Key words

1 Introduction

In the last decade and a half, the ability to isolate human embryonic stem cells (hESCs) and induce pluripotent stem cells (hiPSCs) from somatic cells has driven a surge of interest in the establishment of banks of such cells and the associated primary cells from which the latter can be generated for research , industrial, and clinical application. Not surprisingly for an emergent field the techniques and processes used to generate these cells have varied greatly depending on the starting material along with the resources and expertise available locally. For clinical use, there is also variation in regulatory process and standards, and differing ethical considerations influencing their implementation. In the face of such diversity there is a clear necessity to attempt to define and adopt best practice and standardized methodologies for the characterization of such banks to provide reliable and comparable benchmarks from which to conduct research and develop therapies [1, 2].

There are a few examples of deriving stem cell lines with the intention that these may ultimately be used as the starting material for differentiated cellular therapeutics [3–6]. The standardization of processes and the identification, characterization , and functional testing of such stem cell lines is critical since any final therapeutic product will be intrinsically linked to its starting material. A thorough understanding of how the cells should look and behave is essential to be able to demonstrate control over any subsequent process the cells are used in and any changes made thereafter.

It is essential that any cell bank providing quality assured cells can demonstrate that the cultures have specific characteristics and that they are free from microbial contamination . It is therefore necessary to implement an appropriate testing regime that can establish key criteria for quality control of cell lines such that their characteristics can be demonstrated.

Quality assured (QA) characterization of stem cell banks is also critical to enable robust stem cell research, the outcome of which will ultimately inform the development of safe and efficacious stem cell-based therapies. Robust banking and distribution of cell lines requires defined processes and testing regimes to provide consistency and confidence as well as permitting comparable development across laboratories. Characterization to ensure quality, safety, and efficacy must include testing at critical stages identified throughout the banking process including early outgrowth stages, during culture expansion , pre- and post-cryopreservation and include stability and viability assessment after banking.

Extensive standardization of stem cells as a primary resource provides a baseline from which to conduct efficacious research and development with confidence. The approach that is taken to testing cells must consider the differing stages of the banking process. Testing should permit an analysis of how the cells behave both during their initial expansion and following their recovery from cryopreservation . In this regard, it is important to keep the banking process as consistent as possible, but also to be in control of any changes, be aware of their impact and to consider and evaluate the effect these changes may have on the cells.

Banking processes will vary, particularly in relation to the type and origin of the starting material. There are obvious differences to consider between the procurement of embryos for the derivation of hESCs and the procurement of adult tissue to establish banks of somatic cells.

It is crucial that any laboratory wishing to conduct thorough QA characterization testing does so within the framework of a comprehensive Quality Management System (QMS). This will facilitate control over the establishment and implementation of any test methods and data generated, and will allow for managed changes to be implemented successfully. An established QMS should minimally cover raw materials and other processing components, consumables, equipment (including use, maintenance, and repair), methods (processing and testing) documented as Standard Operating Procedures (SOPs), data recording, and methods for reporting .

Where stem cell lines are being banked with the intention to use them in the development of cell therapies , it is essential that this is performed with an understanding of the relevant legal and regulatory requirements. Under current European regulation , pluripotent stem cell -derived cellular therapeutics are most likely to be regulated as advanced therapeutic medicinal products (ATMP). Current best practice is to qualify raw materials, and to produce and characterize banked cell lines to meet these exacting standards from initiation of the cultures. This will ensure that this part of the regulatory process starts off on a sound footing regardless of the final use of the banked cells.

The characterization data generated for a stem cell line forms a critical component of the complete history of a cell line and should be compiled appropriately to provide documented evidence for this purpose.

This chapter will review some of the key aspects related to the characterization of stem cell banks with a focus on demonstration of their safety and efficacy in the context of their intended use in the manufacture of cell-based therapies. This is structured in terms of describing the overall approach and strategy followed by a description of cellular characterization (identity , purity , potency , viability , and stability) and microbiological testing .

2 Procurement

The ability to clearly demonstrate the provenance of the original material is a critical component that underpins all downstream elements. The quality of any cells used as starting material ultimately for use in clinical application can only be determined where there is a full and traceable understanding of its origin. The potential risk of transmission of infectious agents or genetic abnormalities, the need for comprehensive and fully informed consent , and robust traceability are the primary issues to control. Proper planning, specifications, recording, and documenting of procurement activities are critical.

