PluriFreeze Cryopreservation System

We recommend storing PluriFreeze products in a refrigerator between 2-8°C. This is ideal both for long-term storage and to ensure that the product is at the optimal temperate for use.

PluriFreeze products can remain stable at ambient temperatures during shipping and for short-term storage. If storing at ambient temperatures, keep the products covered to avoid sustained exposure to light.

The efficacy of PluriFreeze does not differ from the labeled expiration following opening (assuming that sterility is maintained following opening to ensure the product remains free of contamination). Internal monitoring has demonstrated comparable performance of PluriFreeze for two years at 2-8°C. 

Most customers employ a wash step prior to cryopreservation that is specific to their process to remove culture media, growth factors, and cellular byproducts. Washing your cells in PluriFreeze Base prior to cryopreservation can be beneficial as it introduces cells to the basal salts and sugars that support the decreased metabolism of hypothermic (i.e., 2-8°C) temperatures. It is also a hyperosmotic solution that can prevent cells from swelling prior to cryopreservation and can be used to dilute cells to a known concentration that can facilitate automated and simpler downstream processing. As always, we recommend that you scientifically evaluate and compare the relative efficacy and need for a pre-storage washing step in your specific process.

It is not likely that frozen storage would influence the effectiveness of PluriFreeze following thaw (either at room temp or 2-8°C). However, we have no validated data specific to that excursion and so cannot offer guidance on the use of PluriFreeze products that have been frozen during storage.

Lactated Ringer’s solution and saline were originally designed as clinical IV fluid replacements. As such, they are composed mainly of sodium salts similar to those found in blood and contain little buffering capacity. PluriFreeze Base, on the other hand, is designed specifically to prepare cells for cryopreservation and is composed of salts, sugars, buffers, and large impermeant molecules for osmotic and oncotic support to maintain cells during periods of low temperature stress when the powerhouse of the cell, known as mitochondria, swell to the point that they cannot recover, causing cells to undergo programmed cell death when they are returned to normal body temperatures. PluriFreeze Base mimics the intracellular environment and provides mitochondrial support compounds that allow the cell (and their mitochondria) to survive the low temperature stress that occurs prior to cryopreservation. While healthy cells can be flushed with isotonic solutions such as Lactated Ringer’s solution or saline, it is likely that marginal or less robust iPSCs or iPSC-derived cells may be better protected against cold ischemia when flushed with an optimized, pH buffered, hypertonic solution such as PluriFreeze Base.

Yes. We recommend rinsing your organoid or biopsy in PluriFreeze Base to remove blood and other extraneous fluids and then resuspending and transporting your tissue sample in fresh PluriFreeze Base at a minimum 1:1 ratio. Since it is important that the tissue be completely bathed in solution, a larger volume of PluriFreeze Base may be necessary to maintain appropriate sample suspension. It is also beneficial to select a container size that limits headroom, air exposure, and mechanical damage due to transport movement.

All PluriFreeze RUO and GMP-grade products are manufactured in ISO 13485 certified facilities and are intended for use in research or further manufacturing only. They are not intended for use in clinical applications, diagnostic procedures, or for injection in, or ingestion by, humans or animals. 

We do not recommend the introduction of a washing step post-cryopreservation. Centrifugation post-thaw represents a significant cellular insult that may reduce cell recovery and increase cell death more so than prolonged exposure to DMSO.

If the cells are to be used in a manufacturing process, we recommend diluting your sample at least 1:10 in warmed culture media (i.e., 37°C) and then allowing the cells to recover for four hours post-thaw. A media change can then be used to remove any residual DMSO. While there are cell washers and microfluidic devices in development for gentler cell washing, many of these devices are not applicable to the large-scale manufacturing of cell products under GMP conditions.

Here are some relevant references for review utilizing cord blood:

http://www.ncbi.nlm.nih.gov/pubmed/21126251

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105472/

It is always advised that you scientifically evaluate and compare the relative efficacy of centrifugation versus dilution within a specific process.

By request, a confidentiality agreement can be executed to disclose the component list to aid in identity testing, risk assessment, and product compatibility. However, because the formulation is proprietary, the concentrations of the components will not be disclosed. The components are all on the recognized excipient list and are at concentrations Generally Recognized As Safe (GRAS) by the US FDA and are at concentrations that have been approved for human infusion. The solutions contain no active ingredients and are composed mostly of salts and sugars. Since the solutions are not drugs or devices, we cannot provide clinical indications for use, and each group must qualify use within their specific process.

