β-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery

Abstract

In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier, our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants. We hypothesized that the autologous origin of human brain microvascular endothelial cells (hBMECs) is the first requirement for the suitable coating to prevent the glial inflammatory response triggered by foreign neuroprosthetics. Therefore, this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurgery patients. Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells. The addition of 10 nM β-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing, supporting the well-known protective role played by estrogens on microvessels. In particular, β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs, while it was not necessary for freshly isolated male-derived hBMECs; however, it did counteract the decay in the viability of the latter after thawing. The tumor-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed before and after two periods of cryopreservation. Despite the thermal stress, the hBMECs remained viable and suitable for re-freezing and storage for several months. This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools, offering the potential to avoid additional surgical sampling for each patient.

Keywords: 17β-estradiol; cryopreservation; gender-specific; gray matter; human brain microvascular endothelial cells; surgical resections; vascular protection; white matter; β-estradiol 17-acetate.

Figure 1

Experimental design. Sequences of fresh isolation and culture of hBMECs in the presence and absence of 10 nM β-estradiol 17-acetate were evaluated. Cell recovery following cryopreservation, thawing, and culture in the presence and after deprivation and re-addition of 10 nM β-estradiol 17-acetate was subsequently tested. Cell culture splits are marked with vertical lines. hBMECs: Human cerebral microvascular endothelial cells.

Figure 2

Effects of donor’s gender, tissue source and 10 nM β-estradiol 17-acetate on hBMECs recovery and growth after fresh isolation from neurosurgical specimens. (A) Representative phase contrast images of morphology and density of female WM-derived hBMECs spiked with 10 nM β-estradiol 17-acetate, after 4 and 22 days in culture from isolation. Scale bars: 20 μm. (B) Representative phase contrast images of male GM-derived hBMECs in the absence of 10 nM β-estradiol 17-acetate, and male WM-derived hBMECs in the absence and presence of 10 nM β-estradiol 17-acetate, after 4 and 22 days in culture from isolation. Scale bar: 20 μm. (C) Comparison between growth curves of GM-derived hBMECs freshly isolated from male patients M1-M4 in the absence of 10 nM β-estradiol 17-acetate (blue squares) and WM-derived hBMECs freshly isolated from male patients M2-M5 in the absence (green circles) and presence of 10 nM β-estradiol 17-acetate (violet triangles) and WM-derived hBMECs isolated from female patients F4 and F5 and spiked with 10 nM β-estradiol 17-acetate (dark pink triangles). Each independent culture was related to GM-derived hBMECs (n = 4) and to WM-derived hBMECs (n = 4) obtained from male patients, and to WM-derived hBMECs (n = 2) obtained from female patients. Each point represents the mean ± SEM of four experiments from individual cell preparations obtained from each donor. Effects of days of culture, gender and/or estrogen treatment on growth curves were analyzed by two-way analysis of variance followed by the Tukey’s multiple comparisons test. *P = 0.001 for male GM-hBMECs vs. male WM-hBMECs; °P = 0.05 for male WM-hBMECs vs. male β-estradiol 17-acetate-treated WM-hBMECs; ¦P = 0.001 for female β-estradiol 17-acetate-treated WM-hBMECs vs. male GM-hBMECs; £P = 0.01 for female β-estradiol 17-acetate-treated WM-hBMECs vs. male β-estradiol 17-acetate-treated WM-hBMECs. GM: Gray matter; hBMECs: Human cerebral microvascular endothelial cells; WM: white matter.