Where donated cells and tissue are used it is essential that procurement is conducted only following ethical review of procedures and with fully informed consent from the original donor (s) [7–12]. It is critical to ensure that the donor specifically is aware of the intended research and clinical uses of the cells derived from their donation and that these cells are likely to be shared with others globally, may be transplanted into humans and animals, and may be exploited commercially. The extent of testing to be performed on the cells/tissue donated and any cells derived thereafter such as human embryonic or induced pluripotent stem cells should be made clear. This will include testing for pathogens, genetic testing including whole genome sequencing and, although challenging, should also make donors aware that future testing methods that may not be currently available, but are feasible in the future. All donations should be anonymized to maintain donor confidentiality; however, they should be coded and traceable. Anonymized copies of the original consent documentation should be obtained as a record of the consent.

Where any specific component of the procurement process is being undertaken by a third party, then suitable specifications and agreements should be in place to describe and control the activity.

Donor selection criteria may be strategically based on specific factors such as ethnic background or immune haplotype [13]. However, from a quality perspective there are a number of other inclusion/exclusion factors that must be considered.

The risk of microbiological contamination (bacterial, fungal, viral, parasitic) should focus on those agents most likely to be contaminants in relation to the geography, donor cohort, and cell or tissue type being procured. Ensuring that donations meet all significant acceptability criteria for local/international blood or platelet donation [14] is one strategy for managing donor eligibility/exclusion criteria. Any approach should include mandatory testing for the most commonly known and transmitted pathogens and indicators of an immunological response such as Syphilis, HBV, HCV, HIV (genome), as reflected by the detection of serological antibodies to these pathogens, as well as any other pathogens classified as at higher risk of prevalence in the country of origin. It is important to ensure that testing for these markers is performed on samples taken at the time of donation and performed using tests having undergone validation for use with donor samples providing suitable specificity, sensitivity and with suitable controls.

Donors should not be considered suitable for donation if, for example they:

  1. 1.

    Were unwell at the time of donation. Deferral periods for specific infections may exist and/or vary in accordance with local regulatory guidelines for other cell and tissue products (e.g., for blood donors ).

  2. 2.

    Have tested confirmed repeat positive for infectious disease (see the list above and section 5 below).

  3. 3.

    Have received a blood transfusion within the previous 12 months (it is advised to check local regulatory guidance for variations and risk factors).

  4. 4.

    Regarding TSE/CJD risk: has had treatment with pituitary extracts of human origin; has received corneal or dura matter transplant, has a personal or family history of CJD.

  5. 5.

    Donors with serious active, chronic, or relapsing disease (e.g., cardiovascular disease, gastrointestinal, genitourinary, hematological, immunological, metabolic, renal, or respiratory diseases).

  6. 6.

    Have been or are intravenous/intramuscular drug users.

  7. 7.

    Has received a xenotransplant.

  8. 8.

    Persons whose sexual behavior puts them at high risk of acquiring severe infectious diseases.

It is important to keep a continued watch over new and emerging infections and consider their potential to be transmitted in the context of the cell type(s), process, and quality control testing implemented. The greatest concern will be for diseases for which there are no validated tests available to identify pathogen presence in the absence of visible symptoms of disease, or technology to remove infectivity if detected. Of these, prion diseases of human origin (Creutzfeld Jacob Disease, Kuru) or animal origin (e.g., Bovine Spongiform Encephalopathy, Ovine Scrapie, Chronic Wasting Disease) evident globally, constitute one of the most significant concerns for which risk assessment-based geographic deferrals have been implemented to date for established human cell and tissue products. Assessments should consider the general donor background as well as the cell type being procured, for example using cells originating from the CNS may pose a higher risk. Potential donors who are clinically ill and those with familial instance of prion disease are assumed to be excluded from eligibility to donate. Similarly, individuals at risk following medical treatments (e.g., surgery) should be identifiable and will have been informed making them identifiable. The largest risk is likely to come from transmission of variant Creutzfeldt-Jakob disease (vCJD) arising from infection from bovine spongiform encephalopathy (BSE) and other cattle sources . Only blood and blood products have so far been implicated in actual cases of transmission. This transmission risk is currently managed by the use of leuko-depleted blood products, e.g., in the UK . Maternal transmission of prion disease has never been established despite cases of pregnancy in affected mothers [15]. Interestingly, limited investigation to date has suggested that if hESC are exposed to an infectious prion inoculum, protease resistant prions are taken up but then rapidly extruded by cells and not perpetuated during further cell growth [16].

