Molecular Cytogenetic Characterization of the Human Cerebral Microvessel Endothelial Cell Line hCMEC/D3

Abstract

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45∼48,XX, –X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13∼14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],–22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

The hCMEC/D3 cell line as a model of the human blood brain barrier

“…They do not show adhesion-independent growth in soft agar but form capillary structures in Matrigel, a characteristic property of cultured endothelium. They were reported to have an apparently normal diploid human karyotype [1], although a high-resolution multicolor fluorescence in situ hybridization (FISH) approach revealed a more complex karyotype at high passages than initially thought [2]. In addition, they stain positively for endothelial markers including CD34, CD31, CD40, CD105, CD144 (VE-cadherin) and von Willebrand factor, but not for CD36, which is absent from brain endothelium.…”

Inflammatory cell trafficking across the blood–brain barrier: chemokine regulation and in vitro models

“…Under those conditions, they can present transudative intercellular junctions and lack paracellular barrier properties, which limit their effective use as an in vitro BBB model (122). Moreover, complex karyotype changes were recently reported in immortalized BMECs, rendering important the genetic testing of cell lines before their application to in vitro studies (123). As a general statement, there are few cell lines that are appropriate for in vitro BBB experiments.…”

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

“…There have been some recently produced brain microvascular endothelial cell lines which have demonstrated some similar characteristics to non-transformed cells 22 . Despite their promising usefulness in research, molecular cytogenetic characterization has recently highlighted complex karyotype changes, which renders genetic testing of cell lines advisable prior to their application in in vitro studies 23 .…”