Edinburgh Research Explorer Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17)

The human embryonic stem cell line RCe021-A (RC-17) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.


Resource details
RCe021-A (RC-17) was received as day 3 embryo that was surplus to requirement or unsuitable for clinical use and was cultivated to the blastocyst stage in medium containing GMP grade granulocytemacrophage colony-stimulating factor (GM-CSF) to improve survival of the inner cell mass (Sjöblom et al., 1999). Human embryonic stem cell (hESC) isolation, expansion and qualification was performed in a facilities whose specification, operation and monitoring complied with GMP standards enabling; i) a fully traceable procurement procedure with informed ethical consent which includes provision for commercial use, ii) detailed medical history and blood borne virus (BBV) M A N U S C R I P T

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screening of donors, and iii) compilation of a cell line history providing details on hESC manufacturing process and quality control testing regime.
Human ESC culture and processing was performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and Distributors -The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (Go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-gooddistribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631).
HESC derivation involved whole embryo outgrowth on mitotically inactivated human dermal fibroblast (HDF) feeder cells. HDFs were derived and manufactured according to GMP and had been approved for clinical use by the Food and Drug Administration, USA. During derivation on HDFs, hESC were grown in a xeno-free cell therapy grade media (XF KODMEM) supplemented with xenofree human recombinant bFGF. The cell line was subsequently expanded in a GMP grade serum-free medium (StemPro hESC Serum Free Medium) on a xeno-free matrix (CellStart). The former contained bovine serum albumin (BSA) from a Transmissible Spongioform Encephalopathy (TSE)-free country of origin. The cell line was cryopreserved in a GMP compliant cryopreservation solution (CryoStor CS10).
A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1). Blood group genotyping gave the blood group O 1 O 1 , expected to give rise to blood group O + (Table 1). The cell line is free from mycoplasma contamination as determined by RT-qPCR.

Verification and authentication
The cell line was analysed for genome stability by G-banding and showed a normal 46XX female genotype (Fig. 4). SNP genotyping was carried out using the Illumina HumanCytoSNP-12 v2.1 BeadChip and revealed a 144kb gain on chromosome12p13.31 as described in Canham et al, 2015. This region contains the genes, SLC2A14; SLC2A3. Duplications and deletions of this region are found commonly in healthy individuals (1 n 25) as documented by the Database of Genomic Variants (MacDonald et al, 2014).

Ethics
Derivation of hESC from surplus to requirement and failed to fertilise/develop embryos was approved by The Scotland A Research Ethics Committee and local ethics board at participating HDF cells were cultured in DMEM (Lonza, Slough, UK), 10% Pharma grade FCS (GE Healthcare (PAA), Buckinghamshire, UK) and 2 mM L-glutamine (ThermoFisher Scientific). HDF were mitotically inactivated using gamma irradiation at 50 Gy using a Gammacell Elite 1000 machine. For use as a feeder layer, irradiated HDFs were plated at 50000 cells/cm 2 in XF KODMEM medium supplemented with 80 ng/ml human bFGF (ThermoFisher Scientific). Cells were cultured at 36.5 -37.5°C, 5.0 ± 0.5% CO 2 , 5.0 ± 0.5% O 2 and 50% medium exchanged 6 days a week.
The established cell line was expanded and banked using CellStart matrix and Stempro hESC Serum Free Medium (cell therapy system quality reagents, ThermoFisher Scientific). This contained BSA from a TSE-free country of origin. Passaging was performed mechanically using an EZ passage tool (ThermoFisher Scientific). hESC lines were expanded to 25-30 wells of a 6-well plate and cryopreserved in 0.5-1 ml Cryostor CS10 (Biolife Solution, Washington, USA) using an EF600-107 controlled rate freezer (Grant Instruments, Cambridge, UK) before being stored in a -150°C freezer (Panasonic Biomedical, Loughborough, UK).

Mycoplasma
In process mycoplasma detection was performed using Applied Biosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and MicroSEQ™ Mycoplasma Real-Time PCR Detection Kit (ThermoFisher Scientific (Applied Biosystems)) according to the manufacturer's instruction.

Endotoxin
Endotoxin levels were determined using the Kinetic-QCL assay (Lonza) and an incubating plate reader (BioTek ELx808) according to the manufacturer's instructions. Briefly, an unknown sample was compared with a standard curve of known levels of control endotoxin. An assay was deemed valid if the coefficient of correlation, r ≥ 0.980 and the CV (%) for the standard curve was ≤10%, and the reaction time of the negative control was greater than the reaction time of the lowest standard on the standard curve.

Flow cytometry
Pluripotency was determined using the Human and Mouse Pluripotent Stem Cell Analysis kit (BD, Oxford, UK). Oct 3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentiation marker. FITC conjugated Tra-1-60 (BD) was used as an additional pluripotency marker. Fixed and permeabilised cells were analysed using a FACS Aria flow cytometer (BD) or a Guava easyCyte flow cytometer (Millipore, Watford, UK). Percentage expression of each marker was compared to isotype control or unstained cells.

Viability
Viability was determined using the Guava ViaCount assay. Briefly, the Guava Viacount reagent (Millipore) containing a nuclear and a viability dye, was mixed with a single cell suspension, incubated for 5 minutes and analysed using the Guava easyCyte flow cytometer (Millipore). Total cell count, viable cell count and percentage viable cells was obtained.

Immunocytochemistry
Cells were fixed in 4% paraformaldehyde (ThermoFisher Scientific (Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisher Scientific) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000; Sigma) and muscle-specific actin (1:50; DAKO, Glostrup, Denmark) and secondary antibody anti mouse IgG-AlexaFluor 488 (1:200; Sigma). Images were acquired using a Zeiss S100 Axiovert fluorescence microscope or Nikon eC1 confocal microscope SNP Genotyping and Analysis DNA samples were assayed using the Illumina HumanCytoSNP-12 v2.1 BeadChip. Genotyping data was initially assessed using GenomeStudio genotyping module (v1.94, Illumina). Karyostudio (v1.4,Illumina) was employed to perform automatic normalisation and to identify genomic aberrations utilising default settings of the built-in cnvPartition algorithm (3.07, Illumina) to generate B-allele frequency and smoothened Log R ratio plots for detected regions. These parameters are designed to detect CNVs greater than 75 kb and CN-LOH regions larger than 1 MB with a confidence value greater than 35. All identified regions were first cross-matched to the Database of Genomic Variants (DGV; http://dgv.tcag.ca) to identify naturally-occurring structural variations in the human. CNVs that were not identified on the DGV were then checked against a list of ES cell-associated culture adaptation genomic variants published by the International Stem Cell Initiative (Amps et al, 2011). See also Canham et al, 2015 for further details.

Genomic analysis and outsourced assays
All outsourced assays were carried out under a Quality and Technical Agreement. DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Manchester, UK) according to manufacturer's recommendations and provided in recommended quantities to the service providers.   Comment DRB1*03 is expressed serologically as DR17, DQB1*03 is expressed serologically as DQ7.