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

Podsumowanie

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Streszczenie

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

Wprowadzenie

The blood-cerebrospinal fluid barrier (BCSFB) is one of the three barrier sites between the blood and the brain1. Its morphological correlate are the epithelial cells of the choroid plexus (CP)2,3, an endothelial-epithelial convolute, which is strongly vascularized and located in the ventricles of the brain. The CP serves to produce the cerebrospinal fluid (CSF) as well as to separate the latter from the blood. In order to achieve barrier function, the CP epithelial cells show a low pinocytotic activity, express specific transporters, and are densely connected by a continuous network of tight junctions (TJs)2,3.

Human choroid plexus papilloma (HIBCPP) cells, derived from a malignant choroid plexus papilloma of a Japanese woman4, were used to construct a functional in vitro model of the BCSFB. HIBCPP cells show a couple of characteristics of a functional BCSFB as the formation of TJ strands, the development of a high transepithelial membrane potential that can be determined as transepithelial electrical resistance (TEER), and minor permeabilities for macromolecules. Moreover, HIBCPP cells express characteristic transporters, which may serve to regulate the ionic microenvironment, and show apical/basolateral polarity5,6,7.

The BCSFB has been shown to function as an entry site for pathogens (bacteria, viruses, and fungi) into the central nervous system (CNS)8. The invasion of pathogens, including Neisseria meningitidis (N. meningitidis), a Gram-negative bacterium, can cause severe diseases like meningitis. Evidence that it overcomes the protective epithelial barrier of the CP is supported by histopathological observations in patients with meningococcal disease exhibiting increased amounts of meningococci in the vessels and CP epithelial cells9,10. To gain entry into host cells bacteria often hijack endocytotic mechanisms, which are mediated or triggered by specific surface receptors located on the host cells. Since interactions of pathogens with these receptors can be species specific11, animal models can only be consulted to a restricted extent. The HIBCPP cell line provides the opportunity to study the invasion process as well as the underlying molecular mechanisms in a human model system. Employing cell culture inserts enables us to analyze interactions of pathogens with host cells from two distinct cell sides. Many bacteria, including N. meningitidis, are strongly subject to the impact of gravity during infection assays. For optimal interaction of pathogens with the HIBCPP cells during the assays, the bacteria are initially added into the upper compartment of the cell culture filter insert system. To enable infection from the apical or the basolateral cell side, respectively, two variations of the in vitro system have been established: In the standard system HIBCPP cells are seeded into the upper compartment of the filter insert, mimicking the situation when microorganisms are located on the CSF-side and get into contact with the apical side of the cells (Figure 1A, C). In contrast, using the HIBCPP cells in an inverted cell culture filter insert system reflects the conditions when bacteria have entered the blood stream. Microorganisms disseminate in the blood and encounter CP epithelial cells from the basolateral side (Figure 1B, D). Noteworthy, in this model system it has been shown that bacteria invade HIBCPP cells in a polar fashion specifically from the basolateral cell side5,7.

Subsequently to infection of the CP, the invaded pathogens can be recognized by the innate immune system through ligation to pattern-recognition receptors (PRRs). Well-described members of the PRRs belong to the Toll-like receptor (TLR) family. TLRs can bind to characteristic structures of infectious microorganisms, which are termed pathogen-associated molecular patterns (PAMPs). Ligation of the receptors leads to activation of host cell signaling cascades that trigger expression of cytokines and chemokines12, which in turn stimulate transmigration of immune cells across the BCSFB13,14. It has been shown that HIBCPP cells express several TLRs at mRNA level and that infection with N. meningitidis results in secretion of several cytokines and chemokines, including CXCL1-3, IL6, IL8 and TNFα15,16.

Here, we describe cultivation and infection of the human cell line HIBCPP in an inverted cell culture insert system that mimics the BCSFB. This model system enables to study interactions of pathogens with the in vivo relevant basolateral cell side as well as the subsequent cellular response.

