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Jul 2021

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Generation of a Human Conditionally Immortalized Cell-based Multicellular Spheroidal Blood-Brain Barrier Model for Permeability Evaluation of Macromolecules    

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Abstract

There is an urgent need for the development of brain drug delivery carriers based on middle-sized or macromolecules, to which in vitro blood-brain barrier (BBB) models are expected to contribute significantly through evaluation of BBB permeability. As part of efforts to develop such models, we have been working on human conditionally immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models), and we herein introduce the model development protocol. Briefly, astrocytes are first seeded in an ultra-low attachment 3D cell culture plate, to make the central core (Day 0). Next, pericytes are added over the core, to form an outer layer (Day 1). Then, brain microvascular endothelial cells are further added to each well, to create the outmost monolayer serving as the BBB (Day 2). Finally, the spheroids cultured for two days (on Day 4) can be used for assays of interest (e.g., antibody permeability assays). Neither special equipment nor techniques are required to produce hiMCS-BBB models. Therefore, the protocol presented here will not only facilitate the model sharing among the BBB community but also provide some technical clues contributing to the development of similar MCS-BBB models using other cell sources, such as primary or iPS-derived BBB cells.


Graphical abstract:




Keywords: Blood-brain barrier, In vitro model, Spheroid, Receptor-mediated transcytosis, Immortalized cell, Central nervous system, Drug development, Microphysiological systems

Background

The blood-brain barrier (BBB) forms a nearly impregnable wall preventing the distribution of numerous blood-borne middle-sized and macromolecules (middle/macromolecules) and low-molecular-weight drugs into the human brain (Sanchez-Cano et al., 2021; Segarra et al., 2021). As such, it often imposes a frustrating barrier to brain drug delivery efforts, thus rendering central nervous system (CNS) diseases among the most difficult to treat pharmaceutically. Brain microvascular endothelial cells (BMECs), which work with the support of pericytes and astrocytes, make up the critical components of the BBB. The pericytes are located in the immediate vicinity of the BMECs, while the astrocytes wrap them through their endfeet. This structural integrity is essential for BBB functions (Ballabh et al., 2004; Abbott et al., 2006; Daneman et al., 2015). The two primary functional characteristics of the BBB are tight/adherens junctions and efflux transporters. The former tightly seals BMEC plasma membranes to limit paracellular transport of various substances, including middle/macromolecules, while the latter are localized on the vascular side of BMECs and actively pump out their substrates into circulation (Ballabh et al., 2004; Abbott et al., 2010). Therefore, as part of efforts to create effective therapeutic approaches to CNS diseases, it is necessary to develop BBB-permeable drug delivery system (DDS) carriers that can overcome this barrier.


From that point of view, receptor-mediated transcytosis (RMT) occurring at the BBB has gained significant attention (Lajoie et al., 2015; Fang et al., 2017). Physiologically, after binding to their middle/macromolecule ligands at the blood side of BMECs, RMT receptors undergo endosomal vesicle formation to travel to the brain side, where they eventually release their cargo into the brain via exocytosis (Freskgård et al., 2017; Kouhi et al., 2021; Zhou et al., 2021). One intriguing idea for facilitating the development of brain DDS carriers involves taking advantage of those RMT processes, as exemplified by antibodies targeting transferrin or insulin receptors expressed in BMECs (Boado et al., 2016; Sonoda et al., 2018), resulting in an ongoing urgent need for effective and high-throughput evaluations of their BBB permeabilities. To this end, there have been a variety of next-generation in vitro BBB models based on microphysiological systems (MPSs), including BBB-on-a-chip, organoids, and spheroids (Cho et al., 2017; Wevers et al., 2018; Simonneau et al., 2021). These models feature high functionality derived based on their reproduction of the structure and surrounding microenvironment of the BBB in vivo (specifically, MPSs).


