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May 2020
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High-throughput 3D Spheroid Formation and Effective Cardiomyocyte Differentiation from Human iPS Cells Using the Microfabric Vessels EZSPHERETM
利用微细血管EZSPHERETM从人类iPS细胞中进行高通量三维球状细胞形成和有效的心肌细胞分化   

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Abstract

High-throughput 3D spheroid formation from human induced pluripotent stem cells (hiPSCs) can be easily performed using the unique microfabric vessels EZSPHERE, resulting in effective and large scale generation of differentiated cells such as cardiomyocytes or neurons. Such hiPSC-derived cardiomyocytes (hiPSC-CMs) or neurons are very useful in the fields of regenerative medicine or cell-based drug safety tests. Previous studies indicated that 3D spheroids arising from hiPSCs are effectively differentiated into high quality hiPSC-CMs by controlling Wnt signals through utilization of the microfabric vessels EZSPHERE. Here, we describe a simple and highly efficient protocol for generating a large number of uniformly sized hiPSC spheroids and inducing them for cardiac differentiation using the EZSPHERE. This method comprises the collection and dissociation of the spheroids from cardiac differentiation medium, in the middle stage of the whole cardiac differentiation process, and re-seeding the obtained single cells into the EZSPHERE to re-aggregate them into uniform hiPSC-CM spheroids with controlled size. This re-aggregation process promotes non-canonical Wnt signal-related cardiac development and improves the purity and maturity of the hiPSC-CMs generated.


Graphic abstract:


Overview of cardiac differentiation from iPSCs by spheroid formation and reaggregation using EZSPHERE.


Keywords: 3D spheroids (三维球状体), Human induced pluripotent stem cells (hiPSCs) (人类诱导性多能干细胞), Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (人类诱导性多能干细胞衍生的心肌细胞 ), Cardiomyocyte differentiation (心肌细胞分化), Microfabric vessels (微结构血管), EZSPHERE (EZSPHERE), Cell aggregates (细胞团), Spheroid re-aggregation (球状体再聚集 ), Regenerative medicine (再生医学 ), Drug safety test (药物安全测试)

Background

Human induced pluripotent stem cell-derived Cardiomyocytes (hiPSC-CMs) are expected to become a cell source for applications in regenerative medicine of heart diseases and drug discovery. Heart diseases remain the leading cause of death worldwide (WHO, 2018). In non-human primates, transplanted hiPSC-CMs improve cardiac contraction function and are sufficient to regenerate the infarcted heart (Shiba et al., 2016). Previous studies, represented by the CiPA Project (Blinova et al., 2017) and the JiCSA Project (Yamazaki et al., 2018), have shown the utility of hiPSC-CMs for prediction of the risk of cardiotoxicity, like Torsades de Pointes. It was also shown that maturity of the generated hiPSC-CMs alters the response to drugs (da Rocha et al., 2017). Thus, the development of a high-throughput and robust protocol for generating high-quality hiPSC-CMs is urgent for the realization of regenerative medicine and drug discovery.


hiPSCs could be directly differentiated into cardiomyocytes in either 2D adhesion culture or 3D cell aggregates/spheroid culture methods. In particular, the spheroid culture method is easier to scale-up and better at promoting maturation of the generated hiPSC-CMs, compared with the 2D culture method (Pal et al., 2013). In the cardiac differentiation process, regulation of canonical and non-canonical Wnt at the right time strongly promotes pluripotent stem cell differentiation into cardiomyocytes, not other mesodermal cell types (Loh et al., 2016). In addition, expression levels of the Wnt signal-related genes Wnt5A and Wnt11 are affected by spheroid size (Hwang et al., 2009, Chen et al., 2015). Therefore, we sought to develop a highly efficient and robust cardiac differentiation protocol by achieving high-throughput spheroid formation with simultaneous active spheroid size control.


In this study, we used the uniquely shaped microfabric dishes, “EZSPHERE,” designed for high-throughput production of uniform spheroids on one culture dish, with its high-density micro-wells (Sato et al., 2016). Initially, a basic spheroid culture protocol, optimized for cardiac differentiation (Yang et al., 2008, Bauwens et al., 2014, Kempf et al., 2014), was combined with the EZSPHERE dishes. For spheroid formation from the hiPSCs, Type 900 EZSPHERE dishes with a diameter of 100 mm were used. Each of them had approximately 14,000 individual micro-wells with regular size (400-500 μm in diameter and approximately 100 μm in depth). On calculation, approximately 14,000 spheroids could be formed in each EZSPHERE dish, and their initial seeding cell number per spheroid ranged 200-220. As per standard protocol, activin A, BMP-4, and FGF2 were added to the spheroids in the EZSPHERE to induce differentiation into Primitive Streak on day 1. On day 4, the spheroids were collected, washed, and transferred onto a flat bottom low cell adhesion dish, followed by treatment with Wnt inhibitor. On day 6, the size uniformity of the spheroids was lost, but 62% of the cell population had differentiated into PDGFR-α/KDR double-positive cardiac mesoderm-like cells (Kattman et al., 2011; Birket et al., 2015). To control spheroid size once more, the differentiated spheroids were collected and dissociated into single cells. Then, the single cells obtained were re-aggregated by re-seeding onto the dishes of Type 903 EZSPHERE (with a diameter of 35 mm), which have approximately 1,000 individual micro-wells with larger size (approximately 800 μm diameter and 400 μm depth) in each dish. The re-aggregated spheroids increased the number of cardiac Troroponin T (CTNT) positive cells faster than that of the non-reaggregated spheroids. In addition, the cardiac maturity (estimated by the gene expression level of MYL2 per MYL7) was much higher in the re-aggregated spheroids compared with that of the spheroids that did not undergo the re-aggregation process. Performing the re-aggregation process also raised the expression levels of the non-canonical Wnt signals Wnt5A and Wnt11. In mice, Wnt5A and Wnt11 are essential for the generation of second heart field progenitor cells (Cohen et al., 2012). Wnt11 is also known for its role in generating myocardial electrical gradient patterns through the regulation of non-canonical Wnt/Ca2+ signaling during zebrafish heart development (Panakova et al., 2010). To reveal the detail of its molecular mechanism, further study is required. However, these data strongly suggest that the cardiomyocyte differentiation process performed on EZSPHERE, including a re-aggregation step for the induced cardiac mesoderm/progenitor, is effective in producing cardiomyocytes with high purity and maturation levels.

