Stem cell laboratories should consistently conduct their research using authenticated, stringently tested, well- characterized cells that are cryopreserved and cultured within a quality framework for quality assurance (Crook et al., 2010; Crook and Stacey, 2014; Stacey et al., 2013; The International Stem Cell Banking Initiative, 2009). At a minimum, all routine laboratory protocols should be well documented, all work traceable, and all critical equipment regularly monitored and maintained. While it may not be achievable for all academic laboratories, researchers should strive to adhere as closely as possible to Good Laboratory Practice Standards (https://www.ecfr.gov/current/title-21/chapter-I/ subchapter-A/part-58). These practices and processes ensure that the quality and integrity of cell lines preserved are assured, affirmed by generations of reliable data regarding cell safety and performance.

Cell Line Authentication

Recommendation 1 .3 .1: Cells for experimental use should be authenticated. Short Tandem Repeat (STR) analysis is recommended for authentication.

Authentication of research materials is important to confirm that investigators are working with the expected material and to demonstrate it is free from cross contamination with cells from another source. Unfortunately, by its very nature, in vitro culture allows opportunity for errors that can lead to the misidentification and/or cross-contamination of cell lines within the laboratory, a long standing and well documented issue and a major contributor to erroneous experimental conclusions and publication retractions (Casadevall et al., 2014; Freedman et al., 2015; Horbach and Halffman, 2017; Souren et al., 2022). This highlights the need for authenticating the identity of cell lines in particular and all cell cultures in general, a practice that will instil confidence in the interpretation and reliability of research data obtained using them (American Type Culture Collection Standards Development Organization Workgroup ASN-0002, 2010; Nelson-Rees and Flandermeyer, 1977).

In principle the need to authenticate research materials is fundamental to good science and this can be achieved by direct testing of the material and rigorous traceability. In the case of cell lines that can be passaged indefinitely, there is a significant risk that misidentified lines could be transmitted widely, potentially leading to corruption of research data on an international basis. Thus, in the case of cell lines, identity testing is strongly recommended. For tissues and low passage materials that will not be distributed, the impact of switched or cross-contaminated cells is lower. Thus, for such materials it may be argued that genetic testing of all cultures is not necessary. However, in these situations the investigators should provide assurance of the provenance of the material they are using, and this may involve enhanced levels of control and traceability.

Funding agencies and journals are increasingly requiring evidence of cell line authentication to receive funds or publish (“Nature Editorial,” 2013; “Guidance: Rigor and Reproducibility in Grant Applications,” 2019). While rigorous documentation and testing by centralized cell banks can reduce the potential for misidentification at the point of sourcing stem cells, the onus still lies with the end researcher to authenticate materials used within the laboratory.

Several methods can be used to authenticate cell lines including short tandem repeat (STR) analysis, single nucleotide polymorphisms (SNP) profiling, whole genome sequencing (WGS), and other DNA profiling technologies. All of these are acceptable authentication strategies which can be used in the laboratory to properly identify cell lines. However, only STR analysis has been formally developed into an internationally recognized and accepted consensus standard for human cell line authentication (Almeida et al., 2016). The STR standards document “Authentication of Human Cell Lines: Standardization of Short Tandem Repeat (STR) Profiling. ANSI/ ATCC ASN-0002-2021” provides information regarding the reasoning behind authentication, and detailed protocols for STR analysis. The advantages of STR are numerous, including cost efficiency, reproducibility, comparability across platforms, and ability to detect multiple cell sources within a culture. For these reasons it is recommended for use in authentication. Regardless of authentication strategy chosen, in order to protect donor privacy, genetic profiles used for authentication should not be made public.

Recommendation 1 .3 .1 .a: When authenticating cells or a cell line, a reference sample from the original donor should be used for confirmation of origin . Where donor material is not available, a profile obtained from the earliest passage stocks available may be used for reference.

It is recommended to use cell materials directly from donors to generate a reference profile to authenticate materials derived from that donor. This allows unambiguous confirmation of identity and clear traceability of consent. Occasionally it is necessary to use materials for derivation or experimental use where no donor profile information exists. When no donor sample is available for existing research materials, the material provider should have available a profile from early stocks that can be used to authenticate laboratory materials.