As part of efforts to facilitate the development of such models, we have been working on human conditionally immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) consisting of human conditionally immortalized BMECs (HBMEC/ci18 cells) (Kamiichi et al., 2012; Ito et al., 2019), astrocytes (HASTR/ci35 cells) (Furihata et al., 2016; Kitamura et al., 2018), and brain pericytes (HBPC/ci37 cells) (Umehara et al., 2018). These cells carry immortalization genes that enable them to extensively proliferate, while their status can be changed simply by varying the cell culture temperature (33 °C for growth and 37 °C for differentiation — for details, please refer to our above-cited previous papers).


In hiMCS-BBB models, HASTR/ci35 cells shape the central core, which is covered by an HBPC/ci37 cell layer. That layer is further surrounded by an HBMEC/ci18 cell monolayer serving as the BBB (Kitamura et al., 2021, 2022). This structural feature allows us to perform several BBB functional analyses, such as transporter activity assessments, and immune cell recruitments (Kitamura et al., 2021, 2022). In particular, the models exhibit RMT functions that are sufficiently high for use in evaluating the BBB permeability of antibodies and peptides (Kitamura et al., 2022). Additionally, the immortalized cells used are both scalable and easy to handle, and neither special equipment nor techniques are required to produce the models (as described herein). Owing to all these advantages, hiMCS-BBB models are expected to have great potential for use in developing novel middle/macromolecule-based DDS carriers.


In this paper, we describe the method used for constructing hiMCS-BBB models together with the culture methods for the human immortalized BBB cells. In alignment with that, we will also introduce an example of a middle/macromolecule permeability assay performed using hiMCS-BBB models. Through this publication, we aim not only to share hiMCS-BBB models among the BBB community but also to provide some technical clues that may contribute to the development of MCS-BBB models using other cell sources, such as primary or iPS-derived BBB cells, although some modifications may be necessary.

Materials and Reagents

Note: Most products can be replaced by those from other suppliers, but it is uncertain whether this is also true for the marked products (*) since we have not tested other materials.

  1. Low binding 200 µL tips (TreffLab, catalog number: 96.11179.4.01)

  2. Normal tips (Nacalai Tesque, catalog numbers: 19166-54 [10 µL], 19167-44 [200 µL], 19168-34 [1,000 µL])

  3. Violamo Disposable Pipette II (VIOLAMO, catalog numbers: 2-5237-12 [2 mL], 2-5237-03 [5 mL], 2-5237-04 [10 mL], 2-5237-05 [25 mL])

  4. Cryotubes (Thermo Fisher Scientific, catalog number: 363401PK)

  5. 15-mL centrifuge tubes (Thermo Fisher Scientific, catalog number: 339650)

  6. 50-mL centrifuge tubes (Thermo Fisher Scientific, catalog number: 339652)

  7. 1.5 mL tubes (WATSON, catalog number: 131-715C)

  8. Protein LoBind 1.5 mL tubes (Eppendorf, catalog number: 22431081)

  9. Collagen I-coated 60-mm dishes (IWAKI, catalog number: 4010-010)

  10. Collagen I-coated 100-mm dishes (IWAKI, catalog number: 4020-010)

    Note: Other collagen type I-coated products may be used.

  11. PrimeSurface 96V plates (96-well V-bottom plates)* (Sumitomo Bakelite, catalog number: MS-9096V)

  12. Cell counting slides (Logos Biosystems, catalog number: L12001)

  13. Slide glass 76 × 26 mm (slide glass) (Matsunami-glass, catalog number: MAS-01)

  14. Cover glass 24 × 60 mm (cover glass) (Matsunami-glass, catalog number: C024601)

  15. 100-mL flask (IWAKI, catalog number: 4980FK100)

  16. Parafilm

  17. Nail polish

  18. Human astrocytes/conditionally immortalized clone 35 (HASTR/ci35 cells)* (Furihata et al., 2016; Kitamura et al., 2018)

