The role of preservation in the variability of regenerative medicine products

Abstract

Regenerative medicine (RM) has the potential to restore or establish normal function of cells, tissues and organs that have been lost due to age, disease or injury. It is common for the site of raw material collection, site of manufacture and site of clinical use to be different for RM products, and at the same time cells must remain viable and functional during transportation among different sites. Freezing products down to cryogenic temperatures along with cold chain transportation has become an effective method of preserving RM products. The quality of RM products along this supply chain represents the cumulative effects of all of the processing steps and all of the reagents used in the process. A variety of sources of variability in the preservation of RM products can result in both cell losses and greater variability in the quality of RM products. The purpose of this article is to review the sources of variability in the preservation process as well as the methods by which variability can be controlled or avoided.

Lay summary

RM products involving the use of allogeneic or autologous cells and tissues are typically cryopreserved before shipping. Each step of the preservation process is a potential source of variability and can probably result in variability in the quality of RM products. This review provides an overview of the sources of variability in the processing of preservation and simple practices that can be used to control or reduce the variability. Future work will focus on incorporation of clinical outcomes with processing that has been performed to identify the root cause of quality inconsistency of RM products, as well as exploring the possibilities of simplifying and automating processing steps of preservation in order to reduce the overall variability that might be present.

Introduction

RM that uses innovative methods to restore damaged function of human cells, tissues and organs has evolved rapidly by translating novel ideas into clinical trials and commercial products with the potential of treating various types of diseases[1–10]. Cellular therapies and RM products have a very unique supply chain. Cells, tissues or organs are typically harvested from a living donor or a cadaver at a clinical facility and then shipped to a specialized site of manufacture and then on to a site of clinical use, which may be a hospital, doctor’s office or even a battlefield. All along this supply chain, the cells must remain viable and functional. The complexity of this supply chain contrasts distinctly from other medical therapies such as drugs or medical devices[11].

Cryopreservation has become an essential part of the logistics process and supply chain for RM products. The ability to successfully cryopreserve RM products enables effective transportation of the products. Furthermore, as live cells that are shipped in culture media only have a very short shelf life[12], cryopreservation of cells also allows for better coordination with patient availability as well as more flexibility in safety and quality control testing. Poor or inadequate methods of preservation have limited the ability to use cells or tissues to treat diseases. Prochymal was a cell therapy used to treat graft-versus-host disease that failed in phase III clinical trial. It was postulated that poor post-thaw function of the cells resulted in failure of the therapy[13].

Variability in the preservation process plays a role in the variability in the quality of RM products. The focus of this article is to review the sources of variability in the preservation process of trypsinized or non-adherent cells and the manner by which this variability can be controlled or reduced. It is important to note that the preservation process is to be based on scientific principles. Understanding of scientific principles are only meaningful when combined with a quality systems approach to ensure that the scientific principles are put into practice properly. With understanding of these principles, the end user can control or reduce the variability in the outcome. Additional background information on the scientific principles to improve the consistency and outcome of the cryopreservation process can be found elsewhere[14].

It is noteworthy that the quality of the end product (the viability, recovery and function of the product post-thaw) for RM products represents the cumulative effects of all of the processing steps and all of the reagents used in the process[15]. For clinical use of a RM product, it is both the final quality and its consistency that are critical for clinical use. The different steps of the preservation process are illustrated (Fig.1). Each step contributes to the overall quality of the end product and will be discussed in terms of scientific principles and recommended practices in this article.

Fig.1.

Different steps of the preservation process

Pre-freeze processing

As described above, cells or tissues used in RM products are collected from patients or healthy donors. The collection of the cells or tissues should be annotated to note factors in the process of collection that may influence product quality. The International Society for Environmental and Biological Repositories (ISBER) has developed Standard Preanalytical Codes (SPREC)[16], an annotation system for both blood and tissue biospecimens. Other organizations that have best practices for annotation of biospecimens include American Association of Blood Banks (AABB)[17] and the Foundation for Accreditations of Cell Therapies (FACT). For bone marrow aspiration, annotation of the sample may include:

• Anesthesia used

• Collection technique (location, etc.).

• Anticoagulant

• Volume collected

• Number of nucleated cells harvested

• Time and temperature between collection and processing

• Centrifugation (duration and g-force used)

• Filtration

Proper annotation of the sample includes a record of these parameters, even if they were not specifically controlled during processing. Reducing the variability in the process will require understanding the factors that influence the quality of the starting material and controlling the parameters that play an important role in the quality and consistency of the preservation process. If the collection parameters that directly correlate to the quality of preservation are unknown, it will be important to carefully annotate the sample and use retrospective analysis to determine the critical quality parameters for the starting material.