When acquiring samples to generate stem cells for clinical application there should be mechanisms by which ongoing donor traceability can be made. Traceability should be considered in terms of tracking back to the original donor and their medical records in a suitably confidential and consented manner, and in relating to forward traceability during banking activity and any subsequent clinical manufacturing and distribution processes. Such mechanisms must be appropriate in scale and suitably coded so as to maintain donor confidentiality. The ability to trace forward and backward from donor records is important mainly from a quality perspective, for example should the donor subsequently be diagnosed with a disease (e.g., CJD or hepatitis C), or in case re-consenting is required to enable unforeseen activities. Guidelines and standards now exist for implementation of suitable coding of cells and tissue intended for clinical application [17, 18] and should be referred to.

For induced pluripotent stem cells there are a number of special considerations.

It is important that the characteristics of the starting material are documented and also that archived material is available should the need arise to perform comparative or confirmatory analysis. To this end, archive samples at suitable volumes for both cellular and genomic material from early tissue or cell populations should be taken and stored appropriately.

Cell storage , packaging, and transport criteria should be clearly defined for the samples that are obtained. Temperature limits, timing restrictions, storage locations and containers, and appropriate alarms should be specified. Biosample containers as well as shipping vessels should be inspected and records of transport must be maintained.

3 Testing Within Defined Cell Banking Procedures

Banking processes will vary and it is essential that each process for different cell types is fully understood. The use of flow diagrams for processes can provide useful tools to identify critical stages and permit the targeting of testing time-points. Any cell bank should ensure that it employs a documented Master and Working Cell Bank system (Fig. 1).

Fig. 1
figure 1

Schematic of a banking workflow . Cell scale-up is performed to generate a Pre-Master Bank (PMB) (or Seed Lot) stage, followed by further expansion to the Master Cell Bank (MCB ) stage. Aliquots from the MCB are then expanded to the final stage of a Working (or Distribution) Cell Bank (WCB ). Additional WCBs are manufactured from aliquots of the MCB

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The procurement process, including the critical stage of providing information and obtaining consent in an ethical and robust manner, should always be considered the beginning of the banking process (see Chapters 3 and 8).

A typical banking process involves the expansion of cells to the Pre-Master Bank (PMB) (or Seed Lot) stage, followed by further expansion to the Master Cell Bank (MCB ) stage (Fig. 1). Aliquots from the MCB are then expanded to the final stage of a Working (or Distribution) Cell Bank (WCB ). Further WCBs are then manufactured by returning to and expanding further aliquots from the MCB . The PMB acts as an original stock from which subsequent MCBs can be derived, although it should be considered a precious resource that is rarely required. It is important to design the banking strategy and scale of each bank stage according to the anticipated scale of production of final clinical cell type to be implemented, taking into consideration, for example, the expansion and attrition rate in downstream processing and the anticipated clinical dose and number of doses required.

In cases where early stage stem cell populations are being derived and subsequently supplied to a cell banking facility, it is essential that there are procedures in place for the receipt and quarantine of such materials until such time as testing can be performed to ensure there is no risk of contamination or infection.

The banking process avails a number of stresses on the cells being expanded, for example age (cell doublings), passaging , and freeze/thaw cycles. This requires that a defined Quality Control (QC ) regime is implemented that monitors the cells for stable continuity of their desired characteristics and to test for the absence of adventitious agents.

Critical control points should be identified in the banking process and suitable tests identified at each point to both characterize the cells and test for the absence of adventitious agents. At each such Sample Point it is very important to consider the quantities of material required for testing, especially where live cells are required, to be sure sufficient cells are available to conduct testing homogenously at a single passage number, or cell doubling stage. A variety of sample types will be needed to conduct all suitable tests. For example, samples of spent media will be required to test for mycoplasma and endotoxin, whereas live cells will be required from both before and after cryopreservation for a number of characterization tests. In addition, samples of cryopreserved cells may need to be used to test for sterility, or to extract nucleic acids for testing, and also to be held as archive samples for future testing.