  19. Human brain pericytes/conditionally immortalized clone 37 (HBPC/ci37 cells)* (Umehara et al., 2018)

  20. Human brain microvascular endothelial cells/conditionally immortalized clone 18 (HBMEC/ci18 cells)* (Kamiichi et al., 2012; Ito et al., 2019)

  21. Blasticidin S (InvivoGen, catalog number: ant-bl-1)

  22. Astrocyte Medium kit (consisting of the basal medium and culture supplements)* (Thermo Fisher Scientific, catalog number: A1261301)

  23. Pericyte Medium kit (consisting of the basal medium and culture supplements)* (ScienCell, catalog number: 1201)

  24. VascuLife VEGF Comp kit (consisting of VascuLife BM and culture supplements) (Lifeline Cell Technology, catalog number: LEC-LL0003)

    Note: EBM-2 (Lonza, catalog number: CC-3162) can be used instead.

  25. Penicillin-streptomycin (Nacalai Tesque, catalog number: 26253-84)

  26. Bambanker (NIPPON Genetics, catalog number: CS-02-001)

  27. Dulbecco’s phosphate buffered saline Ca, Mg free (D-PBS(-)) (Nacalai Tesque, catalog number: 14249-24)

  28. D-PBS(+) preparation reagent (Ca, Mg solution) (Nacalai Tesque, catalog number: 02492-94)

  29. Hanks’ balanced salt solution (HBSS(+)) (Nacalai Tesque, catalog number: 09735-75)

  30. 2.5 g/L-trypsin/1 mmol/L-EDTA (trypsin) (Nacalai Tesque, catalog number: 32777-15)

  31. Trypan blue (Wako, catalog number: 207-17081)

  32. 4% paraformaldehyde (PFA) (Nacalai Tesque, catalog number: 09154-85)

