A novel 4-cell in-vitro blood-brain barrier model and its characterization by confocal microscopy and TEER measurement

Abstract

The blood-brain barrier (BBB) is a protective cellular anatomical layer with a dynamic micro-environment, tightly regulating the transport of materials across it. To achieve in-vivo characteristics, an in-vitro BBB model requires the constituent cell types to be layered in an appropriate order. A cost-effective in-vitro BBB model is desired to facilitate central nervous system (CNS) drug penetration studies. Enhanced integrity of tight junctions observed during the in-vitro BBB establishment and post-experiment is essential in these models. We successfully developed an in-vitro BBB model mimicking the in-vivo cell composition and a distinct order of seeding primary human brain cells. Unlike other in-vitro BBB models, our work avoids the need for pre-coated plates for cell adhesion and provides better cell visualization during the procedure. We found that using bovine collagen-I coating, followed by bovine fibronectin coating and poly-L-lysine coating, yields better adhesion and layering of cells on the transwell membrane compared to earlier reported use of collagen and poly-L-lysine only. Our results indicated better cell visibility and imaging with the polyester transwell membrane as well as point to a higher and more stable Trans Endothelial Electrical Resistance values in this plate. In addition, we found that the addition of zinc induced higher claudin 5 expressions in neuronal cells. Dolutegravir, a drug used in the treatment of HIV, is known to appear in moderate concentrations in the CNS. Thus, dolutegravir was used to assess the functionality of the final model and cells. Using primary cells and an in-house coating strategy substantially reduces costs and provides superior imaging of cells and their tight junction protein expression. Our 4-cell-based BBB model is a suitable experimental model for the drug screening process.

Keywords: Blood-brain-barrier; Central nervous system; In-vitro model.

Figure 1.

Schematic representation of all critical time points and steps in the in-vitro BBB model development. Days represent the total days starting from astrocyte seeding until the completion of the experiment. Abbreviations- hBMEC, Human brain microvascular endothelial cells TEER, Trans Epithelial Electrical Resistances

Figure 2.

Visualization of unstained cells growing on a polycarbonate (a) and a polyester (b) transwell membrane. The TEER in polycarbonate Transwell inserts was measured with an EVOM² meter for eight days (c). Measurement was carried out three times daily, and the average is shown. Data represent values for samples in triplicates. Abbreviations- Fn, Fibronectin; Collagen, bovine Collagen I, and P-Lysine, Poly-L-Lysine; TEER, Trans Epithelial Electrical Resistances

Figure 3.

TEER in astrocyte and pericyte co-cultures seeded in polyester Transwell insert. Data represent values from triplicate wells on each day for six days post-seeding. Star-marked cell density was carried forward for further experiments. Abbreviations-TEER, Trans Epithelial Electrical Resistances

Figure 4.

TEER in 4-cell model containing hBMCEs, astrocytes, pericytes, and neurons in a polyester Transwell insert or with changing various conditions, ± Zn; ± Serum; ± neurons or only hBMECs. At each point, triplicates are used for rigor and TEER measured three times. On day 11 post-cell seeding, cells on the transwell insert membrane were fixed and preserved for confocal microscopy study. Abbreviations-TEER, Trans Epithelial Electrical Resistances; Zn, zinc; hBMEC, Human brain microvascular endothelial cells.

Figure 5.

Expression of TJs, ZO-1, and claudin 5 on human brain cells, layered on polyester transwell insert membrane. ZO-1 (Red) and claudin 5 (Green), and the nucleus (blue) were stained with DAPI (4,6-diamidino-2-phenylindole). TJs were observed on- astrocytes and pericytes in medium alone (a), with the addition of Zn sulfate (b), and hBMECs in medium alone (c) with Zn sulfate (d). Abbreviations- Zn, zinc; TJ, tight junctions; Z0-1, Zona occludens; hBMCE, Human brain microvascular endothelial cells; DAPI, 4',6-diamidino-2-phenylindole.

Figure 6.

Cell identification by detecting s100 beta on astrocytes and CD146 on pericytes and hBMECs as respective cell markers. Astrocytes, pericytes, and hBMEc on a transwell membrane, immunostained with a cocktail of s100beta antibody (red), an antibody against CD146 (green), and the nucleus was stained with DAPI (blue). The basolateral surface of the transwell membrane shows astrocyte and pericyte (a), and the apical part of the same transwell membrane shows hBMECs only (b). Abbreviations- hBMEC, Human brain microvascular endothelial cells; DAPI, 4',6-diamidino-2-phenylindole.

Figure 7.

Serum-free medium, confocal images of TJ protein ZO-1 (red), Claudin 5 (green) expression on astrocytes and pericytes (a) or following treatment with Zn sulfate (b) and for hBMECs (c) following treatment with Zn (d) localized to transwell insert membrane in BBB model. The nucleus was stained with DAPI. Abbreviations- Zn, zinc; TJ, tight junctions; Z0-1, Zona occludens; hBMEC, Human brain microvascular endothelial cells; DAPI, 4',6-diamidino-2-phenylindole; BBB, blood brain barrier.

Figure 8.

In the absence of neuronal cells, expression of ZO-1 (red) and claudin 5 (green) in astrocytes and pericytes (a) and when supplemented with Zn in the medium (b) and the corresponding contact cultured hBMECs (c) supplemented with Zn (d). DAPI was used for nucleus staining. Abbreviations- Zn, zinc; Z0-1, Zona occludens; hBMEC, Human brain microvascular endothelial cells; DAPI, 4',6-diamidino-2-phenylindole.

Figure 9.

Expression of ZO-1 (red), claudin 5 (green) in mono-culture of hBMECs on a transwell membrane (a) and determination of claudin 5 on neuron cell from the non-contact 4-cell model with neuronal cell marker in red (b) and when the medium for the BBB model was supplemented with Zn (c). Abbreviations- Zn, zinc; Z0-1, Zona occludens; hBMEC, Human brain microvascular endothelial cells; DAPI, 4',6-diamidino-2-phenylindole.

Figure 10.

Average fluorescence intensity analysis of claudin 5 for one representative corresponding to the image shown from the triplicate of BBB model types and drug penetration for different sets of BBB representation. (a) Cell surface pixel intensity for claudin 5 measured using FIJI ImageJ-win64 software and (b) evaluation of tight junction integrity and the penetration of HIV-ART drug DTG in the 4-cell model. The color-coded bar pairs show drug distribution in the apical and bottom layers of the indicated in-vitro models and control (membrane only) from a transwell membrane plate. Media from the apical and basal layer of the transwell membrane was collected after 48 hours of drug treatment. Data represent the mean concentrations from 3 replicates. Abbreviations- MFI, mean fluorescence intensity; AP, astrocytes and pericytes; DTG, dolutegravir; hBMEC, Human brain microvascular endothelial cells; Zn, zinc