Materials and Reagents

  1. EZSPHERE Dish 100 mm Type 900 (AGC Techno Glass, IWAKI, catalog number: 4020-900)

  2. EZSPHERE Dish 35 mm Type 903 (AGC Techno Glass, IWAKI, catalog number: 4000-903)

  3. EZ-BindShut II 100 mm Dish (AGC Techno Glass, IWAKI, catalog number: 4020-800LP)

  4. Cell culture T25 Flask (AGC Techno Glass, IWAKI, catalog number: 3100-025)

  5. 5 ml Pipet (AGC Techno Glass, IWAKI, catalog number: 7103-005)

  6. Cell scraper (AGC Techno Glass, IWAKI, catalog number: 9010-320)

  7. 70 μm cell strainer (Corning, Falcon, catalog number: 352350)

  8. Pasteur pipettes (AGC Techno Glass, IWAKI, catalog number: IK-PAS-9P)

  9. Human induced pluripotent stem cell (hiPSC) strains 253G1 and 201B7 (iPS Academia Japan)

  10. Maintenance culture media mTeSR1 (Stemcell Technologies, catalog number: 5850)

  11. Biolaminin 521 LN (LN521) (BioLamina, catalog number: LN521-03)

  12. DPBS (FUJIFILM Wako Pure Chemical, Wako, catalog number: 045-29795)

  13. HBSS, no calcium (Thermo Fisher Scientific, Gibco, catalog number: 14170112)

  14. DMEM (4.5g/L Glucose) with L-Gln, without Sodium Pyruvate, liquid (Nacalai, catalog number: 08459-35)

  15. 0.5 mol/L-EDTA solution (pH 8.0) (Nacalai, catalog number: 06894-14)

  16. Fetal bovine serum, FBS (MP Biomedicals, CELLECT, catalog number: 2916154)

  17. Accutase (Merck, Sigma-Aldrich, catalog number: A6964-100ML)

  18. AccuMax (Innovative Cell Technologies, catalog number: AM105)

  19. TrypLE select Enzyme (1×), no phenol red (Thermo Fisher Scientific, Gibco, catalog number:12563011)

  20. StemProTM-34 SFM (1×) (Thermo Fisher Scientific, Gibco, catalog number: 10639011)

  21. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco, catalog number: 15140122)

  22. L-Glutamine (200 mM) (Thermo Fisher Scientific, Gibco, catalog number: 25030081)

  23. holo-Transferrin human (Merck, Sigma-Aldrich, catalog number: T0665-100MG)

  24. L-Ascorbic acid (Merck, Sigma-Aldrich, catalog number: A92902-100MG)

  25. 1-Tioglycerol (Merck, Sigma-Aldrich, catalog number: M6145-25ML)

  26. ROCK inhibitor Y-27632 (Wako Pure Chemical Industries, Wako, catalog number: 253-00513)

  27. Recombinant Human BMP-4 Protein (Bio-Techne, R&D Systems, catalog number: 314-BP-010)

  28. Fibroblast Growth Factor (basic), Human, recombinant (154 aa) (FUJIFILM Wako Pure Chemical, Wako, catalog number: 064-04541)

  29. Recombinant Human VEGF 165 Protein (Bio-Techne, R&D Systems, catalog number: 293-VE-010)

  30. Recombinant Human/Mouse/Rat Activin A Protein (Bio-Techne, R&D Systems, catalog number: 338-AC-010)

  31. Stemolecule Wnt Inhibitor IWP-4 (REPROCELL USA, Stemgent, catalog number: 04-0036)

  32. DNase I, Bovine Pancreas (Merck, Millipore, catalog number: 260913)

  33. 30 w/v% Albumin D-PBS (-) Solution, from Bovine Serum (BSA), Fatty Acid Free (FUJIFILM Wako Pure Chemical, Wako, catalog number: 015-23871)

  34. 4% Paraformaldehyde Phosphate Buffer Solution (Wako Pure Chemical Industries, Wako, catalog number: 163-20145)

  35. Antibodies used in flow cytometry analysis (Table 1)


    Table 1. Antibodies used in flow cytometry analysis

    Antibodies Distributor   Cat. No.       Dilution ratio
    APC-KDR BioLegend   359915       2.5:100
    APC-Mouse IgG1, kappa Isotype control BioLegend   400121       2.5:100
    PE-PDGFRα BioLegend   323505       5:100
    PE-Mouse IgG1, kappa Isotype control BioLegend   400113       5:100
    PE anti-cardiac Troponin T BD Pharmingen   564767       2.5:100
    PE-Isotype control BD Pharmingen   554680       2.5:100


  36. Cell culture Reagents (see Recipes)

  37. Cytokines and small molecular compounds (see Recipes)

  38. Modified StemPro-34 (see Recipes)

  39. Spheroid formation medium (see Recipes)

  40. Stage-1 medium (2×) (see Recipes)

  41. Stage-2 medium (see Recipes)

  42. Stage-3 medium (see Recipes)

Equipment

  1. Laminar flow hood (Hitachi, model: SCV-Class II type A/B)

  2. Water bath (AS ONE Corporation, model: TR-1AR)

  3. TC10 Automated Cell Counter (Bio-Rad, catalog number: 145-0001)

  4. EVOS FL Auto Cell Imaging System (Life Technologies, EVOS, catalog number: AMAFD1000)

  5. EVOS Onstage Incubator (Life Technologies, EVOS, catalog number: AMC1000)

  6. BD FACSVerse flow Cytometer (BD Bioscience, catalog number: 651154)

  7. Pipet-Aid XPress 110V (Drummond Scientific Company, catalog number: 4-040-135)

  8. Centrifuge (Himac, CF16RX, model: S101812)

  9. Vortex mixer Vortex Gwnie 2 (Scientific Industries, Inc., catalog number: SI-0236)

  10. HERAcell CO2 incubator (Kendro Laboratory Products)

  11. Vacuum pump (Model JN726 FT.18)

Software

  1. ImageJ (NIH, https://imagej.nih.gov/ij/)

  2. AGDRec AG-Desktop recorder (AmuseGraphics, http://t-ishii.la.coocan.jp/hp/ag/index.html)

  3. FlowJo software (FlowJo LLC, https://www.flowjo.com/)

Procedure

Note: Perform the culture operation in a biosafety level 2 laboratory in a dedicated laminar flow hood. All waste (tubes, tips, plates) should be inactivated by autoclaving and then properly disposed of according to the rules of your facility.


