Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels

Abstract

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood-brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

Keywords: blood−brain barrier; brain microvascular endothelial cells; cryopreservation; in vitro modeling; permeability; three-dimensional models.

Figure 1.

Cryopreserved dhBMECs maintain high transendothelial electrical resistance (TEER) for over 1 year. (a) Differentiation and cryopreservation of iPSC-dhBMECs. (b) Subculture of non-frozen, 1-day cryopreserved, and 1-year cryopreserved dhBMECs. (c,d) Cryopreservation is associated with a loss of cell adherence and slight decrease in cell viability. Data collected across n = 3 independent differentiations for each condition. (e) TEER of cryopreserved dhBMECs versus the length of cryopreservation. n = 37 unique thaws across 22 independent dhBMEC differentiations. (f) Fractional TEER (%) displaying the ratio of cryopreserved to fresh TEER over cryopreservation length. Data collected across n = 19 unique thaws consisting of n = 10 unique dhBMEC differentiations. All data represent averages across 2–6 technical replicates (individual transwells).

Figure 2.

Phenotypic characterization of dhBMECs following 1-year cryopreservation. (a) Immunocytochemistry of tight junctions (claudin-5, occludin), nutrient transporters (GLUT-1), efflux pumps (P-gp), and endothelial markers (CD31, VE-cadherin). Representative images at day 2 after seeding are shown across n = 2 independent differentiations for each condition. (b) TEER values for non-frozen (n = 17) and 1-year cryopreserved (n = 7) dhBMECs. n represents the number of unique differentiations. (c) Permeability comparison of four compounds: Lucifer yellow (444 Da), 10 kDa dextran, glucose (180 Da), and rhodamine 123 (380 Da). Data represent independent differentiations for each condition, with n = 7–8 for Lucifer yellow and 10 kDa dextran, and n = 3–5 for rhodamine 123 and glucose. For independent differentiations, data represent averages across 2–6 technical replicates (individual transwells). *p < 0.05.

Figure 3.

Response of cryopreserved dhBMECs to a media switch. (a) Cryopreserved dhBMECs generated from the WTC iPSC line do not display changes in actin or tight junction localization due to a media switch. Representative images are shown across n = 2 independent differentiations for each condition. (b) Percentage drop of TEER immediately following a media change to transport buffer. Data collected across n = 6 independent differentiations for each condition. (c) Time course of TEER following a media switch. Data collected across n = 3 independent differentiations for each condition. Permeability (apical-to-basolateral) for Lucifer yellow plotted versus (d) TEER recorded before the permeability measurement (no media switch) and (e) TEER recorded at the beginning of the permeability assay immediately following the media switch. Data collected across n = 21 individual transwells representing n > 5 independent differentiations of fresh or 1-year cryopreserved dhBMECs. *p < 0.05.

Figure 4.

Gene expression is maintained following 1-year cryopreservation of dhBMECs. (a) Heatmap and hierarchical clustering of gene expression. Pearson correlation coefficients between replicates are shown from 1 (blue) to 0.65 (white). Experiments numbered 1–3 represent biological replicates for each condition. (b,c) Volcano plots depicting significantly (adjusted p < 0.05) upregulated genes (green) and downregulated genes (red): (b) shows iPSCs (n = 3) versus dhBMECs (n = 6) and (c) shows fresh dhBMECs (n = 3) vs cryopreserved dHBMECs (n = 3). Note that raw p-values were adjusted using the Benjamini–Hochberg procedure, leading to no significant DEGs when comparing fresh versus cryopreserved.

Figure 5.

Cryopreserved dhBMECs form BBB microvessels with low permeability. (a) Fabrication of 3D microvessels. (b) Representative phase/fluorescence images of Lucifer yellow permeability for fresh and 1-year cryopreserved BBB microvessels. (c) Average Lucifer yellow permeability across BBB microvessels generated from fresh and cryopreserved dhBMECs. n = 4 for fresh BC1 microvessels and n = 10 for cryopreserved BC1 microvessels, across at least four independent differentiations per condition. (d) Comparison of Lucifer yellow permeability across 2D and 3D models for non-frozen and 1-year cryopreserved dhBMECs. ***p < 0.001.