  33. Fluoro-KEEPER Antifade Reagent, Non-Hardening Type (antifade reagent) (Nacalai Tesque, catalog number: 12593-64)

  34. Methyl cellulose-viscosity: 4,000 cP (methyl cellulose)* (Sigma-Aldrich, catalog number: M0512-100G)

  35. MEM189 [Alexa Fluor 647] (Novus Biologicals, catalog number: NB500-493AF647)

  36. CellTracker Orange CMTMR Dye (Invitrogen, catalog number: C2927)

  37. Red-fluorescent Cytoplasmic Membrane Staining Kit (PromoKine, catalog number: PK-CA707-30023)

  38. CellTracker Green CMFDA Dye (Invitrogen, catalog number: C2925)

  39. AM (astrocyte medium) (see Recipes)

  40. PM (pericyte medium) (see Recipes)

  41. VascuLife medium (see Recipes)

  42. Spheroid medium (see Recipes)

  43. D-PBS(+) (see Recipes)

Equipment

  1. Clean bench (or safety cabinet)

  2. Water bath set at 37 °C

  3. Water bath set at 60 °C

  4. Centrifuge (e.g., Kokusan, catalog number: H-19Rα)

  5. Aspirator

  6. Phase contrast microscope (e.g., Nikon, ECLIPSE Ts2)

  7. Confocal laser scanning microscope (e.g., OLYMPUS, FLUOVIEW FV3000)

  8. Humidified incubator set at 33 °C with 5% CO2/95% air

  9. Humidified incubator set at 37 °C with 5% CO2/95% air

  10. Automated cell counter (e.g., Logos Biosystems, catalog number: L10001SS)

  11. Fridge (4 °C) and freezers (-20 °C and -80 °C)

  12. Liquid nitrogen storage

  13. Electric pipettor (e.g., Greiner Bio-One, catalog number: J847050)

  14. Pipettes (e.g., HTL LAB SOLUTIONS, catalog number: 7904)

  15. Stirring bar (e.g., AS ONE Corporation, catalog number: 1-4206-27)

  16. Magnetic stirrer

  17. Autoclave (e.g., HIRAYAMA, HV-2LB)

Procedure

The HASTR/ci35, HBPC/ci37, and HBMEC/ci18 cell lines carry two immortalization genes: the temperature-sensitive simian virus 40 large tumor antigen (tsSV40T) and the human telomerase reverse transcriptase catalytic subunit (hTERT) genes. The tsSV40T promotes proliferation at 33 °C, while becoming unstable to lose its function at 37 °C. Therefore, for maintenance and differentiation purposes, the cells should be cultured at 33 °C and 37 °C, respectively. Please see our previous reports for the details (Kamiichi et al., 2012 ; Furihata et al., 2016; Kitamura et al., 2018; Umehara et al., 2018; Ito et al., 2019).


  1. Thawing cells

    Notes:

    1. Blasticidin S (final concentration (f.c.) 4 µg/mL) should always be added to each culture medium to maintain the expression of the immortalization genes.

    2. The cell handling procedure should always be performed inside a clean bench.

  1. HASTR/ci35 cells

    1. Warm the astrocyte medium (AM, see Recipe 1) in a water bath set at 37°C.

    2. Add 1 mL of AM into a 15-mL centrifuge tube.

    3. Remove the cryotube containing the frozen cells from the liquid nitrogen and immediately place it in a water bath set at 37°C.

      Note: Cells (0.5 × 106–1.0 × 106) at the logarithmic growth phase (70%–80% confluency) are recommended for frozen storage with 1 mL of Bambanker.

    4. Immediately begin thawing the frozen cells by gently shaking the cryotube in the water bath. However, remove the cryotube from the water bath before the frozen cells are completely thawed.

    5. Transfer the cells into a 15-mL centrifuge tube containing 1 mL of AM, and then perform centrifugation (120 × g) at room temperature for 3 min.

    6. Remove the supernatant using an aspirator and resuspend the cells with 5 mL of AM.

    7. Seed the cells into a collagen I-coated 60-mm dish and then gently shake the dish back and forth and from side to side, to ensure the cells disperse evenly.

    8. Check the cells in the dish under a microscope.

    9. Culture the cells in a humidified incubator at 33 °C with 5% CO2/95% air.

  2. HBPC/ci37 cells

    The handling procedure for HBPC/ci37 cells is essentially the same as that employed for HASTR/ci35 cells, except for the use of the pericyte medium (PM, see Recipe 2) instead of AM.

    Note: For frozen storage, we recommend the cell number of 0.7 × 106–1.0 × 106 in a cryotube with 1 mL of Bambanker.

  3. HBMEC/ci18 cells

    The handling procedure for HBMEC/ci18 cells is essentially the same as that employed for HASTR/ci35 cells, except for the use of VascuLife medium (see Recipe 3) instead of AM.

    Note: For frozen storage, we recommend the cell number of 0.8 × 106–1.0 × 106 in a cryotube with 1 mL of Bambanker.


  1. Passaging cells



    Figure 1. Cell confluency of the human immortalized BBB cells before and after the passaging.

    Upon reaching appropriate cell confluency (>90%) in a collagen I-coated 100-mm dish, as represented in (A), (B), and (C), HASTR/ci35, HBPC/ci37, and HBMEC/ci18 cells can be passaged with 6.0 × 105, 5.5 × 105, and 5.5 × 105 cells, respectively. The representative cell culture images captured one day after passaging are shown in (D), (E), and (F). The images were taken under a phase-contrast microscope (ECLIPSE Ts2, NIKON, Tokyo, Japan).


    Notes:

    1. * Avoid culturing at low cell density levels (you should always maintain confluency at more than 50%).

    2. * Avoid rough pipetting to prevent cell degeneration or loss of cell traits.

    3. * Blasticidin S (f.c. 4 µg/mL) should always be added to each culture medium to maintain the expression of the immortalization genes.

    4. All the above notes (marked with *) are critically important.

    5. The cell handling procedure should always be performed inside a clean bench.

    6. As an example, the procedure shown in this section is used for passaging cells from a collagen I-coated 100-mm dish into a new one.