Most cells and tissues are processed after collection and that processing can be quite extensive. Cells may be isolated from an intact tissue using mechanical or chemical digestion methods. Typically, mixed cell populations are harvested and the therapeutic cell of interest may consist of a specific subpopulation which must be isolated. In particular with the new gene-editing techniques, cells can be genetically modified, most often using a viral vector. Cells may further be expanded in culture by seeding into a matrix or into a specialized culture environment (bioreactor) in order to increase the cell number. Each of the processes above may result in the cells being subjected to various stresses such as mechanical forces (shear or centrifugal), nutrient deprivation and/or viral infection. It is common for viability/membrane integrity to be monitored during processing but some of the stresses experienced during the processing described above may be sub-lethal. Reducing variability and improving post-thaw recovery require monitoring cells after processing to determine if they are stressed (i.e. exhibit early markers of apoptosis) or have shifted phenotype because of poor cell culture practices[18].

Formulation and introduction of the cryopreservation solutions

Cryopreservation of cells and tissues requires the use of specialized solutions containing cryoprotective agents (CPAs) that act to protect the cells from the stresses of freezing and thawing. Not all of the molecular-level mechanisms of action for CPAs have been determined but recent studies have demonstrated that CPAs modify the low temperature behavior of water by strengthening hydrogen bonding. The macroscale changes in behavior resulting from these interactions include: changes in ice crystal morphology[19,20], the freezing temperature and amount of ice present during freezing[21]. In the addition, cryoprotective agents are associated with protection of specific cell structures such as the cell membrane[22] and proteins in the cell[23].

Cryopreservation medium can be purchased pre-formulated from a manufacturer or formulated in-house. Reducing variability for cryopreservation solutions requires using high-quality reagents when formulating cryopreservation solutions in-house or purchasing pre-formulated solution from a manufacturer[14]. Cells or tissues are suspended in a cryopreservation solution prior to freezing and that combination cannot be sterilized or purified after that point. Therefore, an important factor in the quality of the cryopreservation solution is the quality of the reagents that go into the solution. Ideally, components of cryopreservation solutions should be pharmaceutical grade or GMP quality. Residual contaminants resulting from the manufacturing process have been found to influence post-thaw recovery of cells[24].

Cryopreservation solutions are not physiological solutions and most of them are hypertonic. Two independent mechanisms of damage can result from introducing cells to and incubating them in a cryopreservation solution: osmotic stress and biochemical toxicity[25]. Upon transfer from a physiological solution to a cryopreservation solution, cells will initially dehydrate due to higher chemical potential in the extracellular solution, followed by a slow increase in cell volume when small molecules such as DMSO and glycerol that are used in cryopreservation solution diffuse into the cells. If only non-penetrating CPAs (such as dextran) are present in the cryopreservation solution, cells will dehydrate and remain dehydrated[26].

Volumetric changes are also observed post-thaw as cells may need to be washed or diluted prior to use. During washing or dilution, water moves rapidly from extracellular low osmolarity solution to intracellular high osmolarity solution along with small molecular weight CPAs moving slowly in the opposite direction, resulting in expansion of cell volume[27]. Reducing variability and cell losses due to osmotic stress upon introduction and removal of cryopreservation solutions requires validation of methods of introduction and removal of cryopreservation solutions. Common approaches for reducing cell losses resulting from osmotic stresses involve adding the cryopreservation solution to the cell suspension in intermediate steps or using a syringe pump to slowly add the cryopreservation solution to the cell suspension. For the removal of CPA solutions after thawing, a washing solution with slightly higher osmolarity can be utilized to dilute the cells slowly, followed by washing of cells if necessary[28].

Loss in cell viability with time of exposure is commonly observed for cells incubated with certain CPA solutions, in particular solutions containing DMSO. These cell losses result from what is known more commonly as biochemical toxicity, which is distinct from the osmotic toxicity described above. Exposure of cells to DMSO is associated with various cellular alterations including changes to the cytoskeleton, shifts in metabolism and epigenetic alterations[25]. As a result, for cells used therapeutically, exposure to DMSO up to 30min prior to freezing is suggested[29]. In addition to DMSO, a variety of osmolytes including sugars, sugar alcohols and amino acids have been proven of efficiency in cell preservation[19,30]. For the development of novel cryopreservation solutions, assessment of biochemical toxicities of CPAs is crucial, which can be performed by measuring losses of cells that are incubated with cryopreservation solution at several intervals before freezing[31]. Reducing variability and cell losses due to biochemical toxicities of CPAs requires determination of maximum acceptable incubation time of cells with CPAs before freezing.