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Protokół

1. Prepare Cell Culture Filter Inserts for Seeding HIBCPP Cells in an Inverted Model System

  1. Pre-warm DMEM/ F12 (Ham) supplemented with 5 µg/ml insulin, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum (FCS).
  2. Use sterile forceps to place 0.33 cm² growth area cell culture filter inserts with a pore size of 3 µm upside down into a 12-well plate (Figure 1E).
  3. Fill medium into the lower compartment of the cell culture filter insert (about 3 ml) and 100 µl on top of the filter insert. Flood the plate as well as the lower compartment with medium. Aspirate the excessive medium in such a way that the lower compartment of the filter insert stays filled (Figure 1E).
    Note: It is recommended to use a serological pipette for this step.
  4. Cover 12-well plate with the lid and transfer the prepared cell culture filter inserts to the incubator, 37 °C, 5% CO2 until addition of cells.

2. Cultivation and Passaging of HIBCPP Cells

  1. Prepare DMEM/ F12 (Ham) supplemented with 5 µg/ml, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS.
  2. Pre-warm medium and PBS in a 37 °C water bath. Aspirate medium from flask. Add 10 ml PBS to the flask and swirl. Repeat this step once.
  3. Add 3 ml 0.25% trypsin-EDTA to the flask and swirl. Place into the incubator, 37 °C, 5% CO2.
  4. After approximately 20 min remove the flask from the incubator. Ensure that the cells are detached from the bottom of the flask and display a round shape when surveyed with the microscope.
    Note: The cells do not detach completely from each other and are often found in agglomerates. It is recommended to use the cells up to passage 38.
  5. To stop trypsinization add 17 ml medium. Resuspend the cells by pipetting up and down and transfer the suspension into a 50 ml tube. Centrifuge at 50 x g for 10 min at room temperature.
  6. Resuspend the cells in an appropriate volume of medium and count the cells by using a hemocytometer.
    Note: The concentration of resuspended HIBCPP cells should be 1 x 106 cells/ml. For maintenance of HIBCPP cells it is suggested to transfer an amount of 1-6 x 106 cells in 10 ml medium to a T75-flask. Change medium every second day.

3. Seeding Inverted Cell Culture Filter Inserts with HIBCPP Cells

  1. On top of each inverted filter insert (i.e. the bottom side of the filter) add 80 µl of cell suspension (i.e. 8 x 104 cells) (Figure 1E).
    Note: Make sure that the cells are evenly distributed in the suspension by inverting the tube before seeding.
  2. Cover the seeded-cell culture filter inserts with the lid of the 12-well plate and transfer to the incubator, 37 °C, 5% CO2.
  3. On the first day, fill 1 ml medium into the wells of a 24-well plate. Lift the cell culture filter inserts by using forceps out of the 12-well plate, discard the medium inside, turn the filter inserts and place them in the standard orientation into the prepared 24-well plate (Figure 1E).
  4. Place the cells into fresh medium every second day. Prepare 24-wells with fresh medium and transfer the filter inserts to it. Fill inserts with 0.5 ml fresh medium. Check TEER every day as described in section 4.
  5. When TEER values of HIBCPP seeded-cells on cell culture inserts exceeds 70 Ω x cm² (approximately 4 days after seeding), then continue cell culture in medium containing 1% FCS and 5 µg/ml insulin. Prepare 24-well plates with 1 ml medium for each well. Transfer filter inserts to the prepared wells and exchange medium in the upper compartment.
    Note: This serum withdrawal after confluency leads to the formation of a higher membrane potential.
  6. Aspirate medium from filter compartment, transfer to the prepared well and fill with 500 µl medium. Repeat this step once. Put the cells overnight into the incubator, 37 °C, 5% CO2.

4. Measurement of Transepithelial Electrical Resistance (TEER)

  1. Immerse the electrode tips of epithelial tissue voltohmmeter for 15 min in 80% ethanol. Transfer voltohmmeter under the hood and let electrode dry for a moment. Place electrode into respective culture medium used for the cells for another 15 min to equilibrate.
  2. Perform measurements by positioning the longer arm of the electrode so that it touches the bottom of the lower compartment each time, place the shorter arm into the filter insert compartment.
    Note: Resistance values of cell culture filter inserts without cells in medium should be used as blank values ((measured value (Ω) – blank value (Ω)) x filter surface (i.e. 0.33 cm²)).
  3. After measuring place the electrode back into 80% ethanol for 15 min. Store in a dry tube.