  1. HASTR/ci35 cells

    1. Warm AM and D-PBS(-) in a water bath set at 37 °C.

    2. Prepare the cells in a collagen I-coated dish, as shown in Figure 1A.

    3. Remove the AM from the dish using an aspirator.

    4. Wash the cells once with 4–8 mL of D-PBS(-).

    5. After completely removing the D-PBS(-) using an aspirator, add 1 mL of trypsin (one-tenth of the medium volume) to the cells, and then incubate them at 37 °C for 5 min.

    6. Check the cells under a microscope to ensure they have detached from the dish.

    7. Add 1 mL of AM (the same amount of trypsin) to the cells to stop the trypsinization process while loosening any adhering cell lumps via gentle pipetting.

      Notes:

      1. Use of a 1,000 µL tip is recommended for cell pipetting. Take at least 3 s for each suction/discharge operation to avoid imparting excess stress on the cells.

      2. Alternatively, centrifugation can be used to remove the trypsin.

    8. Transfer an appropriate number of cells to a new collagen I-coated 100-mm dish, and then add fresh AM up to 10 mL.

      Note: Do NOT subculture the cells at densities of less than 6.0 × 105 cells in a 100-mm dish.

    9. Gently shake the dish back and forth and from side to side, to ensure the cells disperse evenly.

    10. Culture the cells in a humidified incubator at 33 °C with 5% CO2/5% air.

      Note: Passaging can be performed every three days when using a density of 6.0 × 105 cells in a 100-mm dish (Figure 1D).

  2. HBPC/ci37 cells

    Prepare the cells in a collagen I-coated dish as shown in Figure 1B. The handling procedure for HBPC/ci37 cells is essentially the same as that employed for HASTR/ci35 cells, except for the use of PM and 6 min trypsinization time instead of AM and 5 min, respectively.

    Notes:

    1. Passaging can be performed every four days when using a density of 5.5 × 105 cells in a 100-mm dish (Figure 1E). Do NOT subculture the cells at densities of less than this concentration.

    2. HBPC/ci37 cells are sometimes difficult to detach from collagen I-coated dishes even when following the trypsin treatment method described here. In such cases, try one of the following actions:

      1) Extend the trypsin treatment period up to 9 min.

      2) After 6 min of the first trypsin treatment, collect and transfer the detached cells from the dish into a 15-mL centrifuge tube. Immediately, add another 1 mL of trypsin solution to the dish, and allow it to stand for another 3 min. Then, collect the remaining cells and combine them with those removed earlier.

  3. HBMEC/ci18 cells

    Prepare the cells in a collagen I-coated dish as shown in Figure 1C. The handling procedure for HBMEC/ci18 cells is essentially the same as that employed for HASTR/ci35 cells, except for the use of VascuLife medium and 6 min trypsinization time instead of AM and 5 min, respectively.

    Notes:

    1. Passaging can be performed every four days when using a density of 5.5 × 105 cells in a 100-mm dish (Figure 1F). Do NOT subculture the cells at densities of less than this concentration.

    2. HBMEC/ci18 cells are often difficult to detach from collagen I-coated dishes even when following the trypsin treatment method described here. In such cases, follow the instructions provided in the Note for HBPC/ci37 cells.


  1. Construction of hiMCS-BBB models in a 96-well V-bottom plate



    Figure 2. A diagram of the hiMCS-BBB model construction process.

    This figure shows HASTR/ci35 cells seeded in a PrimeSurface 96V plate (Sumitomo Bakelite, Tokyo, Japan) at 1,750 (cells/well) on Day 0. On Day 1, HBPC/ci37 cells (500/well) are added over the HASTR/ci35 spheroid core. On Day 2, HBMEC/ci18 cells (750/well) are further added to each well. Finally, the spheroids cultured on Day 4 can be used for assays of interest.