In general, more extensive testing is performed near the beginning of the process and, where justified, less testing can be carried out at the latter stages. An example of this kind of approach would be to screen for the presence of any viral contaminants as early in the process as possible. Further viral testing downstream would only be required if there was potential risk of introduction of contaminants from components used in the manufacturing process, for example when products from sources of animal origin are used. This kind of pyramid approach also helps to prevent investment in expanded culture of cells that are contaminated or display undesired characteristics.

Fully traceable documented evidence of the banking procedure, compiled for example as a Cell Line History File, is essential to be able to demonstrate that procedures have been followed and to allow for troubleshooting should any issues arise. Such documentation should be implemented early and record all activities including procurement data and the starting cell material, raw materials and reagents , facility and equipment and qualification status, procedures and protocols followed, testing performed and storage and distribution information. These documents should be updated regularly.

4 Cell Characterization

Cell characterization will primarily be considered an evaluation of cell characteristics indicative of level of safety. In the main, such considerations revolve around microbiological risks, but will also include a number of other factors concerning function and stability. This section and Table 1 describe the methodologies that can be implemented to evaluate cells within a structure cell banking system.

Table 1 Details of appropriate testing technologies
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4.1 Genetic Identity

Establishing and monitoring the identity of cell populations is critical for quality and safety [19], particularly where a specific population of cells is claimed to have been isolated form a heterogeneous starting population. Obtaining a genetic profile of the cells provides evidence that a cell line is monoseptic and can be compared to data from donor material or primary cells to confirm its origin.

Typical forensic methods are carried out using PCR amplification of short tandem repeats (STRs ) and commercial kits are available for this purpose [1]. Each STR represents a short region in the genome that can exhibit variations in terms of length between individuals. These STRs are found across the entire genome. The STR loci and the number of STRs analyzed varies internationally, but some are common. There are a variety of methods used for forensic analysis, but analysis of between 11 and 15 STR loci is considered standard practice. This method is one commonly used internationally and so enables rapid confirmation by other labs that they are using the correct cell line. It is relatively cheap and high-throughput, but requires specific equipment and trained staff to perform the work and analyze the data. Standards are available to ensure a robust approach [20].

Other methods, such as the use of Single Nucleotide Polymorphisms (SNPs ) or Humam Leukocyte Antigens (HLAs ) , can be used, but may not provide data that is easily comparable across laboratories [21].

When conducting such genetic testing on human cell lines it is always important to consider how the data is recorded and distributed to ensure appropriate donor confidentiality is maintained. The publication of data that may allow the identification of specific donor individuals could have implications for them and their families’ future healthcare [22].

4.2 Phenotype

Testing the phenotype of the cell population, by screening for the presence or absence of cell surface or intracellular markers, provides essential characterization data. Phenotype can be established in a number of ways. The most common method is the quantification of multiple known cell surface markers of pluripotency by flow cytometry . Flow cytometry is a high-throughput system that utilizes the principles of light scattering, light excitation, and detection of emission of light from fluorescent dyes to provide multiparametric measurements of a single flow of particles or cells. Information is simultaneously collected on cell size, granularity, and protein expression using antibodies conjugated to fluorescent dyes. Additionally, information can be collected on cellular viability using vital dyes and cell cycle using DNA-binding fluorescent probes. Modern Flow cytometry uses multiple fluorochromes and can therefore allow a number of cell characteristics to be detected simultaneously. A simple set of parameters can be determined to measure cell cycle, viability , and phenotype in a 4–6 color single panel for routine monitoring at various points in the production process (see, for example, Fig. 2). More extensive characterization with a comprehensive marker set can be carried out in the cell banking process. A typical marker profile for pluripotent cells would include positive expression of OCT-4 (POU5F1), Sox-2, Tra-1-60, Tra-1-81, SSEA-3, and SSEA-4, with low or negative expression of SSEA-1 [23, 24]. Immunocytochemistry can also be used to provide additional information on the morphology localization of specific antigens.