  1. High-throughput spheroid formation from hiPSCs

    1. Culture the hiPSCs in the medium mTeSR1 in Flasks coated with Biolaminin 521 LN.

      Note: Subculture the hiPSCs as single cells, not cell clumps, without adding the ROCK inhibitor.

    2. Prepare Spheroid formation medium (see Recipes: Table 5) and pre-warm at 37°C in a water bath.

    3. Use 90% confluent hiPSCs.

    4. Aspirate medium from the cells and wash with 2.5 ml of room temperature DPBS. For aspiration, use the vacuum pump with the pipette and an appropriate tube.

    5. Add 2.5 ml of TrypLE select and incubate at 37°C for 4 min.

    6. Aspirate TrypLE select and add 2.5 ml of Spheroid formation medium.

    7. Detach the cells by flushing the medium to the cells using the Pipet-Aid with a 5 ml pipette. If the cells do not detach, scrape them with a Cell Scraper.

    8. Flush the cells 2-8 times by pipetting, to dissociate the cell clumps into single cells, and collect them into a 15 ml centrifuge tube.

    9. Centrifuge at 100 × g for 4 min at 20°C.

    10. Aspirate supernatant and resuspend pellet with 1-5 ml of Spheroid formation medium.

    11. Measure cell density using the TC10 Automated Cell Counter.

    12. Dilute cells to the density of 3 × 105 cells/ml with Spheroid formation medium, and inoculate 10 ml of the suspended cells to the EZSPHERE Dish 100 mm Type 900 (see Figure 1).

    13. Place the EZSPHERE Dish in a 37°C, 5% CO2 incubator and culture for 24 h.



      Figure 1. Images of the microfabric vessels “EZSPHERE” with a dish type


  2. Stage-1 of cardiac differentiation

    1. Move on to Stage-1 of the cardiac differentiation process, 24 ± 2 h after spheroid formation.

    2. Prepare Stage-1 medium (2×) (see Recipes: Table 6) and pre-warm at 37°C in a water bath.

    3. Take out the EZSPHERE dish from the CO2 incubator and check if spheroids have formed in each micro-well of the EZSPHERE, under a phase contrast microscope.

    4. Carefully add an equal volume (10 ml) of Stage-1 medium (2×) to the spheroids.

      Note: We recommend taking 10 ml of Stage-1 medium (2×) with a 10 ml pipette, placing the tip of the pipette on the liquid surface around the center of the EZSPHERE dish, standing the pipette vertically, and pouring the medium carefully.

    5. Return the EZSPHERE dish to the 37°C, 5% CO2 incubator, and culture for 89 h.


  3. Stage-2 of cardiac differentiation

    1. Move on to Stage-2 of the cardiac differentiation process, 89 ± 2 h after the Stage-1 mentioned above.

    2. Prepare Stage-2 medium (see Recipes: Table 7) and IMEM medium, then pre-warm at 37°C in a water bath.

    3. Harvest spheroids from EZSPHERE dish into a 15 ml or 50 ml tube.

      (Optional)

      1. Transfer spheroids to a 100-mm flat bottom dish and place on the stage of the microscope.

      2. Shake the dish finely in a circular motion to place the spheroids in the center of the dish.

      3. Take a picture of spheroids with the microscope (see Figure 2).

      4. Measure the size of spheroids using the ImageJ software (see Data analysis A).



      Figure 2. Images and size of the hiPSC spheroids generated at Procedure C. Spheroids on the EZSPHERE dish (A) and transferred onto a flat bottom dish (B). Histogram of the size (diameter) of the spheroids (μm) (C).


    4. Loosen the cap of the tube for ventilation, and place the tube in the 37°C CO2 incubator for 10 min to precipitate the spheroids.

    5. Carefully remove the supernatant from spheroids as much as possible using a pipette, and add 5 ml of pre-warmed IMEM medium to wash the spheroids.

    6. Centrifuge spheroids at 50 × g for 3 min at 20°C, removing the supernatant as much as possible.

    7. Add 20 ml of pre-warmed Stage-2 medium to spheroids, and transfer spheroids to a 100-mm low cell adhesion dish (EZ-BindShut II).

    8. Place the dish into the 37°C CO2 incubator.

      Note: We recommend shaking the dish three times back and forth and left and right to distribute the spheroids evenly in the dish.

    9. Shake the dish every morning and evening as described above to equally disperse the spheroids.


  4. Stage-3 of cardiac differentiation and re-aggregation of spheroids

    Note: The re-aggregation of spheroids increases the purity in the cardiac differentiation process. As a negative control, do not re-aggregate the spheroids at this step. To confirm the effect of re-aggregation, analyze the purity of CTNT positive iPS-CMs by flow cytometry after Procedure G.

    1. On day 6 of cardiac differentiation, move on to Stage-3 for re-aggregation of the spheroids.

      Note: While it is recommended that this re-aggregation step be performed on day 6, day 7 is also acceptable.

    2. Prepare Stage-3 medium (see Recipes: Table 8) and pre-warm at 37°C in a water bath.

    3. Collect spheroids from the 100-mm EZ-BindShut II dish into a 50 ml tube and place it in the 37°C CO2 incubator for 10 min to precipitate the spheroids.

    4. Remove supernatant from the collected spheroids and add 5 ml of room temperature DPBS to wash them.

    5. Centrifuge at 50 × g for 3 min at 20°C and remove the supernatant.

      Note: For a negative control, skip Steps D6-D9 and proceed to Step D10.