    Notes:

    1. The cell handling procedure should always be performed inside a clean bench.

    2. Blasticidin S is not necessary in the Spheroid medium (see Recipe 4).

    3. All medium and D-PBS(-) should be pre-warmed in a water bath set at 37 °C.

    4. Only one spheroid should be formed in each well of a 96-well V-bottom plate.

    5. The spheroid development protocol described here is related to our report in 2022 (Kitamura et al., 2022).


    Day 0: HASTR/ci35 cell seeding
    1. Collect the HASTR/ci35 cells from the dish using the same procedure shown in steps B1c–B1g, and transfer them into a 15-mL centrifuge tube.

    2. Centrifuge (500 × g) the tube at room temperature for 3 min.

    3. Remove the supernatant using an aspirator and resuspend the cells with 1 mL of D-PBS(-).

    4. Repeat step 2.

    5. Remove the supernatant using an aspirator and resuspend the cells with 1 mL of the Spheroid medium.

    6. Mix 20 µL of the cell suspension with 20 µL of trypan blue in a 1.5 mL tube.

    7. Apply 10 µL of the trypan blue-mixed cell suspension to a cell counting slide, and count the cell number using an automated cell counter.

    8. Prepare 3.5 × 104 cells/mL (for use at 1,750 cells/50 µL/well) of the cell suspension with the Spheroid medium. (The preparation volume should be calculated based on the number of wells needed.)

    9. Seed 50 µL of the cell suspension to each well of a 96-well V-bottom plate.

      Note: Do not use the outermost wells because the medium in these wells is susceptible to evaporation.

    10. Add 100–200 µL of D-PBS(-) to each of the outermost wells of the 96-well V-bottom plate.

      Note: This step is important to prevent evaporation of the medium.

    11. Place the 96-well V-bottom plate in a humidified incubator set at 37 °C with 5% CO2/95% air.

      Note: A single spheroid will appear.


    Day 1: HBPC/ci37 cell seeding

    1. The seeding procedure for HBPC/ci37 cells is essentially the same as the above-described steps 1–9 and 11, except that the cell concentration for seeding should be set at 1.0 × 104 cells/mL (500 cells/50 µL/well).

      Note: The cell suspension can be added directly to the wells where HASTR/ci35 cells exist. The medium change is not always necessary.


    Day 2: HBMEC/ci18 cell seeding
    1. The seeding procedure for HBMEC/ci18 cells is essentially the same as the above-described steps 1–9, except that the cell concentration for seeding should be set at 1.5 × 104 cells/mL (750 cells/50 µL/well).

      Note: The cell suspension can be added directly to the wells where HASTR/ci35 and HBPC/ci37 cells exist. The medium change is not always necessary.

    2. Place the 96-well V-bottom plate in a humidified incubator set at 37 °C with 5% CO2/95% air, and incubate it for 48 h.

      Note: Spheroids can be kept in culture at least for 48–72 h after HBMEC/ci18 cells are seeded. The medium change is not always necessary during the culture period but may be carried out depending on your assays of interest.


    Note: When conducting a cell localization assay, HASTR/ci35, HBPC/ci37, and HBMEC/ci18 cells can be dyed with something appropriate [e.g., CellTracker Orange CMTMR Dye (541/565 nm), Red-fluorescent Cytoplasmic Membrane Staining Kit (644/665 nm), and CellTracker Green CMFDA Dye (492/517 nm), respectively, as shown in Figure 3]. In each case, follow the original protocol provided for each product.



    Figure 3. Localization of the human immortalized BBB cells in the hiMCS-BBB models.

    These images, which were captured under a confocal laser scanning microscope (FLUOVIEW FV3000, OLYMPUS, Tokyo, Japan), show localization of HASTR/ci35, HBPC/ci37, and HBMEC/ci18 cells in the hiMCS-BBB models. HASTR/ci35 cells are accumulated at the internal core of the model, over which HBPC/ci37 cells form a layer. At the outmost side of the spheroid, HBMEC/ci18 cells create a layer that functions as the BBB. The scale bar indicates 100 µm.


