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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Streszczenie

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-β (oAβ), which is an important contributor to Alzheimer's disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oAβ disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

Wprowadzenie

The blood-brain barrier (BBB) of cerebral capillaries is the largest interface of blood-to-brain contact and plays a central role in the homeostasis of the central nervous system (CNS)1,2. Dynamic processes at the BBB prevent the uptake of unwanted molecules from the blood, remove waste products from the CNS, supply essential nutrients and signaling molecules to the CNS, and modulate neuroinflammation1,2,3,4,5. BBB damage is prevalent during aging and several neurodegenerative disorders including Alzheimer's disease (AD), multiple sclerosis and stroke1,2,3,4,5,6. Therefore, BBB dysfunction may play a key role in neurodegenerative disorders, including as a therapeutic target.

Maintaining vessel coverage is important for the homeostatic functions of the BBB. However, in vivo and in vitro data conflict on whether the processes involved in neurodegenerative disorders cause higher or lower BBB coverage6,7,8,9,10,11,12,13, particularly in AD. Therefore, there is a strong rationale for the development of in vitro models using relevant cell types to assess and more comprehensively understand the dynamics of BBB coverage. Cerebral capillaries are composed of astrocytes, pericytes and brain endothelial cells (BECs)3. All cell types contribute to the function of the BBB through structural support and via the secretion of effector molecules such as angiogenic growth factors, cytokines and chemokines that act in paracrine- and autocrine-like fashion. However, the major effector cells of the BBB are BECs3. In general, the cell culture techniques for assessing BBB function are permeability assays performed on cells grown on filter inserts, or assessing levels of key BEC proteins, both after the addition of stressors14,15,16. Although important, these assays do not focus on the cerebrovascular coverage.

Here, our previous methods17 are detailed to assess BEC coverage and meshwork-like structures using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. The goal was to demonstrate the detrimental effect of oAβ, which is considered an important contributor to AD progression, on BEC coverage. The protective effect of the epidermal growth factor (EGF) highlights the potential of the technique as a therapeutic screening tool. The technique has several broad applications for basic and applied research including: 1) delineating the role of specific pathways on angiogenesis and vessel coverage, 2) evaluating the effects of disease and aging-relevant factors on angiogenesis and vessel coverage, and 3) identifying pharmacological targets.

Protokół

All experiments follow the University of Illinois, Chicago Institutional Animal Care and Use Committee protocols.

1. General Preparation

NOTE: The brain microvascular endothelial cell line (hCMEC/D3) is an extensively characterized immortalized human BEC line14,15,16,18,19. Culture the hCMEC/D3 cells on tissue culture flasks coated with collagen Type I (calf skin, 1:20 dilution of 0.1% solution in Hank's Balanced Salt Solution (HBSS) containing Ca2+ and Mg2+) in basal Endothelial Growth Basal Medium (EBM-2) containing 2-5% Fetal Bovine Serum (FBS), 10% ascorbic acid, 10% gentamicin sulphate, 25% hydrocortisone and 1/4 of the total volume of the supplied growth factor supplements per 500 mL of media [vascular endothelium growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1) and human basic fibroblastic growth factor (bFGF), see Table of Materials].
NOTE: EBM-2 medium with FBS and growth factors is referred to as "EBM-2 complete". EBM-2 without FBS and supplements is referred to as "EBM-2 basal". At full confluence, the hCMEC/D3 cells are ~1 x 105 cells/cm2.

