Where traditional pharmaceutical drugs have failed, cell-based therapies can offer promise and solutions for treatment of diseases, which could revolutionize medicine and human health. Cell-based therapies are referred to as “living pharmaceuticals” because they use viable cells as the final drug product. However, manufacturing processes for these cell therapies are complex and pose unique challenges in comparison with traditional pharmaceutical drug manufacturing. In particular, cell-based therapies typically require a cryopreservation step to ensure that the cell therapy product is preserved without compromising sterility and efficacy. This, however, increases the complexity of cold-chain management for late-stage clinical or commercial products. Cryopreservation of the final product allows for the product to be shipped at times that are most convenient and timely for the patient and clinical staff. It also obviates the need to maintain cells in culture for extended period, minimizing the chances of senescence, genetic drift, and epigenetic changes that could alter the cells’ beneficial characteristics. Cryopreservation of the final product ensures that cell-based therapies are accessible and available on a global scale.


Cryopreservation is the process of lowering the temperature of biological systems (e.g., cells) in order to preserve their structural and functional integrity. The goal of an optimal cryopreservation strategy is to lower the temperature below
–130°C without intracellular ice formation during the transition from aqueous phase to ice phase. Successful cryopreservation will ensure that cells achieve the glass transition temperature (i.e., when liquid begins to behave as a solid), arrest molecular transport, and remain in the state of “suspended animation” without compromising the number and quality of cells [1]. Cryopreservation is preceded by manufacturing steps that include cell wash, cell harvesting, and formulation. The formulation step that involves the addition of cryoprotectant and ancillary materials and/or excipients to the cell suspension, is a time-sensitive and temperature-sensitive step and even minor execution errors in this step can have a negative impact on the final product [2].

Successful cryopreservation strategies are influenced by many factors including cell size, morphology, cell membrane permeability, and composition of organelles. Success is also significantly influenced by external factors such as composition and density of cell culture medium, choice of cyroprotectant, and cooling rates (Figure 1) [3]. To complicate things further, these factors need to be tailored specifically for each final cellular product. Suboptimal cryopreservation can result in loss of viability, insufficient cell number per dose, and dose-to-dose variability that may affect the overall efficacy of the therapy.

Diagram showing the relationship between cooling rates, cryoprotectant choice, and formulation and fill strategies in successful cryopreservation.

Figure 1. Optimal cryopreservation of cell therapeutics is impacted by several external factors, which must be optimized for individual cellular products.

A major challenge in cryopreservation is ensuring that cells not only survive the freezing process, but also maintain safety, efficacy and potency profiles post-thaw. It is critical to optimize the process in order to avoid osmotic shock and membrane damage, which may lead to post-thaw cell death. Inadequate and non-uniform use of freezing parameters can lead to the artificial selection of subpopulations with phenotypic characteristics that are different from the desired population. Cryopreservation-induced stresses arising from various factors, including cryoprotectant toxicity, intra- and extracellular ice crystallization, altered intracellular pH, osmotic imbalance, and suboptimal rates of cooling and post-thaw warming, also represent a major hurdle and contribute to significant loss of cell viability and cellular function. Cryopreservation-induced stress can result in two types of cell death: apoptosis and necrosis. Following cryopreservation apoptosis and necrosis are normally observed 6 to 24 hours into post-thaw culture [4,5]. Necrosis, characterized by swelling and disintegration of cellular organelles, is fast acting, caused by external stressors, and results in massively significant cell loss. In contrast, apoptosis, commonly referred as programmed cell death, is characterized by cell shrinking and formation of apoptotic blebs, affecting single cells or small populations of cells [4,5].

Measuring cell viability post-thaw has its challenges. Immediate post-thaw viability measured by membrane integrity tests such as trypan blue dye exclusion or fluorescent cell imaging is not an accurate measure of cryopreservation process quality [6], highlighting the need for other assays to obtain a realistic viability profile. Preferably, post-thaw assessment of cell viability and cell number should be carried out beyond the 24-hour period [6]. Long-term testing (e.g., over 3–5 years) at multiple post-thaw intervals would be extremely beneficial in evaluating the robustness and stability of the cryopreservation process.