  2. Permeability assay

    Notes:

    1. The series of assay procedures should be performed without stopping.

    2. The procedure for permeability assays using Alexa Fluor 647-labeled MEM189 antibodies (MEM189) is shown below as an example. The underlined parts can be changed appropriately depending on the substance of interest (see Table 1).

    3. Whenever transferring the spheroids, gently place the tip vertically into the bottom of the well or the tube to suck up the spheroids. Always use tips that have been cut at approximately 7 mm from the top of the thinner side so that the entrance hole has a diameter of around 1 mm, to prevent damage to the spheroids.


      Table 1. A summary of experimental conditions used in our permeability assays

      Substrate Final concentration Incubation
      MEM189/13E4 10 µg/mL 180 min
      Lucifer Yellow 5 µM 90 min
      2-NBDG 100 µM 90 min
      Rhodamine123 1 µM 90 min
      Insulin 100 nM 120 min
      Transferrin 100 nM 120 min
      SLS peptide/DNP peptide 10 µM 60 min

      For details, please refer to our previous papers (Kitamura et al., 2021, 2022).


      1. Using a low binding 200 µL tip, transfer 10–20 spheroids from the 96-well V-bottom plate to a Protein LoBind 1.5 mL tube.

      2. Carefully remove the excess supernatant using a pipette until a medium volume of approximately 15 µL remains. Then, allow the tube to stand before the spheroids sink completely to the bottom (Figure 4A).

      3. Wash the spheroids with 500 µL of HBSS(+).

      4. Carefully remove the supernatant using a pipette. Then add HBSS(+) containing MEM189 (f.c. 10 µg/mL) into the tube, until a final total volume of approximately 100 µL is reached.

      5. After placing the tube horizontally to disperse the spheroids, incubate for 180 min at 37 °C or refrigerate at 4 °C (Figure 4B).

      6. Wash the spheroids twice with 500 µL of ice-cold D-PBS(+) (see Recipe 5) as described in steps 2 and 3.

      7. After carefully removing the supernatant using a pipette, incubate the spheroids with 500 µL of 4% PFA at room temperature for 10 min (fixation treatment).

      8. Wash the spheroids twice with 500 µL of ice-cold D-PBS(+), as described in steps 2 and 3.

      9. Using a low binding 200 µL tip, suck the spheroids together with the remaining D-PBS(+) (approximately 10–20 µL) from the tube.

      10. Place a piece of parafilm (from which a square of 6–8 mm on a side has been removed) on a slide glass. The empty area is to provide a protected space for the spheroids to minimize damage (Figure 4C).



        Figure 4. Reference images for the section “D. Permeability assay”.

        Image (A) indicates the amount (approximately 15 µL) of supernatant remaining in a Protein LoBind 1.5 mL tube after step 2. Image (B) shows the position of a Protein LoBind 1.5 mL tube placed horizontally on a table to disperse the spheroids as in step 5. Image (C) shows an example of a slide glass topped by a piece of parafilm with square windows cut out for holding antifade–spheroid mixture, as in steps 10 and 11.


      11. After placing a drop of antifade reagent at the center of the empty square in the glass slide-mounted parafilm sheet, mix the spheroids with the antifade reagent and disperse them into the opening via gentle pipetting.

      12. Place a cover glass on top of the slide and seal it with nail polish.

        Note: To prevent damage to the spheroids, avoid pressing down on the cover glass too firmly.

      13. Immediately detect the fluorescence by observing the spheroids under a confocal microscope (650/665 nm in case with Alexa Fluor 647-labeled MEM189) (Figure 5).



        Figure 5. Permeability assay of anti-transferrin receptor antibody MEM189.

        These images show the representative results of the permeability assays performed using Alexa Fluor 647-labeled MEM189 (MEM189, 10 µg/mL) and its isotype control IgG (CTRL IgG, 10 µg/mL) conducted at 37 °C (RMT-active condition) or 4 °C (RMT-inactive condition). The blank views show spheroids incubated without any antibodies. The scale bar indicates 100 µm. An overview of MEM189 entering the spheroids is also shown.

