title: Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition.
abstract: The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm2, equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm2out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin.
authors.forename: Hannah K
authors.initials: HK
authors.lastname: Wilson
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin.
authors.forename: Madeline G
authors.initials: MG
authors.lastname: Faubion
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Hjortness
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Palecek
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison , Madison, Wisconsin.
authors.forename: Eric V
authors.initials: EV
authors.lastname: Shusta
mesh_terms: D001812:Blood-Brain Barrier; D001921:Brain / Q000166:cytology / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D055806:Microvessels / Q000166:cytology / Q000502:physiology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D047428:Protein Kinase Inhibitors; D054460:rho-Associated Kinases
keywords: ROCK inhibitor; blood–brain barrier; cryopreservation; human induced pluripotent stem cells