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

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.

   
   

2. 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.

      
      

3. 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

   12. 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

   13. 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.

   14. 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.

   
   

4. 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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