  1. Passage the hCMEC/D3 cells at a ratio of 1:5 by first incubating with HBSS for 3 min without Ca2+ and Mg2+ followed by detachment with 5 mL trypsin/EDTA (0.25%) for 5 min. Neutralize the trypsin using EBM-2 complete (1:1 neutralization), and centrifuge at 290 x g for 5 min at 4 °C; discard the supernatant. Resuspend the pellet in EBM-2 complete and re-plate at 1:5 ratio.
  2. Culture the primary human pericytes and astrocytes according to the supplier's protocol [Table of Materials]. Culture the pericytes in pericyte basal medium with FBS and pericyte growth supplement.
  3. Culture the human astrocytes in astrocyte medium containing astrocyte growth factors. Culture both the pericytes and the astrocytes in tissue culture flasks coated with 3 mL of poly-L-lysine (PLL) for cell adherence and utilize the cells between passages 2 and 5.
  4. Euthanize 7 mice and remove the brain stems and cerebella with forceps; detach the meninges by carefully rolling the brains on gauze. Mince the remaining brain tissue in a Petri dish in Minimal Essential Medium (MEM) with HEPES (5 mL per brain) with a sterile razor blade. Isolate the primary mouse BECs from 2-month-old C57BL/6J mice according to the referenced protocol20.
  5. Transfer the minced brain tissue to a 15 mL conical tube. Centrifuge at 290 x g for 5 min at 4 °C. Remove the supernatant and incubate the tissue in a papain (833.33 µL per brain) and DNase (41.7 µL per brain) solution, in a 37 °C water bath for 1 h, to digest the tissue.
  6. Triturate the homogenate sequentially through 19 and 21 gauge needles, mix at a 1:1 ratio with a 22% Bovine Serum Albumin (BSA) solution, and centrifuge at 1360 x g for 10 min at 4 °C.
  7. Resuspend the resulting pellet in 1 mL EBM-2 complete and centrifuge at 290 x g for 5 min at 4 °C. Resuspend again in 1 mL EBM-2 complete and plate the cells (1 mL/well) on 6-well plates coated with collagen (~1 brain per well).
  8. Replace the media 24 h later with EBM-2 complete containing 4 µg/mL puromycin hydrochloride; replace with EBM-2 complete after 2 days. The primary BECs are cultured as for the hCMEC/D3 cells and are at full confluence at 1 x 105 cells/cm2.
  9. Prepare a 100 µM of oAβ, 24 h before the assay.
    NOTE: oAβ is considered the disease relevant form of A . For oAβ, use the well-characterized preparations described by Dahlgren et al.21.
  10. Thaw the basement membrane stock solution overnight at 4 °C and aliquot into sterile PCR tube strips (8 tubes) at 140 µL basement membrane per tube. Refreeze each strip and store at -20 °C. Thaw one strip per 96-well plate at 4 °C, 24 h prior to the assay. Pre-cool the pipette tips to avoid solidification.
    NOTE: All handling of the basement membrane matrix must be carried out on ice to avoid rapid solidification. As 10 µL per well of basement membrane is used on the day of the assay, each strip is sufficient for one 96-well plate.
  11. Incubate fully confluent BECs in EBM-2 basal, 24 h prior to the assay.
    NOTE: The rationale is: EBM-2 complete contains supplemented factors and FBS with the purpose of promoting optimal cell growth; however, the same molecular pathways that promote cell growth are also important for brain endothelial cell coverage and dynamics, both in the presence and absence of a stressor. We therefore serum and supplement starve the BECs to reduce the confounding effect of residual activation of cellular signaling pathways. Of note, the cells are briefly (5 min) exposed to FBS during the neutralization of trypsinized cells prior to the assay. In contrast to BECs, the pericytes and astrocytes are not serum starved, due to the requirement of different factors for growth and the relative instability of these cell types in response to serum starvation (Tai laboratory, unpublished observations).
    NOTE: EBM-2 basal is utilized during the assay for the single and triple culture meshwork formation and disruption assays. This is critical to prevent confounding stressor or treatment dependent signaling.