Cryopreservation-induced delayed-onset cell death (DOCD) is another form of post-thaw cell death has been observed and appears to arise from a combination of necrotic and apoptotic stresses. However, unlike necrosis and apoptosis events, cryopreservation-induced DOCD may not be obvious through one time-point analysis of viable cells during the first few hours in the post-thaw process [7]. Instead cryopreservation-induced DOCD is usually characterized by a significant decrease in viability 12–24 hours post-thaw. DOCD results from permanent damage to cells when the level of oxidative stress is beyond the cells’ ability to sustain or repair [7]. The choice of cryoprotectant and freezing medium formulation is critical to minimize DOCD and improve cell survival after cryopreservation.

Choice of cryoprotectant

Cryoprotectants preserve cells and tissues by minimizing physical and chemical damage during cryopreservation and promoting cell survival and cellular structural integrity. Effective cryoprotectants have a low molecular weight, are nontoxic, and do not influence the behavior of post-thaw cells. Cryoprotectants can be divided into two main classes: intracellular agents and extracellular agents (Table 1). Intracellular cryoprotectants work by penetrating the cell membrane and discouraging ice crystal formation. Extracellular cryoprotectants work by improving osmotic imbalances that can arise during the freezing process. While intracellular cryoprotectants are commonly used in cell-based therapies, interest is growing in the use of a combination of cryoprotectants to reduce toxicity while maintaining structural and functional integrity.

Table 1. Types of cryoprotectants.

Intracellular agents (cell membrane-permeating)Penetrate the cell membrane and prevent the formation of ice crystals that could result in ruptureDMSO, glycerol, ethylene glycol, and propylene glycol
Extracellular agents (nonmembrane-permeating)Act to improve the osmotic imbalance that occurs during freezingSucrose, trehalose, dextrose, methylcellulose, and polyvinylpyrrolidone (PVP)

Intracellular cryoprotectants

The most commonly used cryoprotectant in pharmaceutical manufacturing is the intracellular cryoprotectant dimethyl sulfoxide (DMSO, Me₂SO) (Figure 2) because it offers enhanced penetration, provides long-term stability, and maintains safety and potency of the cells in final formulation [8]. DMSO has been used as an ancillary agent and as an excipient in final formulations.

Diagram showing the arrangement between sulfur, methyl, and oxygen groups in DMSO.

Figure 2. Structure of DMSO, a commonly used intracellular cryoprotectantfor cryopreservation. DMSO is an organosulfur compound that freezes within 18.5°C. It is also a polar aprotic solvent which can dissolve polar and nonpolar compounds and can be easily miscible with water, allowing it to reduce the electrolytic concentration in the residual chilled contents in and around of a biological cell, during cryopreservation.

While DMSO is the most commonly used cryoprotectant, it does have disadvantages. DMSO can adversely affect genomic and proteomic profiles of the cells and cause damage to cellular structures including mitochondria, the nucleus, and the cell membrane. DMSO can also cause a variety of adverse reactions in patients. When used as an excipient, the toxicity associated with DMSO requires that it be used at very low concentrations. Intake of DMSO at <50 mg/day is acceptable, and intravenous administration of up to 1 g/kg/day is common practice in transplantation therapies [9,10]. If DMSO is used as an ancillary material and exceeds ICH and FDA guidelines, it must be removed through cell washes. These additional wash steps are accomplished using traditional centrifugation methods or newer approaches such as filtration by spinning membrane, stepwise dilution and centrifugation using a rotating syringe, diffusion-based DMSO extraction in microfluidic channels, or controlled dilution and filtration through a hollow-fiber dialyzer [11,12] (e.g., CytoMate™ Cell Washer, Sepax™ S-100 Cell Separation System, COBE™ 2991 Cell Processor, Lovo™ Cell Processing System, or Gibco CTS Rotea Counterflow Centrifugation System).