Freezing protocol

Post-thaw cell survival strongly depends on the rate at which cells are cooled[32]. Most types of cells exhibit an inverted U-shaped variation in survival with cooling rate: cells cannot survive cooling rates that are either too high or too low and there is a narrow range of cooling rates corresponding to the maximum cell survival. This relationship also varies with cell type and the composition of cryopreservation solution[33].

Controlled-rate freezing and passive freezing are two basic methods of freezing samples. Controlled-rate freezing ensures high and reproducible post-thaw cell recovery and is commonly used for preserving valuable cell therapies, while passive freezing is typically used for freezing fluid biospecimens when overall recovery and reproducibility are not critical, or used in resource-constrained areas. Passive freezing can be easily accomplished by placing samples in a −80°C mechanical freezer[34]. Controlled-rate freezing can be achieved by the use of a Stirling engine[35] or by adjusting the flow rate of chilled nitrogen gas circulating in a chamber where the sample is placed. As discussed above, there is a strong correlation between post-thaw cell survival and cooling rate, therefore, the freezing profile of a controlled-rate freezer can affect cryopreservation performance substantially. Reducing variability and cell damage during freezing requires a deliberate design of the controlled-rate freezing profile with precise specifications of cooling and holding steps.

Controlled-rate freezing profile in general involves three phases (Fig.2): initial hold period, controlled cooling period with nucleation, and higher cooling rate period to final temperature. Initial hold period should be long enough to allow equilibration of the sample temperature with the chamber temperature, but not exceed the maximum incubation time that might result in cell losses due to biochemical toxicity[36]. Reducing variability in the first segment of the freezing profile involves determining the duration of the hold segment that is sufficient to permit equilibration between the temperature of samples being frozen and the chamber temperature.

Fig.2.

Temperature as a function of time of a controlled-rate cooling profile.

The second segment for the freezing profile starts with cooling at a specific rate, followed by a nucleation step. Cells are most likely injured over this range of high subzero temperatures, therefore, controlling cooling rate is most essential in the second segment. As the sample cools to temperatures below the freezing temperature of the solution, ice will form in the extracellular solution. That process is known as nucleation and the nucleation of ice in the solution is a stochastic process. The temperature at which nucleation occurs may vary from sample to sample, and previous studies have found certain cell types are extremely sensitive to nucleation temperature[37–39]. As a result, precise control of sample nucleation is also crucial to ensure high cell survival. Nucleating the sample can be achieved by manual seeding involving touching the sample with a pre-chilled metal object, or automatic nucleation by a freezer through a rapid cooling of the sample to a low temperature followed by a rapid warming (as shown in Fig.2). Reducing variability in the second segment of the freezing profile involves determining the appropriate cooling rate for the cell type being frozen and inducing nucleation at a subzero temperature(s) that can be tolerated by the cells.

The third segment of freezing profile cools the sample from a low temperature (about −50°C) to the final temperature (about −100°C) at a higher cooling rate than the second segment. The final temperature should be selected appropriately to prevent excessive warming of the sample during the transfer of the sample from the controlled-rate freezer to a storage unit. Commercially available carriers that hold at low temperatures can also be used to transfer samples from controlled-rate freezer to storage unit if the freezer is far away from the storage unit.

The situation can get complicated if a large batch of samples need to be frozen simultaneously, such as for allogeneic cell therapies. Allogeneic cell therapy offers many advantages over the autologous counterpart [40], and has generated promising clinical outcomes[41]. As healthy tissues are collected from unrelated donors and purified cells can be significantly expanded in vitro, allogeneic cell therapies usually are manufactured in large batches, thus requires freezing a lot of samples at the same time. More samples in the freezer will generate more latent heat of fusion that needs to be removed during freezing, which may change the cooling rate experienced by cells, in particular in the temperature range where they are most sensitive. Besides, the orientation of the sample in the freezer may also affect the cooling rate that the sample experiences. As a result, post-thaw recovery and function of cells in different batches might be varied. Reducing variability during freezing also involves mapping the temperature distribution in the freezer during freezing for all the different loading and sample numbers anticipated and comparing those values to the protocol programmed into the controlled-rate freezer to minimize deviations between temperature of the samples and programmed temperature profile. There may be an upper limit in the number/mass of samples that can be frozen using the desired cooling conditions for a given freezer.

