in plants some thirty years ago, and are known to reduce damage to seeds and seedlings that suffer draught and freezing conditions.  Now there are more than 30 known LEA proteins, found in both plants and animals.  Researchers are still working to understand how they function against cellular stress.

Researchers recently described a LEA protein in brine shrimp that protects against both desiccation and freezing[1].  The protein, AfLEA1.3, has a stretch of amino acids in its N-terminal region that targets it to the mitochondria.  By transfecting Drosophila cells, which aren’t adapted to survive freezing or desiccation  the researchers were able to investigate the protective nature of AfLEA1.3.  They found that Drosophila cells expressing AfLEA1.3 tolerated osmotic stress and desiccation better than cells without the protein. In separate experiments, the researchers isolated mitochondria from transfected Drosophila cells and froze them at -1°C/min using the CoolCell freezing container to determine if AfLEA1.3 improves mitochondrial tolerance to freezing[1].  Brine shrimp mitochondria are very tolerant to freezing, and, indeed, so were mitochondria of Drosophila that express AfLEA1.3.

LEA proteins take on a highly organized secondary structure only in conditions of severe dehydration.  These experiments didn’t all involve extreme dehydration, but the protein conferred protection, nonetheless. It appears that relatively disorganized, partially hydrated LEA proteins can stabilize and protect against cellular stress, too.

Reference:

1. Marunde, MR.  Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein.  J Insect Physiol. 2013 Apr; 59(4):377-86.

 

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