    6. Add 1 ml of Accutase to the spheroids and incubate at 37°C in the water bath for 8 min to dissociate the spheroids into single cells. During this incubation, mix the cells vigorously by vortexing the tube for 10 s every 4 min.

    7. If cell aggregates remain after 8 min of incubation, as mentioned above, enforce additional incubation: collect only the remaining cell aggregates from single dissociated cells using a pipette and transfer to a new tube. Then, add double the amount (2 ml) of Stage-3 medium to the single dissociated cells and add 1 ml of Accutase to the aggregates. Incubate the aggregates at 37°C in the water bath for 8 min again. During the incubation, mix the cells vigorously, as mentioned above.

    8. Add double the amount (2 ml) of Stage-3 medium to the single dissociated cells in Accutase, centrifuge at 200 × g for 4 min at 20°C, and then remove supernatant.

    9. Resuspend the collected cell pellet with 5 ml of Stage-3 medium, and count the number of cells with diameter ranging 8-27 μm. If cell aggregates remain, remove them using a 70 μm Cell Strainer.

    10. Dilute the cells with Stage-3 medium to the density of 1 × 105 cells/ml and inoculate 3 × 105 cells into a 35-mm EZSPHERE Type 903 dish.

      Note: For a negative control, add 20 ml of Stage-3 medium to the spheroids obtained from the one 100-mm EZ-BindShut II dish at step 5 and dispense 3 ml each into 35-mm EZ-BindShut II dish.

    11. Change half volume of medium every 2 or 3 days.


  5. Harvest cardiomyocyte differentiated cells

    1. Around day 10 to 14 of the cardiac differentiation process, harvest the obtained hiPSC-CMs or negative control.

    2. Prepare 4 ml of dissociation buffer (0.05% Trypsin-EDTA:AccuMax = 3:1) and 4 ml of quench buffer (DMEM:FBS 1:1 + 200 units ml-1 of DNase I).

    3. Observe the beating state of the cardiac spheroids under the microscope (see Videos 1 and 2).

      Note: Beating of the cardiac spheroids might decay as the EZSPHERE dish grows colder at room temperature. Therefore, we recommend using an on-stage incubator to keep the dish warm at 37°C during microscopic observation. The EVOS FL Auto Cell Imaging System and EVOS on-stage incubator were used for observation, and an AGDRec AG-Desktop recorder was used for capturing videos.


      Video 1. Representative video of the cardiac spheroids re-aggregated in the micro-wells of EZSPHERE. The spheroids were re-aggregated on day 6 and began beating from day 8. This video was recorded on day 14.


      Video 2. Video of a population of beating cardiac spheroids recovered after the re-aggregation process. The cardiac spheroids were transferred from the EZSPHERE dish to a flat dish and then videotaped as they were beating.

    4. Collect the spheroids into a 15 ml tube and let stand for 2-3 min until they settle.

    5. Remove supernatant and add 5 ml of DPBS to wash the spheroids.

    6. Centrifuge the tube at 50 × g for 3 min and remove the supernatant, taking care not to lose the spheroids.

    7. Add 1 ml of dissociation buffer to the collected spheroids and incubate at 37°C in the water bath for 8 min, to dissociate the spheroids into single cells. During the incubation, mix the cells vigorously, as mentioned above.

    8. If cell aggregates still remain after 8 min of incubation, enforce an additional incubation step. Collect only the remaining cell aggregates from single dissociated cells using a pipette and transfer into a new tube. Then, add equal volume (1 ml) of quench buffer to the dissociated cells and add 1 ml of dissociation buffer to the aggregates. Incubate the aggregates again at 37°C in the water bath for 8 min. During the incubation, mix the cells vigorously, as mentioned above.

    9. Add equal volume (1 ml) of quench buffer to the dissociated cells.

    10. Centrifuge the tube at 200 × g for 5 min and remove the supernatant by careful pipetting. Do not use an aspirator to prevent the inhalation of cell aggregates entwined with some broken cell-derived DNA.

    11. For cytometry analysis, resuspend the harvested cells with DPBS. For cultivation in the next step, resuspend the cells with DMEM containing 10% FBS.

    12. Count the number of cells within 8-30 μm in diameter.


  6. Flow cytometry analysis of Cardiac Mesoderm.

    1. Use the cells harvested on day 6 (Step D8).

    2. Prepare two tubes (#1 and #2) and transfer 0.5 × 106 cells into each tube respectively for cell staining and its isotype control.

    3. Prepare 20 ml of flow cytometry buffer [HBSS (-), 3% FBS, 0.03 mM EDTA].

    4. Add 2 ml of flow cytometry buffer to the cells.

    5. Centrifuge the cells at 300 × g for 5 min at 20-25°C and aspirate supernatant.

    6. Repeat Steps F4-F5.

    7. Resuspend 0.5 × 106 cells in 100 μl of flow cytometry buffer.

    8. Add APC-KDR and PE-PDGFRα antibodies into tube #1 for cell staining, and add APC- and PE-isotype control into tube #2 for isotype control (Table 1).

    9. Incubate each tube at room temperature for 45 min in the dark.

    10. Add 2 ml of flow cytometry buffer into each tube.

    11. Centrifuge the cells at 300 × g for 5 min at 20-25°C and aspirate the DPBS.

    12. Repeat Steps F10-F11.

    13. Resuspend the cells in 300 μl of flow cytometry buffer.

    14. Measure the cells using the flow cytometer (BD FACS Verse) and analyze the data using the FlowJo software (Figure 3).



      Figure 3. Representative flow cytometry profile of iPSC-derived cardiac mesoderm on day 6. Events were acquired using the flow cytometer (BD FACS Verse) and analyzed with the FlowJo software. First, the major population was selected by gating in the SSC vs. FSC dot plots (A). Histograms are shown for each antibody: APC-KDR (B) and PE-PDGFRα (C). The plots show isotype control (open histogram) versus stained sample (red histogram). The main population present on day 6 is KDR and PDGFRα positive (5.7% of the cells were KDRhigh, 71.7% of the cells were KDRlow, and 86.7% PDGFRα+). This result suggests that the main population present on day 6 is considered as cardiac mesoderm-like cells.