Storage and shipping of samples

Cryopreserved samples are commonly stored in storage units filled with liquid nitrogen such as dewars or freezers. Storing samples in the liquid phase of liquid nitrogen is less desirable than storing samples in the vapor phase of liquid nitrogen as storage in the liquid phase has been associated with cross contamination of samples[42], as well as entry of liquid nitrogen to vials and bags which may rupture vials or bags upon warming.

Even for samples stored at appropriate temperatures in the vapor phase of liquid nitrogen, there are various factors can affect the stability of the sample. For example, as most racks or boxes contain more than one sample, the retrieval of the desired sample typically involves the transient warming of other samples present in the box that are removed and replaced after the desired sample is removed. These samples are called ‘innocent’ samples as they are not the desired sample but simply a sample adjacent to a desired sample. Repeated warming and cooling of ‘innocent’ samples can result in degradation of the sample over time. The effect of thermal cycling on post-thaw recovery is associated with activation of apoptotic pathways, increasing the fraction of apoptotic cells post-thaw[43,44]. Reducing variability and maintaining stability of samples during storage require limited access to the repository and proper training of individuals who access the repository in order to limit transient warming and cooling of samples. Sample requests should be pooled in order to reduce the times that a repository can be accessed in a specific time period. More advanced automated storage retrieval systems with enhanced sample traceability capability also help reduce the temperature excursions of ‘innocent’ samples.

RM products are typically high value samples. It is also suggested to include a risk-management plan for banking of highly valuable cell products in case of external factors such as severe weather, fire, terrorism, and utility failure etc[45]. Core elements of a risk-mitigation plan include, but are not limited to, the following aspects:

• Two independent methods of identifying a sample

• Splitting of critical samples

• Backup capacity

• Backup power/liquid nitrogen supply

• Monitoring/alarm system for temperature monitoring of the storage units

Cryopreserved samples are frequently transported among sites of collection, processing, storage and use, even crossing national boundaries. Samples during transportation may experience various stresses including temperature fluctuation, mechanical vibrations and shocks, and pressure changes. Reducing variability and maintaining stability of samples during transportation require thorough shipping considerations and development of a verified shipping protocol. Specification of the acceptable range of shipping temperatures is crucial. The temperature of samples should be monitored using temperature recorders and maintained below −135°C for cells banked for nontherapeutic purposes and below −150°C for cells used therapeutically[46]. The verification of a shipping protocol is used to demonstrate the shipping process brings forth acceptable post-thaw recovery at various situations such as wide-ranging environmental conditions (hot summer and low winter temperatures) and unexpected travel delays. It is essential to maintain stability of the dry shipper during shipment as well. An outer container can help dry shipper remain upright to achieve great temperature stability and also provide padding to reduce the vibration of the inner dry shipper. Cryopreserved cells are considered as biohazards, as a result, appropriate training is needed for individuals who handle those goods during shipping. Protocols for shipping must conform to governing regulations and national standards[17].

Thawing and post-thaw processing

Cryopreserved cells need to be thawed at appropriate warming rates from storage temperature to the temperature suitable for downstream clinical and research use. The optimal warming rate associated with highest post-thaw cell survival depends on the cooling rate used to freeze cells and the composition of cryopreservation solution[33], both of which affect the amount and size of intracellular and extracellular ice crystals[47]. In general, slow-cooled cells need to be thawed at a rapid warming rate (above 60°C/min) to avoid damage to the cells.

The most common devices for thawing vials or bags of frozen cells are warm water baths with temperature set at 37°C. Samples are removed from the repository/freezer, immersed in the water bath, and gently swirled until ice disappears. There are several obvious limitations of thawing cells using water baths. Different operators may not perform the thawing procedure in the exact same way due to their skills or habits. For example, the sample might not be swirled in the water bath, which reduces the warming rate. The operator may even continue picking additional samples from the repository while leaving previous samples in the water bath, resulting in different thawing and processing time for the samples. Reducing variability of thawing process requires well-trained operators to establish consistent thawing procedures. Cells thawed in a water bath also have the risk of being contaminated as the water bath is open to the environment. Controlled-rate dry thaw devices using electric heaters have been developed to overcome the limitations associated with thawing samples in a water bath and are in particular suitable for thawing valuable cell therapies.