Recommendation 1 .3 .1 .b: At a minimum, authentication of cell lines should be performed at the establishment of the MCB.

Once the MCB is established (section 1.2), it should be assessed and ensured to match the original donor or early reference sample. For materials that are manipulated or passaged, it is also strongly recommended to assess at significant manipulation points (e.g., gene editing, clonal isolation, etc.) and/or at the end of studies to assure continuity of materials throughout experimental processes. Ideally, materials being provided externally (shared with collaborators or otherwise distributed) should come only from tested, authenticated stocks (MCB or WCBs) and the receiving laboratory should authenticate the cell line upon receipt. Any live cultures transferred between laboratories should be considered to have unknown identity until they have been properly authenticated (for more on the timing of authentication during the experimental process please see Appendix 1, Table A1.1).

Nomenclature/Assigning a Unique Identifier

Recommendation 1 .4 .1: Cell lines should be assigned an unambiguous identifier to safeguard provenance of data associated with that line in the public domain . The identifier should be generated by an international open-sourced registry to ensure it is persistent between laboratories, interoperable, and unique . Published reports should reference this unique identifier.

Cell lines have a physical entity, as well as a digital identity, a unique and persistent online record of the line. Ensuring that the digital identity of the line is unambiguous is an essential part of building confidence regarding the provenance and reproducibility of the physical entity. The recommendation for a unique, persistent, and unambiguous digital reference assigned by a trusted third-party registry assists interoperability of that identifier, as registries maintain a list of existing identifiers and cell lines. Registration of lines is an essential component of good stewardship of the line, as it allows it to be globally findable, and further ensures provenance of derivatives is linked to the originating line (such as genetically modified transgenic reporter lines, gene edited isogenic lines, or subclones that have distinct properties). Examples of such registries are Cellosaurus (https://web.expasy.org/ cellosaurus/) for cells used in biomedical research (Bairoch, 2018), and the human pluripotent stem cell registry (hPSCreg; https://hpscreg.eu) for hPSCs specifically (Kurtz et al., 2018). The hPSCreg links cell lines to established data compliant with these recommendations and to the RRID of the ExPasy-resourced cell line data base ‘Cellosaurus’ (see Appendix 2 for general principles of a registry).

Registration is an important step in adhering to FAIR (FAIR principles of Findable, Accessible, Interoperable and Reusable) principles, even if the lines themselves have restricted availability. It simplifies the process of collating minimal information about the generation, provenance and availability of a line. Line registration assists stem cell researchers seeking to meet local governance requirements and is mandated by some funding bodies and in some national jurisdictions. Note that biobanking a line (Recommendation 1.2) does not preclude the need to register the line. While registration and biobanking are different activities, the biobanking of the physical entity is underpinned by assignment of a unique and persistent identifier for each cell line.

There is currently no international registry designed for primary tissues, or cells that can only be propagated for a finite time from those tissues. Nevertheless, where data is generated and placed in the public domain, there is a need for an unambiguous digital identity to ensure that data generated from the same donor line can be reconciled together, and data from different donor lines can be distinguished from one another. In these instances, we recommend that laboratories working with tissue or primary cells adopt nomenclature rules that allow digital traceability (Kurtz et al., 2018). We encourage the use of ‘common-use’ names associated with primary cells that identify the institute/originating laboratory, cell type, and a unique donor ID that is 3 alpha-numeric digits or more, to reduce the chance of duplicated identifiers. We also encourage the use of tissue, cell and cell line ontologies to reduce ambiguity about the origin or propagation of the material (Sarntivijai et al., 2014). Best practice on publication would include generation of a Research Resource Identifier using webtools at the Research Resource Identification site (https://www.rrids.org).

Reprogramming Transgene Elimination

Recommendation 1 .5 .1: Verification of the elimination of the transgene expression in newly derived human induced pluripotent stem cell (hiPSC) lines should be performed prior to biobanking, distribution, and experimental use.