Fig. 2
figure 2

Flow Cytometric Analysis of the RC9 hESC line [25]. (a) —live cells are selected for analysis by selecting cells with High Forward Scatter (FSC), and cells that exclude the Vital Dye DRAQ7 (measured in the APC-Cy7 channel). (b)—The live gate is activated and doublets are excluded from the analysis by plotting FSC—Area versus FSC-Height, and excluding events that fall below the diagonal. (c)—Live singlet cells highly express the characteristic SSEA-4+ TRA-1-60+ phenotype >98%++ (d)—The phenotype of the cells is confirmed by analysis of SSEA-4 versus SSEA-1, and less than 1% of SSEA4+ cells express SSEA-1. MACSQuant 10 flow cytometer, MACSQuantify software 2.6. Gate line thickness highlighted for presentational purposes, percentages generated using original gates in the analysis software

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Recording the visual morphology of stem cell cultures can provide valuable data to support phenotype determination, particularly where adherent cells are concerned. It can also provide useful information on growth profile and anticipated confluency and processing time points. This type of data should always be used as an additional methodology and not as a stand-alone test.

Phenotype can also be determined using molecular markers of gene expression associated with a specific stem cell type. For human pluripotent stem cells , the expression of known pluripotency genes such as Nanog, Oct-4, DMNT, TDGF, and GABRB3 can provide very useful information [23]. However, such RNA expression data should always be confirmed at the protein level. The use of Protein Ligation Assays (PLAs) can provide a measure of protein expression which can be directly correlated to the mRNA and/or miRNA expression. In these highly sensitive PLA assays a paired set of antibody-oligonucleotide probes bind to the protein target. A third oligonucleotide then hybridizes to the oligonucleotides pair when they are bound in close proximity. This ligation product can then be specifically amplified and detected by PCR to provide a measure of the level of expression of the specific protein. This method can be expensive and require significant optimization.

It is important to remember that it is the overall expression profile of a given set of markers that is important since other cell types may express one or more of the known pluripotency markers.

More recently, the use of micro RNA profiles has been used where such profiles can be linked to specific cells/tissues .

Confirmation that the cell population exhibits all known phenotypic markers for pluripotency should not allow one to allude that the cells will be pluripotent. Additional potency measurements must be made to confirm this.

4.3 Cell Function or Potency

The biological potency of the cell population should be demonstrated. For pluripotent cells the ability to produce cells representative of all three germ layers should be tested for. This has traditionally been performed by testing for the ability of the cells to form teratomas in SCID mice. This technique can be variable, although attempts have been made to standardize the procedure [26], and different results have been seen depending on criteria such as: number of cells injected, site of injection, media /matrix components used as injection vehicle and over numbers of replicates. In this setting, it is important to consider the relevance of any negative results. While a cell line that forms teratomas representative of all germ layers may be chosen for its flexibility, a cell line that consistently differentiates down a single pathway may prove to have considerable utility for making cells of a specific type. In this case, arguments could be assembled to support increased safety due to reduced risk of spontaneous differentiation in undesired ways.

Alternative in vitro methodologies for assessment of pluripotency do exist. For example, formation of embryoid bodies where aggregates of pluripotent cells are cultured so that cells representative of the three germ layers spontaneously arise. These can then be assessed by immunocytochemistry or RT-PCR for detection of markers of differentiated cell types. More recently, protocols for the directed differentiation of pluripotent cells have become available; however, these have been criticized as being less predictive of full pluripotency as they may tend to give rise to differentiated cell types of a more specific pathway as compared to the spontaneous differentiation seen using teratomas or embryoid body techniques. Whilst such directed differentiation methods may provide a better indication of terminal cell or tissue differentiation potency, they vary greatly in methodology, can take time, be costly and difficult to standardise.

Additionally, some tests are available which consider the transcriptome-based profiles of the cells themselves [27, 28] and compare these to a database of other cell lines “validated” in terms of other supportive data. Such approaches can be useful, but in the absence of actual demonstration of in vitro or in vivo differentiation of the cells will always be open to question.