  7. Flow cytometry analysis of the hiPSC-derived cardiomyocytes (hiPSC-CMs)

    1. Use 1 × 106 of the differentiated or negative control cells obtained in Step E10.

      Note: If the cells were harvested in the medium, wash them with DPBS.

    2. Centrifuge the cells at 300 × g for 5 min at 20-25°C and aspirate the supernatant.

    3. Add 200 μl of 4% paraformaldehyde phosphate buffer solution and incubate at room temperature for 20 min to fix the cells.

    4. Add 2 ml of DPBS to the cells and centrifuge at 300 × g for 5 min at 20-25°C.

    5. Remove the supernatant containing paraformaldehyde solution and discard it into an appropriate waste bottle.

    6. Resuspend the cell pellets in 2 ml of DPBS. The cells could be stored at this step for up to 7 days.

    7. Centrifuge the cells at 300 × g for 5 min at 20-25 °C and aspirate the supernatant.

    8. Resuspend the cell pellets in 200 μl (0.5 × 106 cells per 100 μl) of Flow Cytometry Permeabilization/Wash Buffer I and incubate at room temperature for 30 min.

    9. Transfer 100 μl (0.5 × 106 cells) of resuspended cells into one well of a 96-well round-bottom plate as a staining sample. Transfer remaining 100 μl (0.5 × 106 cells) of resuspended cells into one well of another 96-well round-bottom plate as an isotype control.

    10. Add 2.5 μl of PE anti-cardiac Troponin T antibodies to the sample and 2.5 μl of PE-isotype control antibodies to the isotype control (Table 1) and incubate at room temperature for 30 min in the dark.

    11. Add 100 μl of Flow Cytometry Permeabilization/Wash Buffer I to each well and shake the plate gently for 5 min at room temperature in the dark.

    12. Centrifuge the plates at 300 × g for 5 min at 20-25°C and remove the supernatant by decanting or pipetting.

    13. Carefully add 200 μl of Flow Cytometry Permeabilization/Wash Buffer I to each plate without breaking the cell pellets.

    14. Centrifuge the plates at 300 × g for 5 min at 20-25°C and remove the supernatant of each plate by decanting or pipetting.

    15. Repeat Steps G13-G14.

    16. Resuspend the cell pellets with 200 μl of DPBS in each plate and mix well.

    17. Measure the cells in each plate with the flow cytometer (BD FACSVerse) and analyze the data using FlowJo software (Figure 4).



      Figure 4. Representative flow cytometry profile of hiPSC-CMs. Events were acquired using the flow cytometer (BD FACS Verse) and analyzed with the FlowJo software. First, the major population was selected by gating in the SSC vs. FSC dot plots (A). The histograms show the Cardiac Troponin T positive hiPSC-CM populations for no re-aggregated negative control (left) and re-aggregated spheroids (right), respectively (B). The plots show isotype control (open histogram) versus stained sample (red histogram). The histograms show that the purity of cardiac Troponin-T hiPSC-MS in the re-aggregated spheroids increased when compared with the negative control.

Data analysis

  1. Measurement of spheroid size using ImageJ software

    Figure 5 shows the flow of spheroid size analysis.

    1. Open an image of spheroids with the ImageJ software.

    2. If the distance is shown in pixel, set the known distance using the “Set Scale” command in ImageJ. For example, 450 pixels = 1,000 μm.

    3. Convert the image type to 8-bit.

    4. (Option) Filter the image using “Gaussian Blur (Sigma 2.0)” command if the next step of “bipolarization” does not work.

    5. Bipolarize the image using the “Threshold” command.

    6. (Option) Divide overlapping spheroids using the “Watershed” command.

    7. To measure the surface area for each spheroid, use “Analyze Particles” with parameters “Size: 706.5 (Φ30 μm)” – infinity, “Circularity:” 0.50-1.00, “Show:” Outlines; check “Display results,” “Exclude on edges,” and “Include holes.”

    8. (Option) Overlay the Outlines in the original image to ensure the spheroids are recognized correctly.

      1. Change the color of the Outlines using “Lookup Tables > Green” command.

      2. Invert the color of the Outlines using “Lookup Table > Invert LUT” command.

      3. Overlay the Outlines on the original image using “Image Calculator (Image 1: original image, Image 2: outlines)” command.

    9. Analyze the data set consisting of the surface area of each spheroid with Excel.



      Figure 5. Flow of spheroid size analysis with ImageJ at Data analysis A

Recipes

Cell culture reagents and media preparation

  1. Reagents (Table 2)


    Table 2. Reagent preparation

    Reagents Preparation method Stock concentrations
    L-Ascorbic Acid Dissolve L-Ascorbic Acid at 4°C with pre-cooled ultrapure water at 5 mg/ml and mix using a vortex mixer until completely dissolved. Sterilize using a 22 μm syringe filter. Aliquot the solution into 1 ml volumes and freeze at -20°C. Freshly thaw the solution at 4°C or on ice in the dark when preparing the medium. 5 mg/ml
    Monothioglycerol (MTG) Aliquot MTG to 1 ml volumes and freeze at -20°C. Use thawed MTG within 3 months. To prevent oxidation of MTG, minimize the opening and closing of the cap of the stock tube. On the day of media preparation, dilute 13 μl MTG in 1 ml StemPro-34. 100%
    (1.3%(v/v) in use)
    Transferrin Dissolve Transferrin at the concentration of 30 mg/ml with sterilized ultrapure water. Aliquot the transferrin solution to 1 ml volumes and freeze at -20°C. 30 mg/ml
    4 mM Hydrochloric acid (HCl), 0.1% BSA Buffer In a draft chamber, transfer 30 μl of 6.0 N HCl into 50 ml of sterilized ultrapure water. Sterilize using a 22 μm syringe filter. Add 1 ml of 30% BSA into the 30 ml of HCl solution. 4 mM HCl
    0.1% BSA
    DPBS, 0.1% BSA Buffer Add 10 μl of 30% BSA into 3 ml of DPBS. 0.1% BSA


  2. Cytokines and small molecular compounds (Table 3)


    Table 3. Stock preparation of cytokines and small molecular compounds

    Growth factors/small molecular compounds Buffers Stock concentrations
    BMP4 4 mM Hydrochloric acid, 0.1% BSA 10 μg/ml
    bFGF PBS, 0.1% BSA 10 μg/ml
    VEGF PBS, 0.1% BSA 5 μg/ml
    Activin A PBS, 0.1% BSA 10 μg/ml
    IWP-4 Dimethyl sulfoxide 1.2 mM

    Dissolve cytokines or small molecular compounds using their appropriate buffers at the stock concentration shown in Table 2. Aliquot each solution if needed and freeze at -20°C.