Extracellular cryoprotectants

Recent advancements using non-DMSO agents with a combination of osmolytes like sugar, sugar alcohol, amino acids, and proteins show promise by improving post-thaw recovery [13,14]. DMSO-free cryoprotectants are the preferred option because of their lower risk profile, better tolerance by patients, better compatibility with bags and weldable tubing, and the potential of eliminating wash steps prior to infusion.

Currently, extracellular cryoprotectants have limited use in cell-based therapies because their addition typically results in suboptimal cryopreservation performance with poor cell viability post-thaw. However, recent research has shown some promise with trehalose, a nonreducing disaccharide of glucose. Trehalose demonstrates an exceptional ability to stabilize and preserve cells and cellular structures during freezing. Research has shown that the low penetration issue with trehalose can be overcome by addition of P2X7 (an ATP-activated receptor that opens transmembrane pores of the cells) [14]. Some other initial studies suggest the use of PVP in cryopreservation of human adipose tissue–derived adult stem cells resulted in recovery of cells that was comparable to DMSO with animal serum [15]. Methylcellulose either alone or combined with low concentrations of DMSO and human serum albumin (HSA) also demonstrated some promise [16]. While these studies are encouraging, further research is needed to evaluate extracellular cryoprotectants for cell-based therapies.

Formulation and fill


Formulation is the process of combining cells, buffers, proteins, ancillary materials, and cryoprotectants and is carried out immediately after the cells are washed and harvested and following the cell expansion step. Formulation is a temperature-dependent and time-sensitive step because the harvested cells are held in suboptimal conditions without nutrition. Appropriate formulation is needed to stabilize the cells so they can withstand stress factors such as temperature excursions, pH changes, and mechanical stress caused by handling, storage, shipment, and bedside preparation. Because formulation precedes the actual cryopreservation step, optimal formulation is critical to produce a final cryopreserved cellular product that is stable, safe, efficacious, and meets regulatory requirements.

Formulation and final fill strategies involve selection of the appropriate cryoprotectant and other excipients and the final containers (see below). The selection of excipients plays a key role in the maintenance of critical quality attributes (CQAs) of the final product. Human serum albumin is one of the most popular excipients in cell therapy because it is the most ubiquitous protein in blood and is known to create an optimal microenvironment for sustained cell viability. It acts as a scavenger of toxins and other reactive oxygen species, maintains pH, provides insulation, and maintains cell viability during cryopreservation [17]. Additional components of the final formulation include dextran, which serves as an osmotically neutral volume expander and as parenteral nutrition, sodium chloride as a normal saline diluent, and stabilizers such as sodium caprylate and N-acetyltryptophanate that protect proteins such as HSA from oxidative stress [18].

Container choice

The choice of container can greatly impact the success of the overall therapy. Containers provide physical protection and are responsible for the stability over the entire lifecycle of the final product. The design and manufacturing of the containers must adhere to specific standards for storage and shipment reproducibility. Containers must also feature characteristics such as ease of use, stability at below-freezing temperatures, the absence of leachables and extractables, resistance to cryoprotectants (e.g., DMSO), and optimal labeling surface [19]. The types of final containers most commonly used for cell-based therapies are screw-cap cryovials, bags, and plastic or glass vials (Table 2).

Table 2. Advantages and disadvantages of common container types for cell-based therapeutics.