PluriFreeze Base is primarily designed as a pre-freeze wash solution and for short-term 2-8°C non-frozen biopreservation. It is not intended for cryopreservation, as it does not contain the highly effective permeating cryoprotectant DMSO. However, since PluriFreeze Base is composed of the same components as PluriFreeze PF10 without DMSO, it can be supplemented with DMSO at a desired concentration to facilitate efficient freezing.

PluriFreeze Base is intended to be paired with PluriFreeze PF10, which is designed specifically for cryopreservation and is available at a 10% DMSO concentration to simplify the cell culture workflow.

There is a small body of literature detailing the influence of DMSO on cell viability and function. Examples include:

1. Galvao, J. et al. Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J. 28, 1317–1330 (2014).

2. Hanslick, J. L. et al. Dimethyl Sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system. Neurobiol Dis. 34, 1–10 (2010).

3. Yuan, C. et al. Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes. PLoS One 9, e107447 (2014).

The evidence from these studies indicates that long-term exposure to DMSO at normal cell culture or body temperatures increases cellular dysfunction and death. The mechanism of cellular dysfunction and death appears to stem from an impairment of cellular energy production, resulting in the errant movement of ions into and out of the cell. The principal benefit of hypothermic and cryogenic storage is a reduction in metabolism, minimizing the cell’s energy needs and thus diminishing the toxic influence of DMSO. Indeed, cell therapies frozen in DMSO-containing cryogenic solutions have been safely delivered as an excipient agent (without wash) directly into the heart in a preclinical porcine model. Analysis of patient records further demonstrates that stem cells stored in DMSO-containing cryogenic solutions did not correlate with adverse reactions when infused directly into children, suggesting that short-term exposure of cells to DMSO in the frozen state does not lead to long-term impairment or toxicity when administered in clinical settings. The above-referenced studies are:

1. Peral, S. et al., Safety and feasibility for pediatric cardiac regeneration using epicardial delivery of autologous umbilical cord blood-derived mononuclear cells established in a porcine model system. Stem Cells Transl Med. 4, 195-206 (2015).

2. Truong, T.H. et al., Adverse reactions during stem cell infusion in children treated with autologous and allogeneic stem cell transplantation. Bone Marrow Transplant. Jan 11 (2016).

In general, DMSO is the most widely used, clinically tracked, and (for the most part) effective primary cryoprotectant. However, DMSO is often assumed to have a negative impact without specific data conclusively demonstrating impact within a specific cell type and/or clinical model. As such, we encourage all groups to assess the toxicity of DMSO within their particular application. PluriFreeze PF10 utilizes DMSO along with non-DMSO cryoprotectants such as sugars. PluriFreeze PF10 (and PluriFreeze Base) employs a chemical formulation that maintains proper ion balance, and metabolic support molecules to support cells at reduced temperatures, lowering the cell’s energy requirements and negating the effects of DMSO on cellular metabolism.

DMSO has a decades-long quality and regulatory footprint, including known hypersensitivities, for use in multiple administration models. Alternatively, a DMSO-free alternative freeze media necessitates the need to develop a clinical qualification pathway that can be submitted for regulatory consideration. As such, regulatory agents may actually request a higher level of due diligence for the cryopreservation process because there would be almost no pre-existing clinical history. The development of additional qualification pathways is likely to require considerable resources with regard to time and cost. If you still have concerns regarding DMSO toxicity or were to obtain conclusive data demonstrating a detrimental influence of DMSO in your specific application, we could assist in development projects to explore commercially available DMSO-free options or develop custom cryopreservation formulations.

We have not focused specifically on a DMSO-free cryopreservation solution. Although there is discussion in the field regarding DMSO-free options, the DMSO-containing solutions still are most effective. PluriFreeze Base contains the salts and sugars of PluriFreeze PF10 without DMSO but is intended as a pre-freeze wash solution or as a base solution to be used with the addition of DMSO. In general, a DMSO-free freeze media would replace DMSO with another cryoprotectant that may be less effective or have a similar mechanism of action to DMSO (in which case, it should have the same concerns as DMSO). In addition, DMSO has an extensive quality and regulatory history in human applications that can be readily accessed.