In summary, there is no single method that will provide a suitable measure of potency or function and one or more methods will need to be selected based on the specific requirements in each case. For pluripotent cells it is important not only to show ability to differentiate into all three germ layers , but also that the cells can commit to specific mature cell types demonstrating anticipated mature phenotypes and functions .

4.4 Purity

During expansion it is critical that the cell population is monitored for purity . Purity data should be evaluated in the context of the data generated for identity. Purity analysis should check for evidence of unwanted cells, for example differentiating cells in a pluripotent cell culture, as well as for the expected cell type. Flow cytometry can be used here to examine the detail of cell line heterogeneity, and flow cytometry demonstration of the lack of contaminating cells is often of high importance. Where labs are processing multiple cell lines it is prudent to check for evidence of any cross contamination . Limits should be set for acceptable numbers of each undesired cell type, taking into account the level of risk the cell type presents in the context of the intended use of the cell line being processed.

Specific cell types may also require additional tests, for example induced pluripotent stem cell populations should be tested for the absence of any residual vector components used for reprogramming , for example by PCR for detection of exogenous reprogramming factors.

Where a feeder cell population has been used in coculture it is necessary to check that their removal has been successful when the desired population is harvested. In some cases, removal of feeder cells (e.g., by the use of antibody-conjugate magnetic beads) may be desirable or useful.

Purity should also be considered in terms of culture components and excipients used as well as the cells. Purity tests should also consider any potentially harmful components that might reasonably be expected to have been introduced by the manufacturing process. For example, antibiotics, matrix components, breakdown products from raw materials.

4.5 Viability /Growth Profiling

The viability of the cell population should be checked at regular intervals and after changes to cell culture conditions. In particular, viability should be assessed following recovery from cryopreservation , being mindful that cells should be assayed following some time in culture after recovery to avoid false overestimation of viability. Limits should be placed on acceptable percentage of nonviable cells at recovery from cryopreservation and passaging .

As well as simple cell counts alternative methods such as alkaline phosphatise detection [29] or trypan blue exclusion can be used; however, these methods can present inherent variability between operators in terms of performance and experience of assessing results. This can be overcome by introducing automation, but this adds cost and requires larger sample volumes. Methods for assessing cell viability by flow cytometry are extremely accurate and simple to perform—DNA-binding vital dyes such as propridium iodide or DRAQ7 allow for accurate determination of viability. This method is powerful as the cells can be gated to identify debris and cell aggregates in the culture. A variety of dyes that bind to DNA and are not affected by cellular fixation have also now been developed, and these allow accurate viability determination even when local procedure requires cells to be fixed before analysis.

Monitoring the growth profile of the population is important to provide a relatively easy indicator of transformational change in the population. This measurement should be taken regularly and, for example, after adaptation to new culture environments or passaging regimes. Methodologies for measuring growth profiles will vary depending on the nature of the cultures. For example, samples of cells passaged as single cell suspensions can be measured using cell counters. Cell populations growing in colonies or clusters are more problematic. Some automated imaging systems are now available which can be useful for these cells [30].

4.6 Genetic Stability and Karyotype

Cell lines will have some inherent variability in culture, particularly at high passages [31]. However, it is important to maximize stability by minimizing the number of population doublings and passages, being consistent with culture media , passaging regime and process methodology, and controlling and monitoring the culture environment. Although stability should be evaluated using data from all cell testing including proliferation characteristics, genetic profile, phenotype , and response to stresses such as cryopreservation , it is important to regularly karyotype cells by, for example, G-banding . Testing should be performed early to establish a baseline of “normality” for that line. Changes to the genetic makeup of a cell line can lead to loss of function or uncontrolled differentiation . Increased regularity of testing should be performed where there are changes in culture conditions, following recovery from cryopreservation and at high passage numbers and passages beyond that of the MCB and WCB .