Viral systems are commonly used to overexpress the reprogramming factors in somatic cells to generate hiPSCs. Non-integrative reprogramming methods include viruses (Sendai virus (SeV), adenovirus), self-replicative RNA from Equine encephalosis virus (EEV) and episomal vectors which contain sequences from the Epstein-Barr virus (EBV) (Haridhasapavalan et al., 2019). Cells transduced with these engineered viruses or vectors do not produce infectious viral particles, but they transiently express viral sequences containing the reprogramming factors. Clearance of these factors from the cell line is critical, as the persistent expression of the reprogramming factors in hiPSC cells can affect their proliferation and differentiation potential and increase the risk of tumor formation in a mouse model (Okita et al., 2007). Thus, clearance of the reprogramming vectors should be confirmed in newly derived hiPSC lines prior to biobanking or any experimental use.

The transgene expression is retained in newly derived hiPSC lines but can usually be eliminated through multiple cell passages. Viral clearance timing is dependent on the system used. For Sendai virus derived hiPSC lines, the vector clearance should be confirmed by immunostaining with antibodies against the virus or by quantitative PCR (qPCR) for Sendai virus specific sequences. Most of the SeV derived hiPSC lines are transgene free by passage 10. Newly derived hiPSC lines generated with the episomal vector method show faster clearance of the vectors, generally by passage 3-5, however, it was reported that in as many as 30% of the hiPSC lines, the vector was not eliminated, most likely due to genome integration. The clearance episomal vectors can be assessed using qPCR (Schlaeger et al., 2015).

Cell Hygiene

Recommendation 1 .6 .1: Cell cultures should undergo microbiological and viral testing including mycoplasma, sterility, and adventitious agent screening to promote cell competence and technical staff safety.

Recommendation 1 .6 .1 .a: Cell cultures (both primary and stem cell cultures) should be assessed to confirm the absence of mycoplasma upon entering the laboratory and regularly monitored (quarterly at a minimum) to ensure the absence of mycoplasma infection during routine culture . Cultures intended for experimental research should be monitored at the initiation and completion of studies . Any lines shared outside the laboratory should be confirmed mycoplasma negative prior to distribution . If culture lines are found to be contaminated, they should be discarded.

Mycoplasma contamination of cell cultures is well known to be a significant issue in cell repositories, with contamination rates worldwide ranging from 15% to >80% depending on the location and level of monitoring (Chernov et al., 2014; Corral-Vázquez et al., 2017; Drexler and Uphoff, 2002; Hay et al., 1989). The impact a mycoplasma infection can have on a cell culture is significant, compromising both structure and function of the host cells (Cimolai, 2001; Drexler and Uphoff, 2002; McGarrity et al., 1984; Tsai et al., 1995; Zhang et al., 2006). This can affect all measurable parameters of cell morphology and physiology, rendering results obtained using an infected culture unreliable. Mycoplasma is undetectable with standard laboratory equipment, lacks a cell wall making it resistant to most antibiotics, is ubiquitous within the environment, and its size and flexibility permit it to evade filtration devices. These aspects, combined with a rapid expansion rate, allow it to overtake a culture quickly and easily, making screening and exclusion of infected cultures critical to improve rigor.

Maintaining a mycoplasma free culture environment requires both initial vigilance and routine monitoring. All incoming cultures, regardless of origin and testing certification, should be quarantined and tested before being maintained with existing cultures. Once confirmed as mycoplasma negative, the culture can be placed in standard culture areas, and routinely screened as part of an ongoing testing program. When banking cells for future use, each MCB and WCB should be screened and confirmed negative prior to use for experimental purposes or sharing of the culture. If found to be positive at any point, unless the culture is absolutely irreplaceable, it should be discarded (for more on the timing of testing during the experimental process, please see Appendix 1, Table A1.1).

Recommendation 1 .6 .1 .b: Cultures should be screened to ensure that they are free of microbial and viral contamination.

Microbial or viral contamination can alter cellular behavior and integrity, pose an immediate health risk to researchers, and preclude the future therapeutic use of cell products (Barone et al., 2020). Concerns regarding the potential effects, processes for identifying contamination, and potential resolution are common to all culture systems and have been well reported. See Appendix 3 for more detail on identifying and mitigating this risk in culture.