Container typeAdvantagesDisadvantages
Screw-cap cryovials

  • Cost-effective
  • Convenient
  • Long history of use in cryopreservation
  • Open system
  • Increased potential for contamination
  • Labor intensive
  • Increased fill error rates
  • Limited volume per dose

  • Closed system
  • Customizable options
  • Good labeling surface
  • Minimal bedside tasks
  • Increased costs for specialty equipment
  • Specialized operator training required
  • Scale-up issues limiting lot sizes
Plastic or glass vials

  • Closed system
  • Improved manufacturing scale up capabilities
  • Very expensive
  • Specialized training required

Screw-cap cryovials have been extensively used to store many cell-based products, especially for banking of GMP-grade master cell banks. Screw-cap vials are convenient and cost-effective; have a long-standing cryopreservation record; and work well for analytical and stability testing. However, they pose several regulatory challenges. Screw-cap cryovials require open steps for product filling that need to be carried out in a biosafety cabinet (BSC), making the process labor intensive, subject to human error, and more prone to contamination. They are also limited in volume per dose, have a limited labeling surface, and require extensive manipulation at the receiving site prior to delivery into patients.

Use of single-use bags is preferred by manufacturers of cell-based therapies. Single-use bags and kits along with combination of ports and accessory tubing are available in standard and custom sizes based on their usage for various unit operations (e.g., cell washing, cell expansion, volume reduction cell harvest, cryopreservation). Single-use bags offer the advantages of optimal labeling surfaces, multiple sampling ports and minimal bedside manipulation (for final dose delivery). Use of these bags, however, requires investment in specialty instruments such as welders and sealers, specialized training for operators, and carefully planned processes for air removal and specialized packaging to ensure that the bags do not develop cracks and cause leakage of product after thawing. Though multiple bags can be filled using kits or automated systems, scale-up is challenging and lot sizes for a single manufacturing run are typically capped at 150–200 product bags [3].

The use of “ready-to-use” containers such as vials made of cyclic olefin copolymer and a pierceable septum that acts as a sterile barrier offer the advantages and flexibility of a closed system and scale-up for commercial needs [19]. However, they are expensive, require specialized training, and may require filling operations to be conducted inside the BSC unless a substantial financial investment is made in purchasing large and complex multifunctional automated systems or ISO 5 GMP manufacturing suites.

Scientist wearing gloves working with a cryogenic storage device.

Cooling process and rates

The process of lowering the temperature of cells to a frozen state requires a series of steps, which are individualized to cell type. First, in sequential steps, the cryopreservation formulation medium is added to the cells at a controlled rate to prevent cell loss resulting from osmotic stress. It is common practice to prechill the cryopreservation medium and to keep the cell suspension and the admixture chilled using cold packs, a frozen blanket, or a chilled work surface to prevent heat-related cell damage when adding DMSO. After adding cryopreservation medium, the cell suspension is transferred to the precooled chamber of a controlled-rate freezer. During the freezing process, product temperatures are recorded using a probe to generate a freeze curve.

The rate of cooling during cryopreservation has a dramatic impact on cell viability of the final product. Cooling rates control the formation and size of both intracellular and extracellular ice crystals and can impact solution effects during the freezing process. While rapid cooling maximizes intracellular ice formation and minimizes solute concentration effects, slow cooling has the opposite impact. Currently, slow cooling is the most frequently used method of cryopreservation for a variety of cell types [5]. Furthermore, rapid cooling methodologies require a much higher concentration of cryoprotectant, resulting in toxicity-induced cell loss and/or addition of a washing and reformulation step at the clinical site [5].

Scientist wearing gloves working with a cryogenic storage device.

Figure 1. Controlled rate freezers, such as the Thermo Scientific CryoMed freezer, help manage the cryopreservation process to ensure optimal parameters are met. For more details on the use of the CryoMed freezer, please read this Smart Note


The process of cryopreserving final product for cell-based therapies is critical, as sub-optimal cryopreservation process can lead to failed product lots and ultimately failure to treat patients. While some point-of-care facilities for early stage clinical trials continue to deliver non-cryopreserved or “fresh” final product to the patient’s bedside, this is not a sustainable option. As the field of cell-based therapies matures, delivery of cryopreserved final product that is standardized, scalable, reproducible, in compliance with global regulatory agencies, and has a maximized shelf life for an “on-demand distribution” will prove to be the best option.

— Author: Rupa Pike, PhD, Thermo Fisher Scientific.

For more details on the cryopreservation process, please check out this white paper


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