Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties

Abstract

Background: Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity.

Methods: Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.

Results: The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.

Conclusions: The data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Fig. 1

Establishment and characterization of in vitro BBB models. a Monoculture consisting of brain capillary endothelial cells (BCECs, yellow), a non-contact co-culture of BCECs and astrocytes (green) and a triple culture of BCECs, pericytes (purple) and astrocytes. Astrocytes and pericytes were cultured for 21 and 10 days, respectively. The BCECs were cultured for 3 days until 80% confluent. Puromycin was added to the media for the first 2 days. On day −1 the pericytes and/or endothelial cells were passaged to each side of the inserts and left to adhere for 24 h. On day 0, the inserts used for co- and triple culturing were moved to a plate containing astrocytes, and the BCECs were stimulated with hydrocortisone, cAMP and RO. b The cells were identified based on their expression of the tight junction protein ZO1 (BCECs), alpha smooth muscle actin (α-SMA) (pericytes) and glial fibrillary acidic protein (GFAP) (astrocytes). The cell nuclei were counterstained with DAPI (blue). Scale bars 10 µm. c The maximal TEER values were reached on day 2. Co-cultures (red), and triple cultures (green) displayed TEER values of 299 ± 17 and 331 ± 28 Ω cm² respectively, while the monoculture (blue) only showed a slight increase in TEER (128 ± 9 Ω cm²). Data are presented as means ± SEM (n = 24). Statistical differences were analysed using a 1-way ANOVA with Tukey’s multiple comparisons post hoc test (***p < 0.001). There was no ignificant difference between co- and triple cultures. d The apparent permeability (Papp) of mannitol in cultured BCECs. Data are calculated based on measurements from 18 inserts with TEER values ranging from 72.4 to 321 Ω cm². The permeability to mannitol decreases as TEER values increase around 150 Ω cm², which can be obtained by co-culturing the BCECs with pericytes and/or astrocytes.

Fig. 2

Gene expression analysis of the hall mark proteins related to brain capillary endothelial cells (BCECs). RNA was obtained from BCECs grown in mono- (blue), co- (red) and triple- (green) culture conditions at day 1 and analysed for the expression of BCECs hallmark genes (claudin-5, occludin, PECAM-1, ABCG2, ABCB1 and Transferrin receptor 1 (TfR1)). The relative gene expression among the three culture conditions was statistically analysed using 1-way ANOVA with Tukey’s multiple comparisons post hoc test. Data are presented as sample means ± SEM (n = 8). p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Analysis of the cell proliferation of brain capillary endothelial cells (BCECs) from barrier culture days −2 to 3 using a 5-and 6-carboxylfluorescein diacetate succinidyl ester (CFDA SE) assay. BCECs were isolated on day −3 and seeded directly onto inserts. 1 µM CFDA SE was added to the cells at day −2. After 30 min of incubation all the cells were labelled with CFDA SE and the first group were terminated (T₋₂) (red). Every day for 5 days one group (T₋₁ to T₃) (blue, orange, green, black and purple, respectively) were terminated. The BCECs were microscopically visualised to be confluent on day 0, and subsequently co-cultured with astrocytes and stimulated with hydrocortisone, cAMP and RO-201724. The cells were examined on BD FACS canto™ and analysed with the FlowJo v10 software. The cells were gated using forward and side scatter to eliminate cell debris. Unlabelled BCECs (purple) were used as a negative control.

Fig. 4

The effect of transfection on the integrity of the brain capillary endothelial cells (BCECs). a The experimental design used for in vitro transfection of BCECs. BCECs (yellow) were isolated on day -3, seeded directly onto the inserts. Barrier properties were induced by co-culturing with astrocytes (green) in the presence of hydrocortisone, cAMP and RO-201724. BCECs were transfected at two different stages of barrier maturity: T₋₁, an immature state, defined by dividing BCECs without barrier properties, and T₁, a mature state defined as the BCECs being confluent and having barrier properties. b The integrity of the transfected BCECs (T₋₁ and T₁) (black) was monitored daily by measurements of TEER and compared to non-transfected cells (T_CTRL) (grey). Left non-confluent BCECs transfected on day −1 (T₋₁). No attempts were made to increase tight junction formation. Right on experimental day 1, barrier properties were present (TEER above 150 Ω cm²) in both transfected and control BCECs and lasted for at least 2 days. Significant differences among the two states and their respective controls were analysed using 1-way ANOVA with Tukey’s multiple comparisons post hoc test (***p < 0.001). No significant difference was found between T₁ and T_CTRL. Data are presented as means ± SEM (n = 34–44). c To investigate the origin of the HcRed1-C1 positive cells, an immunocytochemical analysis was performed for the tight junction protein ZO1. The cells illustrated were transfected at day 1 (T₁) and examined 48 h after transfection. The illustrations depict a BCEC containing both the HcRed1-C1 protein and the ZO1 protein (green). Nuclei are counterstained with DAPI (blue). Scale bar 10 µm.

Fig. 5

The transfection efficiency of brain capillary endothelial cells (BCECs) at different stages of barrier maturity. a Expression of HcRed1-C1 fluorescent protein as seen in BCECs 48 h after transfection. HcRed1-C1 positive cells were expressed at both stages of barrier maturity. The HcRed1-C1 protein distributed to both the cell nucleus and cytoplasm. Scale bar 10 µm. b The relative gene expression of HcRed1-C1 was investigated with RT-qPCR at the two different states of barrier maturity: T₋₁ (blue) and T₁ (green). Non-transfected cells (T_CTRL) (purple) showed no HcRed1-C1 gene expression. The gene expression of claudin-5 was included to evaluate the origin of the transfected cells and to measure the degree of tight junction formation at the various stages of barrier maturity. The relative gene expression among the three culture stages was statistically analysed using 1-way ANOVA with Tukey’s multiple comparisons post hoc test (**p < 0.01). Data are presented as sample means ± SEM (n = 6–8). No significant differences were found in the expression pattern of claudin-5 among the three groups. c The transfection efficacy of BCECs was additionally assessed by flow cytometry. A transfection efficacy of about 4% was found in both the immature highly diving stage (T₋₁) and in the mature non-dividing stage (T₁). Results were analysed using the FlowJo V10 software. The cells were gated using forward and side scatter to eliminate cell debris. Additionally, the cells were corrected for auto fluorescence using unlabelled BCECs (purple) to ensure that less than 1% of the HcRed1-C1 positive cells were false positive. Scale bar 10 µm.