2. Meshwork-like Formation and Disruption Assays

  1. Single BEC culture assays:
    NOTE: Three different paradigms for the single cultures of BECs are shown in Figure 1A. Each paradigm is designed to assess the response of BECs in different models of vessel coverage. Paradigm 1 is meshwork formation. This paradigm is designed to examine the effects of stressors and/or treatments on angiogenesis and meshwork formation. BECs, oAβ, and treatments are all added to the plate at the 0 h time point. Paradigm 2 is prevention of meshwork disruption. Cells are plated in the presence of a growth factor or drug and incubated for 4 h to form meshwork-like structures. A stressor, oAβ in this case, is added after 4 h. This paradigm assesses the ability of a treatment to prevent stressor-induced damage to preformed meshwork-like structures. Paradigm 3 is simultaneous treatment of meshwork disruption. Cells are plated and allowed to form meshwork-like structures for 4 h. Treatments and oAβ are then added simultaneously at the 4 h time point, assessing the ability of the preformed cell network to respond to various treatments. All the paradigms follow similar common steps, except with differences in the timing of treatment addition.
    1. On the day of the assay, pipette the basement membrane matrix at 10 µL/well into the bottom of 96-well plates and allow to set for 1 h at 37 °C.
    2. Suspend the lyophilized green cell tracking dye (see Table of Materials) in 10 µL DMSO to generate a 10-mM stock solution, and dilute to 10 µM (1:1000) in EBM-2 basal. Remove the media from the fully confluent flask and replace with EBM-2 basal containing the green cell tracking dye (5 mL for a 75 cm2 flask). Preload BECs with the green cell tracking dye, 20 min prior to the start of the assay.
    3. Incubate the cells for 20-30 min at 37 °C, remove the medium with cell tracking dye, and detach the cells as described in Step 1.1. Neutralize the media with EBM-2 containing 10% FBS and centrifuge at 240 x g for 5 min at 4 °C. Resuspend the pellet in 1 mL of EBM-2 basal.
    4. Plate the BECs in the 96-well plate at 10,000 cells/well in EBM-2 basal (0 h time point in all paradigms).
      NOTE: Depending on the paradigm being utilized, treatments, stressors, or vehicle controls are added at the specified time points. In all paradigms, the medium is harvested for subsequent analysis (e.g. ELISA analysis) and cells are fixed at 24 h with freshly prepared 4% paraformaldehyde in Phosphate Buffered Serum (PBS). As an alternative approach to determine the term effects of oAβ on mesh formation, the protocol could be modified to pre-incubate confluent cell culture flasks with oAβ, and then perform the meshwork formation paradigms (+/-oAβ).
      NOTE: The final volume in each well for all paradigms is 70 µL. All treatments (oAβ, EGF, etc.) are added to their respective wells at a 1:20 dilution i.e. 3.5 µL per treatment. For Paradigm 1, the cells are added in a cell suspension solution at 63 µL/well. Immediately oAβ, desired treatments, and controls are added at 3.5 µL/well each, giving a total of 70 µL. For Paradigm 2, 63 µL of cell suspension and 3.5 µL of treatment (e.g. 100 ng/mL EGF) are added at the 0 h time point. After 4 h incubation, 3.5 µL of an oAβ stock is added (e.g. for 100 nM final oAβ concentration, 3.5 µL of 2 µM oAβ stock). For Paradigm 3, 63 µL of cell suspension is added at the 0 h time point and at 4 h, oAβ and desired treatments are added at 3.5 µL/well each, giving a total of 70 µL. These volumes can be adjusted according to treatments. For example, if only one treatment is required, the cell suspension volume can be adjusted to 66.5 µL (maintaining 10,000 cells/well) and the treatment volume can remain at 3.5 µL.
  2. Triple culture assay:
    NOTE: In addition to the single culture assays of BECs, we have developed a triple culture assay paradigm (Figure 1B) to determine the response of BECs, pericytes, and astrocytes within preformed networks to relevant stressors (e.g.oAβ). BECs are plated in the presence of desired treatments at 0 h. At 4 h, pericytes are gently added to the plate. At 7 h, astrocytes are added to the plate, followed by the addition of a stressor at 11 h. Cells are then incubated until the 24 h time point.
    NOTE: For the triple culture assays, it is important to consider timing to ensure all cells are confluent at the same time. Human astrocytes and pericytes should be cultured 1 week before the assay, whereas the hCMEC/D3 cells need to be plated or passaged 4-5 days before the assay date. Further, it is important to use low passage (2-5) primary cells to avoid phenotypic drift.
    1. Add the green cell tracking dye working solution to the hCMEC/D3 cells 20 min prior to starting the assay. Plate the hCMEC/D3 cells at 10,000 cells/well in EBM-2 basal (0 h time point). The volumes used are 45 µL of cell suspension and 3.5 µL of treatment (i.e. final EGF concentrations of 50 ng/mL, 100 ng/mL, or 1,000 ng/mL from stocks that are 20 times more concentrated).
    2. Following the 3.5 h incubation at 37 °C, add the blue cell tracking dye working solution (10 µM cell tracker blue in EBM-2 basal) to the human primary pericytes. At the 4 h time point (~30 min incubation with cell tracking dye), gently add 2,000 pericytes/well to the plate containing the hCMEC/D3 in a volume of 6 µL/well.
    3. Following an additional 2.5 h incubation (total 6.5 h from 0 h time point), add the orange cell tracking dye working solution (10 µM cell tracker orange in EBM-2 basal) to the human primary astrocytes. At the 7 h time point, gently add 10,000 astrocytes/well to the plate in 12 µL volume. Therefore, the overall cellular ratio for endothelial cells:pericytes:astrocytes is 5:1:5. Further, the volume achieved to this point is 66.5 µL.
    4. Add oAβ (3.5 µL of 100 µM stock) 4 h later (total 11 h from 0 h time point).
    5. Fix the cells 13 h later in freshly prepared 4% paraformaldehyde in PBS.
      Caution: Wear protective clothing, gloves and eyeglasses while handling paraformaldehyde.