As discussed above, cryopreservation solutions are not physiological solutions and usually contain DMSO, as a result, removing or diluting these solutions is typically necessary prior to downstream use of the cells. Post-thaw washing is the most common method of removing cryopreservation solutions: cells are centrifuged, supernatant is removed, and then cells are re-suspended in a wash solution or culture media. However, manual methods of post-thaw washing are not only labor-intensive but also result in significant cell losses (10-30%) when used with bags[48]. Commercially available washing devices such as CytoMate, Sepax and Lovo have been developed to automate the washing procedure. New and emerging methods of post-thaw washing are also being investigated such as a hollow fiber bioreactor[49], a dead-end filtration device[50] and a microfluidic device[51]. Dilution of post-thaw cells is also commonly performed in order to prevent the toxicity from DMSO by decreasing final DMSO concentration.

Thawed cells are added to culture media and cultured for 24h, after which, media is removed and replaced to further dilute the cryopreservation solution present in the sample. Reducing post-thaw variability requires performing post-thaw processing (dilution, infusion or washing) at a consistent post-thaw time. Post-thaw washing or dilution solutions should be designed such that post-thaw osmotic stresses are minimized.

Post-thaw assessment

The objective of cryopreserving cells is to maintain the critical biological properties of cells to ensure proper post-thaw function for the appropriate downstream use. Appropriate quantification and characterization of the critical biological properties of post-thaw cells are essential. Unfortunately, post-thaw assessment is done incorrectly or incompletely on a regular basis. A variety of assays have been developed for post-thaw assessment of cells, including assays of mechanical integrity, metabolic activity, mechanical activity, mitotic activity, differentiation potential, and transplantation potential[52].

The most common method of determining cell viability is using a membrane integrity dye such as trypan blue which is compatible with conventional light microscopy, as well as other fluorescent dyes including 7-aminoactinomycin, acridine orange, and propodium iodine. Cells that have been cryopreserved and thawed were subjected to various types of stresses, which resulted in changes in cell membrane[53] and cytoskeleton, making membrane integrity as a measure of cell viability, function and persistence unreliable[52]. Calculation of cell viability using membrane integrity dyes can also bring with it a measurement bias. A common calculation error is to ignore the cells that have lysed after freezing and thawing, as a result, the viability of cells will be estimated to be higher than actual and efficiency of the freezing protocol will be artificially elevated. It is more accurate to calculate the recovery of cells, defined as the ratio of total number of viable cells pre-freeze to total number of viable cells post-thaw. It is also common for some cells to experience post-thaw apoptosis, especially with the presence of DMSO in the cryopreservation solution, as a result, the viability of a cell population may vary with time post-thaw[54]. For instance, the viability measured at 30min post-thaw might be different from that measured at 1h. Reducing variability in post-thaw assessment requires using post-thaw recovery instead of viability and performing the measurement at consistent time post-thaw.

Although physical integrity of cells measured using membrane integrity dyes is most widely implemented for post-thaw assessment, this method provides inadequate information for many cell types and cannot reflect the actual post-thaw function of cells. Characterization of other critical biological properties such as metabolic activity, mechanical activity, mitotic activity, or the activities reflecting the desired function and application of the cells should also be performed. Metabolic activity can be estimated using a variety of assays but is most commonly assessed by two basic methods: measure of oxygen consumption[55] and detection of a product of metabolism such as Nicotinamide adenine dinucleotide phosphate (NADP) and its reduced form (NADPH)[56]. Mechanical activity is another common post-thaw measure of function typically achieved by examining the ability of a specific type of cell to adhere to a substrate. Cells are seeded and cultured at normal conditions, after a given period of time, unattached cells are removed with the supernatant and counted, and thus the percentage of cells attached can be calculated. The ability of cells to proliferate post-thaw is also an important assessment metric, especially for cells used therapeutically as it may require high-fold expansion of cells after being infused to patients. Proliferation can be easily tested by monitoring the number of cells cultured at normal conditions for a given period of time and an increase in cell number with time indicates effective cell proliferation. Stem cells such as mesenchymal stem cells (MSC) have the potential to differentiate into many other types of cells and treat various types of diseases[57–66], as a result, a differentiation potential assay is crucial to verify cryopreserved stem cells still capable of differentiating post-thaw. Reducing variability in post-thaw assessment requires using multiple measures of post-thaw assessment, especially the measures reflecting the desired function and application of the cells. It is noteworthy that optical methods of characterization such as flow cytometry may need to be recalibrated for analyzing cells that have been frozen and thawed as the forward and side scattering as well as complexity of cells can be affected by cryopreservation.