Analysis of the karyology of cell lines by G-Banding is the most common method routinely applied in cell banking . It is a well-practiced and cheap method used to assess the chromosomal status of a cell line in terms of the number and appearance of the chromosomes. This method requires that mitotic cells are arrested in the metaphase stage of division when the chromosomes are at their most condensed. The arrested cells are then treated to release the chromosomes from the nucleus and are fixed to a glass slide. The fixed chromosomes are then treated with an enzyme (e.g., trypsin ) to degrade chromosomal proteins and “relax” the chromosomes that can then be more easily stained and evaluated under a microscope. Usually, Giemsa (lending the “G” to G-banding ) stain is then applied which binds to A T-rich areas. Once visible the stained bands make it easier to identify chromosomes that are arranged as an ideogram for presentation. The final stage of evaluation of the chromosomes requires the skills of an experienced cytogeneticist. More recently, individual elements of the preparation processes have been automated leading to increased standardization of chromosome quality, important in assays such as these where operator to operator variability can be high. See example in Fig. 3. Flow-cytometry-based DNA analysis can also detect abnormalities in the DNA content of cells, is easily standardized, and has the advantage of rapidly examining thousands of cells, but also requires experienced operators for accurate results.

Fig. 3
figure 3

Karyogram image of iPSC line RCi002-A. G-banding image of iPSC line RCi002-A demonstrating normal 46, XX chromosome complement, and banding pattern from 20 of 20 spreads

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Similar to G-banding , but with increased resolution, is spectral karyotyping (SKY ) . SKY utilizes fluorescent probes for DNA sequences on specific chromosomes to label the sample. The fluorochromes are then visualized for analysis.

It is important to be aware that the sample size and the sensitivity of the method chosen will impact the sensitivity of detecting abnormal clones. With hESCs for example, at least 20 metaphase spreads should be prepared for routine in-process testing. For assessment of banked material intended for clinical application this number should be much higher (ca. 50-100 spreads). All spreads analyzed for human cells should show diploid chromosomes with no structural abnormalities detected. When an abnormality is detected it may be necessary to repeat testing of the same culture, or at subsequent passages to determine if the result is spurious, or representative of the population.

There are many new technologies for genetic analysis which provide rapid analysis and provide unprecedented levels of sensitivity and resolution. The cost and speed of performing these tests are rapidly improving. Techniques such as SKY , Comparative Genome Hybridization (CGH ) [32], and analysis of Single Nucleotide Polymorphisms (SNPs ) can provide high resolution. Additionally, whole genome sequencing is becoming more cost efficient. These methods can produce significant quantities of data and it is important to ensure that sufficient resources and expertise are available to manage the results correctly. Also, the extensive data generated will be challenging to handle and raises concerns for the maintenance of donor confidentially.

The analysis of single nucleotide polymorphisms (SNPs ) is an alternative method that increases resolution compared to G-banding and performs copy number analysis of specific genomic regions. This method has been suggested for use in clinical applications [33, 34]; however, the significance of any gain or loss is challenging to determine. This approach would be useful where predetermined SNPs in regions of known risk, e.g., tumor suppressing genes, were targeted.

It is important to remember that with all these techniques the number of cells analyzed will directly affect the sensitivity of the assay performed and how representative of the results generated are of the population sampled.

5 Microbiological Tests

The risks of microbiological contamination from a number of sources need to be considered. It is important to consider the risk from donor individuals providing primary tissue for cell derivation , the risk from manufacturing components used, and the risk from the manufacturing environment. In most cases, it will be possible to test raw materials, the cell populations, supernatants, or spent media for the presence of adventitious agents; however, in some cases, a risk-based approach may be required which leans on data from parametric testing and information.

The presence of microbial contamination presents obvious risks in the context of manufacture of clinical products. It is also necessary to be aware of the impact such contamination can have on the biological characteristics of the cell population being cultured. For example, a low level viral infection may not have a significant impact on cell death, but could dramatically influence biological activity. This type of contamination can impact not only the suitability of the cells for clinical use, but also any research data being generated.

Taking a holistic view of processes from donation and procurement, through preparation of culture reagents and equipment, to maintenance and cryopreservation of cultures it is important to establish microbiological testing at critical points in the process. In addition, it is advisable to introduce procedures to minimize risk to other established cultures. For example, it is good practice to maintain quarantine procedures for primary tissues or cell lines newly brought into the laboratory. Such cultures should ideally be maintained in a dedicated laboratory, but minimally in segregated equipment until sufficient data is available to justify their relocation.

Test methods used for microbiological testing should always be qualified and it is important to be sure that appropriate levels of sensitivity, specificity, and robustness are being used in respect of testing cell cultures. It is equally important to be sure that sampling regimes ensure results are representative of the cell culture, or cryopreserved cell bank as a whole.