  3. Modified StemPro-34 (Table 4)


    Table 4. Preparation of Modified StemPro-34

    Compositions Final concentration Volume
    StemPro-34 - 48.5 ml
    Pen/Strep (100×) 500 μl
    L -glutamine (100×) 500 μl
    Transferrin (30 mg/ml) 150 μg/ml 250 μl
    Ascorbic Acid (5 mg/ml) 50 μg/ml 500 μl
    1.3% (v/v) MTG 0.0039% (v/v) 150 μl


  4. Spheroid formation medium (Table 5)


    Table 5. Preparation of Spheroid formation medium

    Compositions Final concentration Volume
    Modified StemPro-34 - 50 ml
    BMP4 (10 μg/ml) 1 ng/ml 5 μl
    Y-27632 (10 mM) 10 μM 50 μl


  5. Stage-1 medium (2×) (Table 6)


    Table 6. Preparation of Stage-1 medium (2×)

    Compositions Final concentration Volume
    Modified StemPro-34 - 50 ml
    BMP4 (10 μg/ml) 20 ng/ml 100 μl
    bFGF (10 μg/ml) 10 ng/ml 50 μl
    Activin A (10 μg/ml) 12 ng/ml 60 μl


  6. Stage-2 medium (Table 7)


    Table 7. Preparation of Stage-2 medium

    Compositions Final concentration Volume
    Modified StemPro-34 - 50 ml
    VEGF (5 μg/ml) 10 ng/ml 100 μl
    IWP-4 (1.2 mM) 2.5 μM 104 μl


  7. Stage-3 medium (Table 8)


    Table 8. Preparation of Stage-3 medium

    Compositions Final concentration Volume
    Modified StemPro-34 - 50 ml
    VEGF (5 μg/ml) 10 ng/ml 100 μl
    bFGF (10 μg/ml) 5 ng/ml 25 μl

Acknowledgments

A part of this work was supported by the “Network Program for Realization of Regenerative Medicine” from the Japan Agency for Medical Research and Development (AMED). This protocol was adapted from a previously published work (Miwa et al., 2020).

Competing interests

There are no conflicts of interest or competing interests.

References

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简介

摘要]使用独特的微结构血管 EZSPHERE 可以轻松地从人类诱导多能干细胞 (hiPSC) 形成高通量 3D 球体,从而有效和大规模地生成分化细胞,例如心肌细胞或神经元。这种 hiPSC 衍生的心肌细胞 (hiPSC-CM) 或神经元在再生医学或基于细胞的药物安全测试领域非常有用。先前的研究表明,通过利用微结构容器 EZSPHERE 控制 Wnt 信号,由 hiPSC 产生的 3D 球体可有效分化为高质量的 hiPSC-CM。在这里,我们描述了一个简单而高效的协议,用于生成大量统一大小的 hiPSC 球体,并使用 EZSPHERE 诱导它们进行心脏分化。该方法包括在整个心脏分化过程的中期从心脏分化培养基中收集和分离球体,并将获得的单细胞重新接种到 EZSPHERE 中,以将它们重新聚集成均匀的 hiPSC-CM 球体尺寸。这种重新聚合过程促进了非典型 Wnt 信号相关的心脏发育,并提高了生成的 hiPSC-CM 的纯度和成熟度。

