Freezing of peripheral hematopoietic progenitor cells (PHPCs) for clinical transplants

In a clinical setting, recovering a high percentage of cells, and having those cells function normally, is of very high importance. A study that’s been published electronically ahead of print in the journal Transfusion sought to minimize both cost of storage and toxicity due to DMSO exposure while maintaining hematopoietic and immunologic reconstitution of autologous peripheral blood stem cell transplantations.

The researchers tested a new method for freezing of peripheral hematopoietic progenitor cells that involved a lesser concentration of DMSO (just 3.5%) and using a -80°C freezer instead of storage in a liquid nitrogen container. The group concluded that “Transplantation with PHPCs cryopreserved at -80°C for no more than 6 months is satisfactory for long-term hematopoietic and immunologic reconstitution.”

Clinical cell freezing of PHPCs – now easier and cheaper.

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Additional Considerations when Freezing Cells

Using an optimized, reproducible protocol is of great importance when looking to freeze cells for later research or clinical use. However, freezing cells involves more than just the protocol.

Especially in unregulated research laboratories, maintaining complete records of samples is easy to overlook. When storing frozen cells or tissue, the location of each sample should be documented and linked to information about the sample (for example, when the sample was collected, what has been done to it, what experiment is it a part of, etc.) Simply writing on the cryotubes is often not sufficient, as cryogenic temperatures can cause ink or adhesives to lose their useful physical properties. Software is sometimes used to help document this information and track samples, and there is specialized software made specifically for this purpose.

The protocol is also only as good as the equipment used to perform it. For any cell freezing application, reproducibility is of great concern. Variability in the method can cause decreased viability or aberrant function after thawing. By using high-quality cell freezing equipment reproducibility can be increased and such negative effects can be avoided.

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PromoCell Recommends Freezing Cells with the CoolCell

PromoCell, a Germany-based manufacturer of human primary cells, stem cells, blood cells, and cell culture media, will be recommending the CoolCell for freezing cells! Dr. Ute Liegibel, who runs PromoCell’s “Cell Culture Trouble Shooting Course” told us that he’ll be using the CoolCell to demonstrate cell freezing during the course. He also mentioned that he’s successfully used the CoolCell for freezing keratinocytes and is “very pleased with the result.”

Thanks, PromoCell!

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Freezing Cells and Tissue: How Cold is Cold Enough?

Colder is often better for freezing cells and tissue, but sometimes there are practical considerations that make us not want to store everything in liquid nitrogen (imagine if every time you wanted to retrieve an antibody or enzyme you had to pull it out of liquid nitrogen). The same goes for freezing cells for cryopreservation. While -80°C is not cold enough for long-term cryopreservation of cells, how cold is cold enough?

The key is the glass transition temperature of water. There’s no agreed upon value, but the glass transition temperature is roughly between -135°C and -125°C. Anything below -135°C and you’re safe.

Now you may be thinking: “What’s the difference? That’s well below the range of any laboratory freezer!” However, that temperature requirement offers us one nice convenience. The temperature of liquid nitrogen is -196°C. However, the vapor phase that’s found in a dewar or most other cryogenic containers is around -150°C – easily cold enough to offer good cryopreservation. Storing frozen cells and tissue in the vapor rather than in liquid nitrogen is much safer, and also prevents cryotube explosions that occur when using low quality cryotubes or not sealing them properly. (The explosions occur because liquid nitrogen enters the tube. That’s why we always recommend high-quality cryotubes such as TruCool™.)

How cold is cold enough for cryopreserving frozen biological samples? The vapor phase above liquid nitrogen is cold enough so long as it’s in a proper cryogenic container.

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Flash Freezing Cells & Tissue for Microscopy

Cryopreservation certainly isn’t the only reason one would want to freeze cells or tissue. In many laboratory applications, sample quality is better preserved by standardized, rapid freezing of samples.

