Edinburgh Research Explorer Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15)

The human embryonic stem cell line RCe019-A (RC-15) 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 cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available


Resource details
RCe019-A (RC-15) was derived from a 3-4 cell cleavage stage embryo that was surplus to requirement or unsuitable for clinical use and cultivated to the blastocyst stage.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) 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 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, hESCs were grown in a xeno-free cell therapy grade media (XF KODMEM) supplemented with xenofree human recombinant bFGF.HESCs were 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 (Table 1).

Verification and authentication
The cell line was analysed for genome stability by G-banding and showed a mixed female genotype.
A normal 46XX genotype was present in 21 of 30 cells analysed, but an extra copy of chromosome 8 (47XX, +8) was present in a subpopulation (8 of 30 cells) of cells (Fig. 4).The cell line is free from mycoplasma contamination as determined by RT-qPCR.

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 fertility clinics and conducted under licence no R0136 from the UK HFEA with informed donor consent.The processing and storage of hESC cells for human application was conducted under licence number 22631 from the UK Human Tissue Authority.

Cell culture
Fresh embryos were cultured in Sydney cleavage medium (Cook Medical, Hertfordshire, UK) until day 3 and Sydney blastocyst medium (Cook Medical) after day 3 of development.Embryos were cultured at 36.5 -37.5°C, 5.0 ± 0.5% CO 2 , 5.0 ± 0.5% O 2 in drops under paraffin oil (Cook Medical) and transferred to fresh medium at least every 2-3 days.
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.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT
In vitro differentiation hESCs were pre-treated for 1 h with 10 µM ROCK inhibitor in Stempro hESC SFM (ThermoFisher Scientific) and embryoid bodies (EBs) generated in ultra low attachment plates (Corning) for 7 days before being transferred into EB medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1 mM Lglutamine, 3.5 µM -mercaptoethanol, 1 % nonessential amino acids (all ThermoFisher Scientific)), on glass slide tissue culture chambers (Nunc, ThermoFisher Scientific) coated with 0.2% gelatin (Sigma Aldrich, Dorset, UK) at 0.1 ml/cm 2 for 14 days.

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.
Microsatellite PCR, or Short Tandem Repeat analysis, was used to determine cell line identity and was carried out by Public Health England.A profile was obtained for the following core alleles: vWA, D16S539, Amelogenin, THO1, CSF1PO, D5S818, D75820, D135317 and TPOX.
Human Leukocyte Antigen (HLA) tissue typing was carried out by the Scottish National Blood Transfusion Service.
Blood group genotyping was carried out by the Molecular Diagnostics laboratory at NHSBT.
Karyotype analysis was carried out by the Western General Cytogenetics Laboratory (Edinburgh, UK).Live cells at 60-70% confluency were shipped in warm containers, fixed and analysed by standard Gbanding analysis.For clinical grade lines, 30 spreads were analysed.
hpscreg.eu/cell-line/RCe019-A Ethics Informed consent obtained.Scotland A Research Ethics committee approval obtained (07/MRE00/56).Conducted under the UK Human Fertilisation and Embryology Authority licence no R0136 to centre 0202 and UK Human Tissue Authority (HTA) licensing number 22631.
with ISO9001:2008 standards.HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631).

Fig. 1 .
Figures and tables

Fig. 4 .
Fig. 4. RCe019-A (RC-15) was analysed by Giesma staining of 30 metaphase spreads a normal 46XX female karyotype in 21 spreads and a subpopulation of 8 cells with an extra copy of chromosome 8.