Often, I get asked about cryopreservation; the safety of material used to cryopreserve, and the viability of cells that pass sub-zero temperatures during storage and transport.

Cryopreservation is a means to slow down and bring to a halt, metabolic functions within a living organism and prevent the decomposition of various organic compounds that make up that living organism. This is achieved by safely transitioning the organism into a sub-zero frozen state. The lower the temperature, the firmer the paused life system. Even today, safely achieving this state is no less than a miracle.

Modern cryogenic technology allows precise control over temperature and materials to preserve structural integrity and halt osmotic changes to achieve the ultimate cryopreserved state. However, the biochemical complexities of specific tissue or cells govern how well they can be preserved in sub-zero temperatures and for how long. Parameters for temperature, pressure, transition timelines, gaseous exposure, light spectrums, and contamination safety measures are specific for different cells or tissue entering or exiting a cryopreserved state (thawing).

Thawing is the process of safely recovering cryopreserved cells where ideally, the cell structural integrity and cell mortality can be minimized during the restoration of the cells back to life. The level of environmental control required to transition between these phases makes handling post-cryopreservation thawing particularly a challenge during human therapeutic rendering.

Primary factors impacting the thawing process are inferior clinical environments, clinicians who lack training, understanding, or experience in cell biology, the complete lack of regulatory oversight concerning clinical handling of biologic material meant to be injected into human subjects, the lack of handling precision on the clinical side (sound scientific protocol), the unreliability of measures to ascertain the success of the thaw, as well as the impact from materials used during the cryopreservation process itself.

The objective of any cell therapy is to overcome disease processes by reconstructive division, differentiation, and targeted paracrine activity by the cells being administered. Administration into the recipient’s body adds tremendous amounts of stress to cells that are yet to readjust to the post-thaw environment, let alone having been frozen for long periods with possible exposure to temperature fluctuations during transport.

Therefore not only does post-thaw cell integrity matter but the impact to live cells from cell debris (dead, decaying organic matter), damage from exposure to cryopreservation material as well as the post-administration stress sustained by the last living cells, govern what the end product will be, entering a patient’s body. Currently, the only method used by some clinicians is a ‘cell count’ to determine the percentage of cells remaining with an intact cell wall and a nucleus within their circumference, suggesting that they haven’t exploded yet.

A cell count is not the same as cell viability. A cell counter is a microscope with software to count cells from an image of a sample (slide). A cell counter, although defines an estimate of cells still maintaining their physical structure, does not determine if those cells have any remaining life whatsoever. Needless to say, the viability (living cells) will always remain far less than a general cell count which also accounts for dead cells still having a cell wall.

The safety of the end product being administered to human subjects is of utmost importance to any health authority in the world. In the conventional pharmaceutical industry, the minimum criteria to receive a new market registration for any generic chemical entity (with prior regulatory approval) is a Bioavailability / Bioequivalence (BA/BE) study wherein the chemical integrity of the product is analyzed for safety in-vitro (pharmacokinetics) and in-vivo (toxicology) using Mass-Spectrometry on a very large sample size (number of subjects).

How a product transforms from its last known state suggests the associated risks. Unlike chemical entities that have higher stability, cell products quickly die and decompose from minor environmental shock. This poses a hazard of toxic biological transformation. It is therefore essential to run a tight cryopreservation / thawing protocol and biologically characterize a cell product as close to human administration as possible, preferably after the thawing process. This is by far the most common safety checkpoint imposed by health authorities over companies conducting human clinical trials on their experimental therapies.

None of this is possible for the unregulated mediocre setups known as ‘stem cell clinics’ as they do not seek any regulatory approval or assume medicolegal accountability for the products they administer.

Countries with health regulatory authorities missing a biological drug legislation, are practically inept in satisfying safety parameters for the manufacturing, handling, or human administration of cell products the way they can, pharmaceuticals products. Clinicians procuring cryopreserved cells for their experimental treatments have a thin line between losing cell count, and viability or administering cryopreservation chemicals such as Dimethyl sulfoxide (DMSO) into patients which can cause serious allergic reactions.

Lapses during the secondary processing are not completely avoidable considering infrastructural and time limitations during procedures. Lost viability equals dead cells. Due to its cytotoxic nature, DMSO begins to kill cells it preserves while frozen, the moment the vial is thawed. For patient safety, a clinician must either swiftly wash the mix several times to get rid of most debris and the DMSO to lose significant viable count along with it, or they end up leaving traces of DMSO as well as decaying cell protein to cause various immunogenic reactions. Injections containing decomposing proteins are a massive immune hazard. Cell debris poses a serious risk of decaying into unwanted proteins which can lead to complicated immune disorders such as autoimmunity.

Using fresh cells that I like to call ‘off-the-incubator’, is not an option for clinicians that procure their material from central laboratories, usually beyond national borders. Culture labs solely targeting unregulated or experimental therapy providers do not comply with local health regulatory (GLP/GMP) good laboratory/manufacturing practices due to the non-existence of institutional review oversight. Such labs generally cryopreserve their cells immediately post-harvest to minimize contamination risks and transport over cold-chain logistics, to locations beyond a four-hour journey.

Since the risks associated with DMSO began surfacing, some clinics have resorted to portraying the supposed ‘advantages’ of DMSO, through social media to hide therapeutic lapses that have triggered immune disorders in numerous subjects who received therapies at such clinics. While there have also been incidents reported to the US FDA about ‘stem cell’ vials introducing new infections in subjects, from cell death caused by cold-chain transit without cryopreservation material and possible contamination. These incidents shed further light on the complete lack of safety compliance, accountability, and knowledge concerning the biological characteristics of the products used and the danger such initiatives pose for the recipient of infectious dead cell debris in the name of ‘stem cell therapies’.

Pharmaceutical regulatory guidelines in most countries, including the US FDA emphasize the safety and biochemical integrity of any medicinal product during storage, transportation, clinical handling, and post-administration (in-vivo characterization). For cell products that are categorized under biological drugs, the Center for Biologics Evaluation and Research (CBER USA) provides good research pathways.

Many countries do not have guidelines for handling biologics and biosimilars like the USA does. This regulatory loophole allows for a booming ‘stem cell’ market, now flourishing at the cost of patient safety, driven by unregulated culture labs and clinics alike. The promise of cell therapy is well-set to transform the way disorders are treated. But it will not be such entities that promote vague therapies as all-rounder miracle medicine.

As we see a rise in unregulated cell therapies being targeted toward all possible conditions, thanks to the inherent safety of specific cells, lapses in handling and storage are now becoming major risk factors. For an individual seeking cell therapy in an unregulated market, it is essential to evaluate safety before anything else.

Specific multipotent cell lines may inherently trigger immunogenic reactions in subjects while certain cell lines do not. Despite the choice of cells, cell culture phase issues such as mishandling or contamination can induce unwanted dynamics to an existing disorder. Risks associated with cryogenic preservation, cold-chain transport, and thawing protocols are well understood about live cell biologics, tissue, and blood-derived products. If a patient is not informed of known risks during baseline counseling sessions, it is less likely that a patient will be notified of the less known risks before receiving therapy.