5. Determination of Paracellular Permeability

  1. Dissolve 1 g FITC-Inulin in 200 ml culture medium supplemented with 5 µg/ml insulin 1% FCS. Apply 50 µg/ml of the solution into the upper filter compartment before infection of the cells.
  2. After infection collect medium samples from the lower well to determine how much Inulin passed from the filter compartment through the cell layer.
  3. Prepare a FITC-Inulin standard solution and perform 1:2 dilutions, 10 times (100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%, 0.39%, 0.2% and 0%). Determine fluorescence by measurement of all samples in microplate reader.

6. Preparation of Bacteria for Infection of HIBCPP Cells on Cell Culture Filter Inserts

  1. One day prior to the experiment, scrape the frozen N. meningitidis strains off a glycerol-stock and streak out on Chocolate Agar with Vitox. Grow overnight in the incubator, at 37 °C, with 5% CO2.
  2. Extract 20-30 colonies from the overnight culture and transfer into a tube containing 8 ml Proteose Peptone Medium supplemented with 0.042% NaHCO3, 0.01 M MgCl2 and 1% Polyvitex.
  3. Shake the bacteria for 1.25 hr at 220 rpm, 37 °C. Pellet the bacteria by centrifugation at 2,684 g for 10 min at room temperature. Discard supernatant and resuspend the bacterial pellet with 8 ml serum-free medium. Vortex to ensure an even suspension.
  4. To determine culture density, measure a dilution of 1:10 at an optical density (OD) of 600 nm. Adjust the bacterial suspension to an OD600 of 0.1.
    Note: This suspension contains approx. 1 x 108 CFU/ml.
  5. To confirm the bacterial cell concentration, dilute the suspension stepwise (1:10) up to a dilution of 10-5 and plate onto Chocolate Agar plates.
    Note: Similar serial dilutions need to be performed to calculate bacterial growth curves.

7. Infection of HIBCPP Cells on Cell Culture Filter Inserts and Determination of Bacterial Invasion by Double Immunofluorescence

  1. After continuing cell culture in medium containing 1% FCS and 5 µg/ml insulin, measure cells every day using an epithelial tissue voltohmmeter. If cells have reached a TEER of around 500 Ω x cm² , carry out the infection.
  2. Infect the cells with the prepared bacterial suspension at a multiplicity of infection (MOI) of 10. Store the infected cells in the incubator, at 37 °C, with 5% CO2 for the indicated period of time.
    Note: The MOI can be calculated by taking into account the number of cells per cell culture filter insert at confluence (1.21 x 106 cells/cm2).
  3. Stop the infection by washing three times with 500 µl serum-free medium (SFM) containing 1% bovine serum albumin (BSA) applied to the filter compartment, 1 ml to the lower compartment.
  4. Block with 500 µl SFM containing 1% BSA buffer in the filter compartment and 1 ml in the lower compartment for 20 min at room temperature to prevent adherence of antibodies to unspecific binding sites.
  5. Incubate with 100 µl primary antibody anti N. meningtidis α-OMP (1:200) in the filter compartment and 500 µl in the lower compartment for 20 min at room temperature.
    Note: If required, the antibody concentration needs to be titrated.
  6. Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml SFM containing 1% BSA and fill the emptied filter compartment with 500 µl SFM containing 1% BSA. Repeat this step twice.
  7. Fix cells with 500 µl 4% formaldehyde in the filter compartment, 1 ml in the lower compartment for 10 min at room temperature.
  8. Wash the cells by aspirating the medium from the filter compartment, transfer the filter insert to a prepared well with 1 ml PBS and fill the emptied filter compartment with 500 µl PBS. Repeat this step once.
    Note: Samples can be stored overnight in PBS at 4 °C.
  9. Cut fixed cell culture filters out of the inserts and wash with 250 µl PBS containing 1% BSA. Incubate cells for 15 min at room temperature with 250 µl fluorescent labeled (excitation wavelength 594 nm) secondary antibody chicken anti rabbit (1:500) to stain extracellular bacteria.
    Note: If required, the antibody concentration needs to be titrated.
  10. Permeabilize cells with 250 µl PBS containing 1% BSA and 0.5% Triton X-100 for 1 hr at room temperature. Wash cells three times with 250 µl PBS containing 1% BSA.
  11. Incubate with 250 µl primary antibody anti N. meningitidis α-OMP (1:200) for 30 min to stain extra- and intracellular bacteria. Wash cells three times with 250 µl PBS containing 1% BSA.
  12. Apply 250 µl of fluorescent labeled (excitation wavelength 488 nm) secondary antibody (1:500), fluorescent labeled (excitation wavelength 660 nm) Phalloidin (1:250) and 4´,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000) for 1 hr at room temperature to stain extra- and intracellular bacteria, actin cytoskeleton and nuclei.
    Note: If required, the antibody concentration needs to be titrated.
  13. Wash cells three times with 250 µl PBS containing 1% BSA. Embed the cells in mounting medium and store at 4 °C until examination via microscope.
  14. Determine number of invaded bacteria per predefined field. Do this by counting 20 fields per filter membrane. Calculate the percentage of invaded bacteria.
    Note: Multiply the mean bacterial count of the 20 microscopic fields with an area coefficient. The result expresses the amount of total bacteria present in a 0.33 cm² cell culture filter insert. Divide this value by the amount of bacteria grown in media during the duration of infection.