3. Quantification

  1. Capture fluorescence images of the whole well of the 96-well plate at 1.6X magnification with a 2 s exposure time at 50% maximum power using a dissecting microscope.
    NOTE: Equivalent microscopes can be utilized; however, it is critical to capture the entire well for comprehensive analysis.
  2. For quantitative analysis, process the images using the ImageJ angiogenesis analyzer plugin22 to quantify the number of branches, number of meshes, and total cell length (these readouts are automatically tabulated by the software) as described below. A detailed visual protocol of this step is provided in Figure 2.
    1. Open fiji ImageJ by clicking on the software icon.
    2. Click on the double red forward arrows located at the end of the toolbar, then click "Angiogenesis Analyzer".
    3. Select the folder with image files, click "Open".
    4. When the "settings for analysis" box appears, click "OK".
    5. When the "processing images" box appears, which indicates the number of images to be processed, click "OK".
    6. When the "batch progress window" box appears, which indicates the estimated processing time. During this time the program quantifies the outputs including the number of branches, number of meshes, and total cell length.
      NOTE: Once all the images are quantified, the results file will appear in the original image folder as a spreadsheet document.
    7. Select the spreadsheet file containing results and compare the number of branches, meshes and total cell length between groups.
      NOTE: In the triple culture assay ImageJ is further utilized to analyze pericyte/astrocyte coverage and number. Images are thresholded by a blinded experimenter and the "analyze particles" function utilized to generate readouts. Fixed cells and tube-like structures can also be immunostained for Aβ17 or other markers.

Wyniki

In single cultures, both the hCMEC/D3 cells (Figure 3A) and the primary mouse BECs (Figure 3B) form meshwork-like structures throughout the well. The structures are characterized by a meshwork of interlinked nodes (Figure 3). In all the paradigms described (Figure 1), the meshwork-like structures are similar after 24 h in the control groups, forming ~20 meshwork-like str...

Dyskusje

The methods described can be utilized to address several fundamental biological questions surrounding cerebrovascular coverage24. Specifically, they can identify which receptors and signaling pathways play a role in angiogenesis, vessel coverage in cancer tissue, and peripheral endothelial cells relevant to the brain. Examples include angiogenic growth factor receptors, nitric oxide, mitogen activated protein kinase signaling and calcium signaling25,

Ujawnienia

The authors have nothing to disclose.

Podziękowania

Leon Tai is funded by University of Illinois Chicago start-up funds.