During treatment for skin cancer, for example, multiple thin sections of tissue are often examined to determine which areas contain residual cancer cells. The tissue that is examined for this determination is normally frozen in a cryostat, however this can lead to the drying of the tissue and drying artifacts, as well as increasing overall turnaround time. Newer methods are being developed which use flash freezing / snap freezing to achieve better sample preservation and reduce turnaround time, but such methods are still in need of standardization. Continue reading here for more info.

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Neural Cell Freezing for EM

Electron Microscopy is often a go-to method for ultra-high resolution structural investigation of neurons, however the sample preparation required makes it questionable whether the neurons are really in a physiologically relevant state after sample prep.

A new study examines the fine structure of hippocampal mossy fiber synapses, comparing standard electron microscopy methods with rapid high-pressure freezing. The research team notes that “There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts,” and theses artifacts interfere with neuroscientists’ ability to understand the structure and function of finer synapses. Their results were positive, with high-pressure neuronal freezing effectively preserving the ultrastructural detail of the cells.

The study can be found here: PubMed link. For more information on cell freezing, including products and protocols, visit us at BioCision.

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Freezing Nucleofection™ Competent Cells

cryopreservation

CoolCell cryopreservation containers

We’re really happy that Lonza, with their vast knowledge of cell culture products, have recommended the BioCision CoolCell for freezing of their Nucleofection™ competent cell lines! Of course, Lonza had done their homework on cell freezing before, testing multiple devices against each other and going with the CoolCell!

Step 9 in the freezing guidelines section from Lonza’s “Protocol for Cryopreservation of Nucleofection™ Competent Cells” states:

Freeze the cell aliquots in a controlled manner by decreasing the temperature by 1°C per minute down to at least to -80°C (recommendation: use a manual device e.g. CoolCell®, Biocision for this purpose). The cells can be stored at -80°C for up to 24 hours prior to the transfer to the cryogenic storage. If you use a controlled rate freezer, decrease the temperature to at least below -125°C before you transfer the vials to the cryogenic storage.

Thanks, Lonza! We appreciate the nod to our CoolCell!

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Freezing Induced Pluripotent Stem Cells

A recent publication in the journal Stem Cell International briefly discussed the freezing of induced pluripotent stem cells derived from adult equine fibroblasts. While assessing the effectiveness of the induced pluripotent stem cell freezing method was not the focus of the study (rather, the generation of the iPS cells was the focus) it does provide an at least workable protocol.

Of course, induced pluripotent stem cells can differ based on the cells that they are derived from and the method used to induce pluipotency, so this is by no means an endorsement of the cell freezing protocol for all types of iPS cells. Nor is it necessarily an optimized cell freezing protocol. It may, however, serve as a good starting point, and we at least know the protocol works for iPS cells derived from adult equine fibroblasts.

The researchers used an isopropanol-based cell freezing container equilibrated to 4°C. The iPS cells were dissociated into clumps of approximately 100-200 cells, collected in 15 ml tubes and washed with iPS cell medium. The medium is described as a “conventional medium containing α-minimum essential medium (α-MEM) with deoxyribonucleosides and ribonucleoside (Invitrogen, Mulgrave, Australia), supplemented with 2 mmol/mL glutamax (Gibco, Invitrogen, Mulgrave, Australia), 0.1% (v/v) Mercaptoethanol (Gibco), 1% (v/v) nonessential amino acid (NEAA) (Gibco), 1% (v/v) ITS (10 μg/mL insulin, 5.5 μg/mL 125 transferrin, 6.7 ng/mL selenium; Gibco), 5 ng/mL human LIF (Millipore, North Ryde, Australia), 10 ng/mL βFGF (Millipore), 10 ng/mL EGF (Invitrogen), 0.5% (v/v) penicillin-streptomycin (Gibco), and 20% (v/v) FBS.” The cells were then centrifuged at 400g for 3 minutes, the supernatant was discarded, and cells were resuspended in iPS cell medium. 500μL of cell suspension was added to cryovials and freezing medium was then added, which consisted of 80% FBS and 20% DMSO. The vials were frozen to −80°C overnight and then transferred to a LN2 tank at minus 196°C for long-term storage. To thaw the cells, the cryovials were placed in a water bath at 37°C for 1 minute. Cells were then transferred to a 15 ml tube and 10 mL of iPS cell culture medium was slowly added. The cells were centrifuged at 400g for 3 minutes, the supernatant was discarded, and the cells were resuspended in iPS medium for use.

