Abstract
The blood–brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.
Highlights
The blood–brain barrier (BBB) regulates the passage of soluble and cellular substances from the blood into the central nervous system (CNS) [1]
Because cell culture conditions are crucial to maintain reliability and reproducibility of experimental data it was important to establish that the cryobiological response of Human cerebral microvascular endothelial cells (hCMECs)/D3 cells was not affected by the vessel surface on which they were cultured
We describe here improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension that yielded ~90% post-thaw viability, and a newly established protocol for cryopreservation of endothelial monolayers applied to hCMEC/D3 and astrocyte single cultures and co-cultures
Summary
The blood–brain barrier (BBB) regulates the passage of soluble and cellular substances from the blood into the central nervous system (CNS) [1]. The blood vessels of the BBB are composed of continuous endothelial cells which are joined together by intracellular tight junctions and lack pores in their plasma membranes [2]. The BBB is a dynamically regulated partition that is permeable but highly selective, allowing the entry of small essential molecules like oxygen, amino acids, and glucose, but preventing the passage of pathogens and toxins [1, 3]. Because the interactions of microvascular endothelial cells with astrocytes are necessary for the formation, maintenance, and regulation of the BBB [1, 5], this work describes the cryopreservation of these cells in suspension, and in monolayers as single cultures and co-cultures
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