Protocol Drift

Detailed procedures of a freezing protocol should be clearly documented, and operators should conform to the protocol strictly while performing duties of freezing and thawing cells. Homogeneity of freezing and thawing procedures is critical to the quality consistency of samples. In reality, it is very common for people to omit steps of a protocol that are burdensome or do not make sense to them. A common example can involve checking liquid nitrogen level in a storage freezer. For high-value biospecimens, the liquid nitrogen level should be checked daily. However, individuals in charge of monitoring storage units may get tired of checking levels daily and their practice drifts to every other day and then once a week and so forth. One day, the individual may check the unit and find that it has run out of liquid nitrogen, as a result, the samples are thawed and destroyed. Strong quality and audit systems must be in place to monitor compliance with protocols. In addition, individuals must receive proper training and proficiency testing to verify that they are properly trained in both the protocol and best practices. Preventing protocol drift requires a quality control system ensuring procedures performed do not deviate from those described in approved protocols as well as policies of proficiency testing for personnel.

Summary

The unique supply chain for RM products results in preservation being a critical aspect in the supply chain. Each step of the preservation process is a potential source of variability. In most situations, variability in outcome can be reduced and controlled through the application of fairly straightforward practices based on scientific principles and quality systems to ensure that technicians are following protocols. Training, education, and proficiency testing of those involved in the preservation process are also critical in reducing variability. A summary of the steps where variations can occur from raw material collection to shipping (cold chain) has been illustrated (Fig.3).

Fig.3.

Steps in the preservation process where variations can occur from raw material collection to shipping

Cells may be subjected to various pre-freeze processing steps such as selection of subpopulations, genetic engineering, in vitro culture and expansion. These steps may impose stresses on cells, making them vulnerable to downstream freezing and thawing processes. Cells are frozen in specialized cryopreservation solutions which are not physiological, and introduction/removal of solutions can result in cell losses independent of the freezing process. Formulation of cryopreservation solution requires using defined and high-quality reagents as it is not practical to sterilize cells after processing.

The cooling rate at which samples are cryopreserved from physiological temperatures to cryogenic temperatures is critical and can be controlled by using a controlled-rate freezer. Each segment in the freezing profile can be rationally designed and optimized based on cell type and sample loads. It is noteworthy that the nucleation temperature of extracellular solution may influence post-thaw cell recovery substantially and needs to be determined for a given solution at a given cooling rate.

After freezing process is completed, samples need to be transferred from the freezer to a storage unit, during which, samples can warm rapidly, resulting in partial melting of the sample and thus inducing cell losses. Commercially available carriers that hold at low temperatures can be used to transfer samples safely. Cells that have been successfully frozen and transferred to the storage unit can be stored for years to decades. However, retrieval of a sample can involve transient warming of other ‘innocent’ samples due to unskilled rack/box handling techniques, leading to a larger portion of apoptotic cells post-thaw. An inventory management system can be used to enable access and use of samples in storage.

Cryopreserved samples are typically used at a different location other than the storage facility. Maintaining low temperature of the sample during shipping is crucial to preserve its critical biological properties. Cryopreserved samples can be shipped in a dry shipper placed in an outer container that helps the dry shipper remain upright and maintain temperature stability. A shipping protocol should take both the quality of the sample and safety of all personnel into consideration and be verified by sending test shipments before sending real samples.

Samples need to be warmed at appropriate rates, which are more than an order of magnitude greater than the cooling rates, in order to prevent cell damage during thawing[67]. Conventional method of thawing samples in a warm water bath set at 37°C is associated with many issues. Instead, a programmable dry thaw device can be utilized, especially for thawing valuable cell therapies. Post-thaw samples may need to be washed or diluted prior to the downstream use.

Post-thaw cells need to be properly assessed to determine if the cell integrity and function have been impaired by any previous processing steps. The design of a post-thaw assessment protocol should be fit-for-purpose based on cell type and intended downstream use. Assessment of integrity of cell membrane is common, but not sufficient to provide enough information about post-thaw cells. Multiple measures of post-thaw assessment, especially the measures reflecting the desired function and application of the cells, should be performed as well.

In conclusion, a variety of sources of variability can occur in the preservation of cell therapies and RM products, and the variability can be controlled or reduced by carefully designing processing protocols based on scientific principles, and strictly conforming to these protocols by operators. If robust protocols from pre-freeze processing to post-thaw assessment can be achieved, the critical biological properties of a product can be preserved.