All in all it’s a pretty standard cell freezing protocol, aside from the cell-specific medium. One thing that we found that was noteworthy was their use of an alcohol-based cell freezing container. The team could have saved themselves the expense and hassle of using alcohol if they opted for a BioCision CoolCell to freeze their induced pluripotent stem cells. The CoolCell’s design ensures reproducible freezing without ever needing alcohol, making cell freezing cheaper and easier than ever.

The research article that we discuss, “Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC” can be found here.

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Freezing Bacteria

The bacteria that many researchers use in the lab have a reputation for being hardy and growing like wildfire on just about anything. Many bacteria species, however, are quite finicky, and the finicky ones don’t often take well to freezing. Freezing methane-oxidizing bacteria, for example, can be quite difficult. While many bacteria can be lyophilized, most methane-oxidizing bacteria do not survive lyophilization. Applying “general purpose” cell freezing procedures to methane-oxidizing bacteria is similarly ineffective and the bacteria can only be frozen for short periods of time if viable recovery is desired. Despite the importance of having optimized cell freezing protocols for bacteria (for research, biodiversity and biotechnological applications), such protocols are scarce.

A team of researchers from Belgium took up the task of finding a workable cell freezing protocol for methane-oxidizing bacteria and published the results in a recent paper: “Survival or Revival: Long-Term Preservation Induces a Reversible Viable but Non-Culturable State in Methane-Oxidizing Bacteria.” Interestingly, they found that the key issue in recovery of bacteria after freezing is not viability, but rather culturability. While many bacteria freezing protocols resulted in acceptable levels of viable bacteria, these bacteria would not proliferate in culture to form colonies. The key to culturability was found in the preservation medium and cryoprotectant. 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant was the only combination tested that did not result in a significant loss of culturability in all strains.

One aspect that is often critically important that this group did not test, however, is the freezing rate. Even slight differences in freezing rate can effect cell viability or other post-thaw properties. In order to ensure that bacteria are frozen at a highly reproducible rate, a cell freezing container, such as the BioCision CoolCell, can be used. Such cell freezing containers are far more economical than programmable freezers, and the CoolCell, whose technology is based on highly conductive metal alloys, does not require any consumable such as alcohol in order to operate.

The open-access article can be found in PLoS ONE.

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Cryotubes & Cell Freezing

barcoded cryotubes

Cryotubes

One of the important but often overlooked aspects of cell freezing is the cryotube that is used. If a low-quality cryotube is used, liquid nitrogen can enter the tube during storage. While this may not damage the samples while in storage, as they are in a cryogenic environment anyway, it is a problem when you go to thaw the cells.

Since frozen cells are generally thawed quite rapidly, the liquid nitrogen in the tube will rapidly vaporize, causing a rapid increase in pressure inside the cryotube that most often results in the explosion of the tube. Such a situation can arise due to the use of poor quality cryotubes or due to user error. In the case of user error, the cap of the cryotube is generally either left too loose or over-tightened, damaging the threading.

Another important aspect of how cryotubes impact good cell freezing technique is sample organization. Markers and pens do not always prove to be a reliable method of marking a cryotube. To ensure that samples are accurately managed, some cryotubes employ bar codes. Bar codes that are built into the cryotube design cannot wear or rub off like ink, and are the required in many biobanking facilities.

For barcoded cryotubes that are consistently high-quality, we invite you to view our TruCool Cryotubes. TruCool CryoTubes are leak-proof, having a screw-cap with a thermally fused gasket. Instead of the usual silicone o-ring, which is usually not affixed to the tube or cap, this gasket is a co-molded thermoplastic elastomer that is actually fused onto the cryotube.

TruCool Cryotubes come in sizes from 1ml to 5ml and in either internal or external threading. All TruCool cryotubes are sterile and DNA-, DNase-, RNase-, Pyrogen- and ATP-free.

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