图文摘要: 使用 EZSPHERE 通过球体形成和重新聚集从 iPSC 的心脏分化概述。

[背景] 人类诱导多能干细胞衍生的心肌细胞 (hiPSC-CMs) 有望成为用于心脏病再生医学和药物发现的细胞来源。心脏病仍然是全球主要的死亡原因(世卫组织,2018 年)。在非人类灵长类动物中,移植的 hiPSC-CM 可改善心脏收缩功能,足以使梗塞的心脏再生(Shiba等,2016)。以 CiPA 项目(Blinova等人,2017 年)和 JiCSA 项目(Yamazaki等人,2018 年)为代表的先前研究已经表明 hiPSC-CM 可用于预测心脏毒性风险,如 Torsades de Pointes。还表明,生成的 hiPSC-CM 的成熟度会改变对药物的反应(da Rocha等,2017)。因此,开发用于生成高质量 hiPSC-CMs 的高通量和稳健的协议对于实现再生医学和药物发现是紧迫的。 hiPSC 可以在 2D 粘附培养或 3D 细胞聚集体/球体培养方法中直接分化为心肌细胞。特别是,与 2D 培养方法相比,球体培养方法更容易扩大规模,并能更好地促进生成的 hiPSC-CM 的成熟(Pal等,2013)。在心脏分化过程中,在正确的时间调节经典和非经典 Wnt 会强烈促进多能干细胞分化为心肌细胞,而不是其他中胚层细胞类型(Loh等,2016)。此外,Wnt 信号相关基因 Wnt5A 和 Wnt11 的表达水平受球体大小的影响(Hwang等,2009;Chen等,2015)。因此,我们寻求通过实现高通量球体形成和同时活动球体大小控制来开发一种高效且稳健的心脏分化方案。 在这项研究中,我们使用了形状独特的微纤维培养皿“EZSPHERE”,该培养皿设计用于在一个培养皿上高通量生产均匀球体,并具有高密度微孔(Sato等人,2016 年)。最初,针对心脏分化进行了优化的基本球体培养方案(Yang等人,2008 年,Bauwens等人,2014 年,Kempf等人,2014 年)与 EZSPHERE 培养皿相结合。为了从 hiPSC 形成球体,使用了直径为 100 毫米的 900 型 EZSPHERE 培养皿。它们中的每一个都有大约 14,000 个独立的微孔,它们具有规则尺寸(直径 400-500 μm,深度约 100 μm)。根据计算,每个 EZSPHERE 培养皿中可以形成大约 14,000 个球体,每个球体的初始接种细胞数为 200-220。按照标准方案,在第 1 天将激活素 A、BMP-4 和 FGF2 添加到 EZSPHERE 中的球体中,以诱导分化为原始条纹。第 4 天,收集、洗涤球体并转移到平底低细胞粘附培养皿,然后用 Wnt 抑制剂处理。在第 6 天,球体的大小均匀性丧失,但 62% 的细胞群已分化为 PDGFR-α/KDR 双阳性心脏中胚层样细胞(Kattman等人,2011 年;Birket等人,2015 年) )。为了再次控制球体大小,收集分化的球体并将其分解成单个细胞。然后,通过重新接种到 903 EZSPHERE 型(直径为 35 毫米)的培养皿上来重新聚集获得的单个细胞,该培养皿具有大约 1,000 个较大尺寸(直径约为 800 微米,深度约为 400 微米)的单独微孔) 在每道菜中。重新聚集的球体比非重新聚集的球体增加心脏肌钙蛋白 T (CTNT) 阳性细胞的数量更快。此外,与未经历重新聚集过程的球体相比,重新聚集的球体的心脏成熟度(通过 MYL2/MYL7 的基因表达水平估计)要高得多。执行重新聚合过程还提高了非规范 Wnt 信号 Wnt5A 和 Wnt11 的表达水平。在小鼠中,Wnt5A 和 Wnt11 对于第二心脏野祖细胞的产生至关重要(Cohen等,2012)。Wnt11 还因其在斑马鱼心脏发育过程中通过调节非典型 Wnt/Ca 2+信号而产生心肌电梯度模式的作用而闻名(Panakova等,2010)。为了揭示其分子机制的细节,需要进一步研究。然而,这些数据强烈表明,在 EZSPHERE 上进行的心肌细胞分化过程,包括诱导心脏中胚层/祖细胞的重新聚集步骤,可有效产生具有高纯度和成熟水平的心肌细胞。

关键字:三维球状体, 人类诱导性多能干细胞, 人类诱导性多能干细胞衍生的心肌细胞 , 心肌细胞分化, 微结构血管, EZSPHERE, 细胞团, 球状体再聚集 , 再生医学 , 药物安全测试

材料和试剂

EZSPHERE Dish 100 mm Type 900AGC Techno GlassIWAKI,目录号:4020-900

EZSPHERE Dish 35 mm 903 型(AGC Techno GlassIWAKI,目录号:4000-903

EZ-BindShut II 100 mm 碟(AGC Techno GlassIWAKI,目录号:4020-800LP

细胞培养 T25 烧瓶(AGC Techno GlassIWAKI,目录号:3100-025

5 ml PipetAGC Techno GlassIWAKI,目录号:7103-005

细胞刮刀(AGC Techno GlassIWAKI,目录号:9010-320

70 μm 细胞过滤器(CorningFalcon,目录号:352350

巴斯德移液器(AGC Techno GlassIWAKI,目录号:IK-PAS-9P

人类诱导多能干细胞 (hiPSC) 菌株 253G1 201B7 (iPS Academia Japan)

维持培养基 mTeSR1Stemcell Technologies,目录号:5850

Biolaminin 521 LNLN521)(BioLamina,目录号:LN521-03

DPBSFUJIFILM Wako Pure ChemicalWako,目录号:045-29795

HBSS,无钙(Thermo Fisher ScientificGibco,目录号:14170112

L-Gln DMEM4.5g/L 葡萄糖),不含丙酮酸钠,液体(Nacalai,目录号:08459-35

0.5 mol/L-EDTA 溶液(pH 8.0)(Nacalai,目录号:06894-14

胎牛血清,FBSMP BiomedicalsCELLECT,目录号:2916154

Accutase(默克,Sigma-Aldrich,目录号:A6964-100ML

AccuMaxInnovative Cell Technologies,目录号:AM105

TrypLE select Enzyme1 ×),无酚红(Thermo Fisher ScientificGibco,目录号:12563011

StemPro TM -34 SFM)(Thermo Fisher ScientificGibco,目录号:10639011

青霉素-链霉素(10,000 U/ml)(Thermo Fisher ScientificGibco,目录号:15140122

L-谷氨酰胺(200 mM)(Thermo Fisher ScientificGibco,目录号:25030081

全转铁蛋白人(默克,Sigma-Aldrich,目录号:T0665-100MG

L-抗坏血酸(默克,Sigma-Aldrich,目录号:A92902-100MG

1-硫代甘油(默克,Sigma-Aldrich,目录号:M6145-25ML

ROCK抑制剂Y-27632Wako Pure Chemical IndustriesWako,目录号:253-00513

重组人 BMP-4 蛋白(Bio-TechneR&D Systems,目录号:314-BP-010

成纤维细胞生长因子(碱性),人,重组(154 aa)(FUJIFILM Wako Pure ChemicalWako,目录号:064-04541

重组人 VEGF 165 蛋白(Bio-TechneR&D Systems,目录号:293-VE-010

重组人/小鼠/大鼠激活素 A 蛋白(Bio-TechneR&D Systems,目录号:338-AC-010

Stemolecule Wnt Inhibitor IWP-4REPROCELL USAStemgent,目录号:04-0036

DNase IBovine PancreasMerckMillipore,目录号:260913

30 w/v% 白蛋白 D-PBS-)溶液,来自牛血清(BSA),无脂肪酸(FUJIFILM Wako Pure ChemicalWako,目录号:015-23871