5.1 Primary Cells and Tissue

As with any application of human-derived products for use in therapy , the risk of transmission of viral contamination from the original donor is the most obvious. It is critical that planning is done well in advance of procuring any primary tissue to determine relevant inclusion/exclusion criteria for donors that may influence risk of infection as well as the types of medical and lifestyle information that will be useful for selection of donors and risk mitigation.

Medical screening of donors should be performed at the point of donation. Where medical screening information is available it is still necessary to take caution with any donated tissue as there it will likely only be possible to test for the most common infections and a certain level of unknown infection may remain, or have been obtained between screening and donation. The use of lifestyle questionnaires, such as those used in blood donation centers, may be useful to help reduce risk. Such questionnaires must be used in the context of what is ethically and morally acceptable to ask. This is especially true where tissue is being donated in special circumstances, for example where embryos are being donated from couples undergoing assisted conception procedures it might not be appropriate to ask questions around individual sexual history .

5.2 Viruses

Early and comprehensive testing of material is critical to preventing investment in contaminated product. This is especially true for viruses as subsequent testing can justifiably be excluded based on process controls and use of suitably sourced and tested raw materials.

It will not be possible to test for all viruses and a risk-based approach should be taken to test those of highest risk given the origin and history of the cells to be tested. Typically, viruses of blood-borne origin should be prioritized. In addition, it is necessary to consider the risk from raw materials used, for example the use of fetal calf serum, animal sourced growth factors, or enzymes can pose a risk of transmission.

If possible, the expertise of clinical and microbiological specialists should be drawn on to enable a risk-based approach to be taken with regard to selection of the most relevant viruses to test for. In general, when testing established cell lines some of the most common viruses to test for include HIV I/II, HepB, HepC, hCMV, HHV 6-8, CMV, HTLV I/II.

It is critical that procedures are established which describe actions to be taken in case of a positive result for any virus. These procedures should also be complimented by routine practices to be taken by operational staff to minimize the risk of transmission .

5.3 Mycoplasma and Sterility

Tests for the presence of bacteria, yeast fungi, and mycoplasma should be conducted routinely. The use of antibiotics should be eliminated as soon as possible; however, where they are used in the culture medium , these should be removed prior to sampling.

Mycoplasma is recognized as a common contaminant of cell cultures due to the risk of contamination from numerous sources . Three major sources of contamination are (1) other cell cultures introduced to the laboratory; (2) humans (M.orale, M.fermentans, M.salivarium, or M.hominis); and (3) cell culture reagents such as bovine sera (M.arginini, A.laidlawii) or porcine trypsin (M.Hyorhinis). Mycoplasma can be very difficult to remove from cell culture as their small size limits filtration and they can be difficult to detect without establishing routine testing procedures.

A pharmacopoeial method for the detection of mycoplasma is available, but this culture method is lengthy and results can take a number of days to complete. There are many alternative methods such as PCR -based assays and biochemical assays measuring specific mycoplasma enzymes [35]. The PCR methods are becoming increasing sophisticated and do provide a screen for many mycoplasma species and can be used to identify specific species present.

Enzymatic methods are inexpensive and provide relatively rapid turnaround of results, but can give inconclusive results.

It is advised that Pharmacopoeial (USP/EP) methods for sterility are used. In general, these tests are often outsourced to specialist labs due to the large investment required to set up the procedure in-house.

5.4 Prions

The risks posed by transmissible spongiform encephalopathies (TSEs) should be considered irrespective of the origin or history of the cell line. There are a number of TSE diseases present across the globe and their ability for transmission to humans, while unknown in many cases, is plausible.

There exists a lack of clarity on the risks of prion diseases in cell culture and variability in terms of uptake, persistence and expulsion can be seen across different cell types. There are currently no qualified assays that can be applied robustly in the context of cell cultures; however, it is important to be aware of new developments in this area where assays are being developed from those designed for analysis of human tissues .

As well as the risks from cell sources it is important to also be aware of the risks posed by reagents and raw materials used in culture. The use of low-risk reagents is highly advised in the context of clinical applications [36].