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  • Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

Current protocols in stem cell biology (2020-09-22)
Aya Shibamiya, Elisabeth Schulze, Dana Krauß, Christa Augustin, Marina Reinsch, Mirja Loreen Schulze, Simone Steuck, Giulia Mearini, Ingra Mannhardt, Thomas Schulze, Birgit Klampe, Tessa Werner, Umber Saleem, Anika Knaust, Sandra D Laufer, Christiane Neuber, Marta Lemme, Charlotta Sophie Behrens, Malte Loos, Florian Weinberger, Sigrid Fuchs, Thomas Eschenhagen, Arne Hansen, Bärbel Maria Ulmer
ABSTRACT

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Insulin solution human, sterile-filtered, BioXtra, suitable for cell culture
Sigma-Aldrich
Deoxyribonuclease II from bovine spleen, Type V, essentially salt-free, lyophilized powder, ≥1,000 units/mg protein
Sigma-Aldrich
Sodium selenite, BioReagent, suitable for cell culture, ≥98%
Sigma-Aldrich
Pluronic® F-127, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Transferrin human, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Accutase® solution, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Dimethyl sulfoxide, Molecular Biology
Sigma-Aldrich
Lipid Mixture (1000×), liquid, sterile-filtered, BioReagent, suitable for insect cell culture
Sigma-Aldrich
1-Thioglycerol, liquid, BioReagent, suitable for cell culture, ≥97% (titration)