    Recipes

    Note: All the complete medium prepared here should be stored at 4 °C before use. Please follow the storage guideline provided by each supplier.


    1. AM (astrocyte medium)

      1. Add N-2 supplement and FBS, which are included in the Astrocyte Medium kit, to the basal astrocyte medium.

      2. Add penicillin-streptomycin at 1% (v/v) (e.g., Nacalai Tesque, as shown at No. 25 in the material section) to the above-prepared medium.

    2. PM (pericyte medium)

      Add FBS, PGS, and P/S solution, which are included in the Pericyte Medium kit, to the basal pericyte medium.

    3. VascuLife medium

      1. Add rh FGF-b, ascorbic acid, hydrocortisone, FBS, L-glutamine, rh IGF-1, rh EGF, rh VEGF, and heparin, all of which are included in the VascuLife VEGF Comp kit, to the VascuLife basal medium.

      2. Add penicillin-streptomycin at 1% (v/v) (e.g., Nacalai Tesque, as shown in the material section at No. 25) to the above-prepared medium.

        Note: Although we do not routinely use, gentamicin-amphotericin provided by the medium supplier may be alternatively used.

    4. Spheroid medium

      1. After adding 1.2 g of methyl cellulose (viscosity: 4,000 cP) and a stirring bar to a 100-mL flask, sterilize the flask in an autoclave.

      2. Add 100 mL of VascuLife medium to the above-sterilized 100-mL flask.

      3. Incubate the flask in a water bath set at 60 °C for 20 min.

      4. Completely dissolve the methyl cellulose by rotating the stirring bar in the flask for an hour.

      5. Aliquot 100 mL of the methyl cellulose solution into two 50-mL centrifuge tubes.

      6. Centrifuge (2,500 × g) the two tubes at room temperature for 30 min.

      7. Transfer the supernatant into a sterilized container. This solution is referred to as the methyl cellulose stock solution.

      8. Prepare the Spheroid medium containing 0.48 mg/mL of methyl cellulose by mixing the methyl cellulose stock solution together with the VascuLife medium at a ratio of 1:24.

      Note: To ensure spheroids with good reproducibility, use a freshly-prepared Spheroid medium whenever possible. The Spheroid medium should not be used if it is more than three weeks old. To create an additional supply of Spheroid medium, follow the procedure above starting from step h.

    5. D-PBS(+)

      Add D-PBS(+) preparation reagent (Ca, Mg solution) [1%(v/v)] to D-PBS(-).

    Acknowledgments

    This work was supported by grants from JSPS KAKENHI (19K07214, 22Kxxxxx), Eisai (Tokyo, Japan), Ono Pharmaceuticals (Osaka, Japan), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Tokyo, Japan), and partly by AMED under grant no. JP17be0304322h0001. The authors would like to express our sincere appreciation to Dr. Takafumi Komori (Eisai), Dr. Saki Izumi (Eisai), Dr. Yoshiyuki Yamaura (Ono Pharmaceuticals), and Dr. Ryo Ito (Ono Pharmaceuticals) for their kind supports. This protocol is essentially derived from Kitamura et al. (2022).

    Competing interests

    Conflict of interest statements related to research funds are provided in the acknowledgments section, and the model development method herein has been applied for a patent (No. 2020-007041). There is another related patent application (No. 2020-065670). The authors declare that they do not have any other conflicts of interest.

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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Isogai, R., Morio, H., Okamoto, A., Kitamura, K. and Furihata, T. (2022). Generation of a Human Conditionally Immortalized Cell-based Multicellular Spheroidal Blood-Brain Barrier Model for Permeability Evaluation of Macromolecules. Bio-protocol 12(15): e4465. DOI: 10.21769/BioProtoc.4465.
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