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Wyniki

Here we describe culturing and infection of HIBCPP cells in an inverted cell culture insert system. This model allows us to study invasion mechanisms and the underlying molecular signaling pathways from the basolateral cell side, reproducing a physiological situation of bacteria disseminating and entering epithelial cells via the blood stream (Figure 1).

The HIBCPP cells display certain barrier functions, which ...

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Dyskusje

The epithelial cells of the CP form the BCSFB that separates the CSF from the blood2,3. We recently established the HIBCPP cell line as a functional human model of the BCSFB. The cells display important barrier functions of the BCSFB in vitro, including the development of a high membrane potential, a low permeability for macromolecules, as well as the presence of continuous strands of TJs5. The TJ proteins contribute to an apical/basolateral polarity of the cells. The polarity is of high im...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

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Materiały

NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGibco25200-056
4´,6 diamidino-2-phenylindole (DAPI)Life TechnologiesD1306
12-well platesStarlabCC7682-7512
24-well platesStarlabCC7682-7524
Anti Neisseria meningitidis α-OMPThis antibody was a gift from Drs. H. Claus and U. Vogel (University of Würzburg, Germany)
Alexa Fluor 488 (chicken anti rabbit)InvitrogenA21441
Alexa Fluor 594 (chicken anti rabbit)InvitrogenA21442
Alexa Fluor 660 PhalloidinInvitrogenA22285
Bovine serum albumine (BSA)Calbiochem12659
Chocolate agar platesBiomerieux43109
Cytochalasin DSigmaC8273
DMEM/F12 + L-Glut + 15 mM HEPESGibco31330-095
DMEM/F12 + L-Glut + 15 mM HEPES w/o PhenolredGibco11039-047
Dimethyl sulfoxideSigmaD2650
Fetal calf serum (FCS)Life Technologies10270106
FITC-InulinSigmaF3272
InsulinSigma19278
MgCl2Sigma2393
NaHCO3Sigma55761
PBS + Mg + CaGibco14040-174
Penicillin/StreptomycinMP Biomedicals1670049
PolyvitexBiomerieux55651
Proteose peptoneBD211684
Serum-free mediumGibco10902-096
Thincert cell culture inserts for 24-well plates, pore size 3 µmGreiner662630
Tissue culture flask 75 cm² red cap sterileGreiner658175
Triton X-100SigmaT8787
Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrodeMilliporeMERSSTX00

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Choroid Plexus Epithelial CellsBlood cerebrospinal Fluid BarrierBacterial InfectionBasolateral SideCentral Nervous SystemHIBCPP CellsCell CultureFilter InsertsTranswell SystemTripsyn EDTACell SuspensionCell Culture Method

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