Materiały

NameCompanyCatalog NumberComments
hCMEC/D3 cellsMiliporeSCC066
EBM-2 basal mediaLonzaCC-3156
Collagen Type 1ThermoFisherA1064401
HBSS, calcium, magnesium, no phenol redThermoFisher14025092
HBSS, no calcium, no magnesium, no phenol redThermoFisher14175095
Trypsin-EDTA (0.25%)ThermoFisher25200056
Final concentrations of the SingleQuot growth factor supplements for EBM2 mediaLonzaCC-4147
5% FBSLonzaCC-4147
10% Ascorbic acidLonzaCC-4147
10% Gentamycin sulphateLonzaCC-4147
25% HydrocortisoneLonzaCC-4147
1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factorLonzaCC-4147
Puromycin hydrochlorideVWR80503-312
MEM-HEPESThermo Scientific12360-038
Papain cell dissociation system (papain and DNase1)Worthington BiochemicalLK003150
Human pericytesSciencell1200
Pericyte basal mediaSciencell1201
Pericyte growth supplementSciencell1252
Human AstrocytesSciencell1800
Astrocyte mediaSciencell1801
Astrocyte growth supplementSciencell1852
Basement membrane (Matrigel Growth Factor Reduced)Corning356231
Angiogenesis m-plates (96-well)ibidi89646
Human Epidermal growth factorShenendoah Biotechnology100-26
CellTracker greenThermoFisherC7025
CellTracker orangeThermoFisherC34551
CellTracker blueThermoFisherC2110
Poly-l-lysineSciencell0403
10% Neutral Buffered FormalinSigma-AldrichHT5012-60ML
C57BL miceJackson Laboratoryna
PCR tube stripsGeneMateT-3014-2
Zeiss stereo discover v.8 dissecting microscopeZeissna

Odniesienia

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  5. Pardridge, W. Targeted delivery of protein and gene medicines through the blood-brain barrier. Clin Pharmacol Ther. 97 (4), 347-361 (2014).
  6. Tai, L. M., et al. The role of APOE in cerebrovascular dysfunction. Acta Neuropathol. 131 (5), 709-723 (2016).
  7. Biron, K. E., Dickstein, D. L., Gopaul, R., Jefferies, W. A. Amyloid triggers extensive cerebral angiogenesis causing blood brain barrier permeability and hypervascularity in Alzheimer's disease. PLoS One. 6 (8), e23789 (2011).
  8. Cameron, D. J., et al. Alzheimer's-related peptide amyloid-beta plays a conserved role in angiogenesis. PLoS One. 7 (7), e39598 (2012).
  9. Boscolo, E., et al. Beta amyloid angiogenic activity in vitro and in vivo. Int J Mol Med. 19 (4), 581-587 (2007).
  10. Paris, D., et al. Impaired angiogenesis in a transgenic mouse model of cerebral amyloidosis. Neurosci Lett. 366 (1), 80-85 (2004).
  11. Kitaguchi, H., Ihara, M., Saiki, H., Takahashi, R., Tomimoto, H. Capillary beds are decreased in Alzheimer's disease, but not in Binswanger's disease. Neurosci Lett. 417 (2), 128-131 (2007).
  12. Jantaratnotai, N., Ryu, J. K., Schwab, C., McGeer, P. L., McLarnon, J. G. Comparison of Vascular Perturbations in an Abeta-Injected Animal Model and in AD Brain. Int J Alzheimers Dis. 2011, 918280 (2011).
  13. Donnini, S., et al. Abeta peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEB J. 24 (7), 2385-2395 (2010).
  14. Tai, L. M., Holloway, K. A., Male, D. K., Loughlin, A. J., Romero, I. A. Amyloid-beta-induced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation. J Cell Mol Med. 14 (5), 1101-1112 (2010).
  15. Tai, L. M., Loughlin, A. J., Male, D. K., Romero, I. A. P-glycoprotein and breast cancer resistance protein restrict apical-to-basolateral permeability of human brain endothelium to amyloid-beta. J Cereb Blood Flow Metab. 29 (6), 1079-1083 (2009).
  16. Tai, L. M., et al. Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123. Brain Res. 1292, 14-24 (2009).
  17. Koster, K. P., Thomas, R., Morris, A. W., Tai, L. M. Epidermal growth factor prevents oligomeric amyloid-beta induced angiogenesis deficits in vitro. J Cereb Blood Flow Metab. 36 (11), 1865-1871 (2016).
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