Stem cell-based therapies are promising tools for regenerative medicine and require bulk numbers of high-quality cells. Currently, cells are produced on demand and have a limited shelf-life as conventional cryopreservation is primarily designed for stock keeping. We present a study on bulk cryopreservation of the human iPSC lines UKKi011-A and BIONi010-C-41. By increasing cell concentration and volume, compared to conventional cryopreservation routines in cryo vials, one billion cells were frozen in 50 mL cryo bags. Upon thawing, the cells were immediately seeded in scalable suspension-based bioreactors for expansion to assess the stemness maintenance and for neural differentiation to assess their differentiation potential on the gene and protein levels. Both the conventional and bulk cryo approach show comparative results regarding viability and aggregation upon thawing and bioreactor inoculation. Reduced performance compared to the non-frozen control was compensated within 3 days regarding biomass yield. Stemness was maintained upon thawing in expansion. In neural differentiation, a delay of the neural marker expression on day 4 was compensated at day 9. We conclude that cryopreservation in cryo bags, using high cell concentrations and volumes, does not alter the cells' fate and is a suitable technology to avoid pre-cultivation and enable time- and cost-efficient therapeutic approaches with bulk cell numbers.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.
© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.

Antibodies are instrumental tools in stem cell identification, purification, and analysis. Most commonly, cell samples are either dissociated to obtain a single-cell suspension suitable for FACS analysis or cell sorting, or fixed in situ for immunostaining and fluorescence microscopy imaging. This unit describes an alternative method in which live adherent cells are stained and imaged in situ without the need for cell dissociation, fixation, or fluorescent reporter genes. This minimally invasive method is particularly useful for identification and distinction of fully and partially reprogrammed induced pluripotent stem cells (iPSCs). The unit also describes the use of mCD49e and hCD29 antibodies in live-cell (vital) imaging. mCD49e strongly stains mouse embryonic fibroblast (MEF) feeder cells in human pluripotent stem cell cultures, whereas hCD29 recognizes an antigen expressed on undifferentiated and many differentiated cells. A distinguishing feature of hCD29 in live-cell staining is that its antigen is precluded from detection wherever cells have formed tight epithelial junctions (e.g., in the center but not the periphery of pluripotent stem cell colonies) due to basolateral location. A non-fluorescent fixed-cell staining protocol is also provided for medium- to high-throughput quantification of stem cell experiments without an automated microscope. The discussion addresses technical limitations, pitfalls, troubleshooting, and potential applications, such as identification of emerging bona fide human iPSC colonies in reprogramming experiments.