Figure 3

Effect of donor’s gender, tissue source and 10 nM β-estradiol 17-acetate on hBMECs recovery and growth after thawing from cryopreservation and splitting. (A) Representative phase contrast images of morphology and density of male GM-derived hBMECs and of female WM-derived hBMECs subcultured for 6 days and 12 days, respectively, after thawing from cryopreservation both in the presence and in the absence of 10 nM β-estradiol 17-acetate. Scale bars: 20 μm. (B) Comparison between growth curves related to GM-derived hBMECs isolated from male patients M1-M4, thawed and subcultured in the absence (blue triangles) and in the presence (violet squares) of 10 nM β-estradiol 17-acetate, and growth curves related to WM-derived hBMECs isolated from female patients F4–F5, thawed and subcultured in the presence (pink triangles) and in the absence (red circles) of 10 nM β-estradiol 17-acetate. (C) Effects of withdrawal (blue circles) and new addition (violet triangles) of 10 nM β-estradiol 17-acetate on recovery and growth after thawing of GM-derived hBMECs isolated from male patients M1-M5 and effects of withdrawal of 10 nM β-estradiol 17-acetate on recovery and growth after thawing of WM-derived hBMECs isolated from female patients F4–F5, each subcultured after thawing. The inserted graph represents the magnification of the failed recovery after withdrawal and re-addition of 10 nM β-estradiol 17-acetate in WM-derived hBMECs from female patients F4–F5. Each point represents the mean ± SEM of four experiments from individual cell preparations obtained from each donor. Effects of thawing, gender and/or estrogen treatment on growth curves were analyzed by two-way analysis of variance (P = 0.0001) followed by the Tukey’s multiple comparisons test. (B) Male GM-derived hBMECs with estrogen vs. male GM-derived hBMECs without estrogen, *P = 0.0001; female WM-derived HBMECs with estrogen vs. female WM-derived hBMECs without estrogen, °P = 0.0001. (C) Male GM-derived hBMECs withdrawal of estrogen vs. male GM-derived hBMECs re-addition of estrogen, *P = 0.0002; male GM-derived hBMECs re-addition of estrogen vs. female WM-derived hBMECs readdition of estrogen, **P = 0.0007. GM: Gray matter; hBMECs: Human cerebral microvascular endothelial cells; WM: white matter.

Figure 4

Cell viability. (A) Phase contrast images of bright fields of the hemocytometer and (B) quantification of the live/dead trypan blue exclusion assay, referred to male GM- and WM-derived hBMECs and female WM-derived hBMECs, each of them splitted after thawing. Orange arrows indicate dead cells. Scale bar = 50 μm. No statistical differences (NS) in viability resulted from one-way analysis of variance (P = 0.1035) followed by Bonferroni’s multiple comparisons test, performed on three aliquots of cells deriving from three independent cultures (n = 3) of each male GM- and WM-derived hBMECs and female WM-derived hBMECs, respectively. GM: Gray matter; hBMECs: Human cerebral microvascular endothelial cells; WM: white matter.

Figure 5

Visualization and quantification of immunofluorescence staining of CD31, CD54 and CD62E markers in representative cultures of male GM- and WM-derived hBMECs and female WM-derived hBMECs. (A) Constitutive expression of the endothelial marker CD31 or PECAM-1 was shown for all the types of hBMECs both after fresh isolation and after thawing from cryopreservation. TNF-α activated hCMEC/D3 cells were used as positive control for staining of CD31. CD31 primary antibody was stained in green by fluorochrome-conjugated goat anti-mouse secondary antibody Alexa Fluor 488 and nuclei were stained by the blue Hoechst 33342 dye. (B) Immunofluorescence intensity quantification (in arbitrary units; A.U.) of CD31-stained hBMECs in comparison to the TNF-α activated hCMEC/D3 cells, CD31 positive control. Scale bars: 50 μm. **P = 0.0075, ***P = 0.0002, ****P = 0.0016, *****P = 0.0003. (C) CD54 or ICAM-1 exhibited a low expression in male GM-derived hBMECs and female WM-derived hBMECs compared with the positive control TNF-α activated hCMEC/D3 cells, as shown by fluorescence intensity quantification. CD54 primary antibody was stained in red by fluorochrome-conjugated goat anti-mouse secondary antibody Alexa Fluor 633 and nuclei were stained by the blue Hoechst 33342 dye. Scale bars: 50 μm. *P = 0.0167, ***P = 0.0004, ****P = 0.00001. (D) CD62E or E-Selectin was clearly seen as being negative both for male GM-derived hBMECs and female WM-derived hBMECs in comparison with the TNF-α activated hCMEC/D3 cells, as shown by fluorescence intensity quantification. CD62-E primary antibody was stained in orange-yellow by fluorochrome-conjugated goat anti-mouse secondary antibody Alexa Fluor 568 and nuclei were stained by the blue Hoechst 33342 dye. All the endothelial markers were merged with the nuclear Hoechst 33342 dye. Scale bars: 50 μm. Each point represents the mean ± SEM of four experiments from individual cell preparations obtained from each donor. Significance was calculated by one-way analysis of variance followed by Dunnet’s post hoc test, comparing each marker staining in hBMECs with its staining in hCMECs/D3 positive control. AT: After thawing; FI: Fresh isolation; GM: gray matter; hBMECs: Human cerebral microvascular endothelial cells; WM: white matter.