Overall, despite considerable interest, there is a relatively small market for DMSO-free cryopreservation products, especially for raw materials such as iPSCs. DMSO alternatives have been tested and developed for decades but have yet to see widespread commercialization and clinical adoption. While DMSO-free products would be attractive from a regulatory perspective, we view DMSO-free cryopreservation not as a replacement technology, but rather as an additional tool for certain sensitive cell types such as neurons.

PluriFreeze PF10 performs competitively with the industry reference. Both are intracellular-like solutions that provide osmotic support during the freezing process and have comparable post-thaw results in multiple iPSC lines. However, PluriFreeze PF10 has several advantages that make it superior as a cell therapy development and manufacturing solution. PluriFreeze PF10 is completely animal-origin free, contains metabolic support compounds that maintain mitochondrial health, and is comprised of components that are all on the recognized excipient list and are at concentrations Generally Recognized As Safe (GRAS) by the US FDA at concentrations that have been approved for human infusion. PluriFreeze PF10 is less viscous than the industry reference and can be paired with the PluriFreeze Base wash solution to enable efficient manufacturing scale-up and process automation. PluriFreeze PF10 formulations are also available in an RUO version that facilitates process development at a lower cost.

PluriFreeze PF10 is not formulated for cell storage at -20°C, and we have no data for use at that temperature. However, it is possible that PluriFreeze may be effective at -20°C for a very short period of time if tested and validated as part of your process. Optimal cryopreservation of frozen cells necessitates the use of liquid nitrogen to maintain temperatures below the glass transition temperature (approximately -130°C). At this temperature, cellular enzymatic activity and DNA degradation are virtually halted. At -20°C, it is possible that cells may only survive for a few weeks. In addition, the freezers that are generally used for -20°C storage are often frost-free and more variable in their temperature cycling. Long-term storage of viable cells at -20°C would only be possible with a cryoprotectant concentration high enough to raise the glass transition temperature to above -20°C. If short-term storage of cells is necessary, PluriFreeze Base is formulated to maintain cellular viability at 2-8°C for hours to days depending on the cell type.

Short-term sample storage of cells at -80°C is possible for several months. However, -80°C storage does not halt biological reactions, and the cells will degrade over time. The physics of cryobiology dictate that successful long-term storage must utilize temperatures below the glass transition temperature of cells and media (approximately -130°C). The glass transition temperature of PluriFreeze PF10 was directly measured at approximately -75°C. However, that value can fluctuate based on cell concentration. Cells frozen in PluriFreeze PF10 should be stored below the glass transition temperature (vapor phase liquid nitrogen recommended) to maintain stability.

Cells stored in liquid nitrogen using optimized cryopreservation protocols should last for decades. A decline in cell viability over time following liquid nitrogen storage is usually a function of improper temperature maintenance. Several things can contribute to this:

1. If the cells are in vapor nitrogen, the air above the liquid has gradients. As the liquid sublimates, the location of the sample in the vapor may have a variable temperature that rises too high.

2. The key to effective cryopreservation is maintaining the cells below the glass transition temperature, where activity is virtually stopped. This is usually below -130°C or -135°C. If the temperature around the sample periodically rises above that, then the sample experiences some thawing (even though still frozen). Repeated transient events that rise above the glass transition temperature (thermal cycling) are likely to reduce stability.

3. If your samples are grouped together in the dewar and you require a specific individual sample, the remaining samples are likely experiencing thermal cycling as you remove the box from storage and repeatedly select individual vials. Thus, those samples stored for extended time periods are likely to experience multiple thermal cycling events and often may demonstrate a linear decline in stability.

4. Samples should not be stored directly in liquid nitrogen, as it can lead to liquid nitrogen ingress into the container and the possibility of viral or mycoplasma contamination.

The filing is in process, and the Master Files will be available soon. Since the formulation of PluriFreeze is proprietary and not freely disclosed to customers, the Master Files will allow the FDA to view confidential, detailed information about our facilities, processes, and products. It can be cross-referenced by the FDA in their evaluation of your clinical trial. By request, a Confidentiality Agreement can also be executed to disclose the component list (but not the component concentrations) to help with your identity testing, risk assessment, and product compatibility studies.