4%多聚甲醛磷酸盐缓冲液(Wako Pure Chemical IndustriesWako,目录号:163-20145

流式细胞术分析中使用的抗体(表 1

1. 流式细胞术分析中使用的抗体抗体经销商猫。

稀释比例APC-KDR生物传奇3599152.5:100APC-小鼠 IgG1kappa 同种型对照生物传奇4001212.5:100PE-PDGFRα生物传奇3235055:100PE-小鼠 IgG1kappa 同种型对照生物传奇4001135:100PE抗心肌肌钙蛋白TBD Pharmingen5647672.5:100PE-同型对照BD Pharmingen5546802.5:100 细胞培养试剂(见配方)

细胞因子和小分子化合物(见食谱)

改良的 StemPro-34(见配方)

球体形成介质(见配方)

阶段 1 培养基()(见配方)

Stage-2 培养基(见食谱)

3 阶段培养基(见食谱)

设备 层流罩(日立,型号:SCV-Class II type A/B

水浴(AS ONE Corporation,型号:TR-1AR

TC10 自动细胞计数器(Bio-Rad,目录号:145-0001

EVOS FL 自动细胞成像系统(Life TechnologiesEVOS,目录号:AMAFD1000

EVOS Onstage 孵化器(Life TechnologiesEVOS,目录号:AMC1000

BD FACSVerse流式细胞仪(BD Bioscience,目录号:651154

Pipet-Aid XPress 110VDrummond Scientific Company,目录号:4-040-135

离心机(HimacCF16RX,型号:S101812

涡流混合器 Vortex Gwnie 2Scientific IndustriesInc.,目录号:SI-0236

HERAcell CO 2培养箱(Kendro 实验室产品)

真空泵(型号 JN726 FT.18

软件 ImageJNIHhttps: //imagej.nih.gov/ij/

AGDRec AG-桌面记录器(AmuseGraphicshttp ://t-ishii.la.coocan.jp/hp/ag/index.html

FlowJo 软件(FlowJo LLChttps ://www.flowjo.com/

 

程序

注意:在生物安全 2 级实验室的专用层流罩中进行培养操作。所有废物(管子、吸头、盘子)都应通过高压灭菌灭活,然后根据您的设施规则妥善处理。 hiPSCs 的高通量球体形成

在涂有 Biolaminin 521 LN 的烧瓶中培养培养基 mTeSR1 中的 hiPSC

注意:在不添加 ROCK 抑制剂的情况下,将 hiPSC 作为单个细胞进行亚培养,而不是细胞团块。准备球体形成介质(参见配方:表 5)并在 37°C 的水浴中预热。

使用 90% 融合的 hiPSC

从细胞中吸出培养基并用 2.5 ml 室温 DPBS 洗涤。对于抽吸,请使用带有吸管和适当管子的真空泵。

加入 2.5 ml TrypLE select 并在 37°C 下孵育 4 分钟。

抽吸 TrypLE 选择并添加 2.5 ml 球体形成培养基。

使用带有 5 ml 移液管的 Pipet-Aid 将培养基冲洗至细胞,从而分离细胞。如果细胞没有分离,用细胞刮刀刮掉它们。

用移液器冲洗细胞 2-8 次,将细胞团解离成单个细胞,并将它们收集到 15 ml 离心管中。

20°C 下以 100 × g离心4 分钟。

吸出上清液并用 1-5 ml 的球体形成培养基重悬沉淀。

使用 TC10 自动细胞计数器测量细胞密度。

用球体形成培养基将细胞稀释至 3 × 10 5 个细胞/ml的密度,并将 10 ml 悬浮细胞接种到 EZSPHERE Dish 100 mm Type 900 (见图 1)。

EZSPHERE 培养皿置于 37°C5% CO 2培养箱中培养 24 小时。

1. 具有培养皿类型的微纤维容器“EZSPHERE”的图像 心脏分化的第一阶段

进入心脏分化过程的第 1 阶段,球体形成后 24 ± 2 小时。

准备 Stage-1 培养基 (2x)(参见配方:表 6)并在 37°C 的水浴中预热。

CO 2培养箱中取出 EZSPHERE 培养皿,并在相差显微镜下检查 EZSPHERE 的每个微孔中是否形成了球体。

小心地将等体积 (10 ml) Stage-1 培养基 (2x) 添加到球体中。

注意:我们建议用 10 ml 移液器吸取 10 ml Stage-1 培养基 (2x),将移液器的尖端放在 EZSPHERE 盘中心周围的液面上,垂直放置移液器,然后小心地倒入培养基. EZSPHERE 培养皿放回 37°C5% CO 2培养箱,培养 89 小时。

 心脏分化的第 2 阶段

在上述第 1 阶段后 89 ± 2 小时,进入心脏分化过程的第 2 阶段。

准备 Stage-2 培养基(参见配方:表 7)和 IMEM 培养基,然后在 37°C 的水浴中预热。

EZSPHERE 盘中收获球状体到 15 毫升或 50 毫升管中。

(可选的)将球体转移到 100 毫米的平底培养皿中,并放置在显微镜的舞台上。

以圆周运动精细地摇动盘子,将球体放在盘子的中心。

用显微镜拍摄球体照片(见图 2)。

使用 ImageJ 软件测量球体的大小(参见数据分析 A)。

  2. 在程序 C 中生成的 hiPSC 球体的图像和大小。EZSPHERE (A) 上的球体并转移到平底盘 (B) 上。球体 (μm) (C) 大小(直径)的直方图。 松开管盖进行通风,将管置于 37°C CO 2培养箱中 10 分钟以沉淀球体。

使用移液器尽可能小心地从球体中取出上清液,并加入 5 ml 预热的 IMEM 培养基以洗涤球体。

20°C 下以 50 × g离心球体3 分钟,尽可能去除上清液。

20 ml 预热的 Stage-2 培养基添加到球体中,并将球体转移到 100 毫米的低细胞粘附培养皿 (EZ-BindShut II)

将培养皿放入 37°C CO 2培养箱中。

注意:我们建议来回和左右摇动盘子 3 次,使球体均匀地分布在盘子中。如上所述,每天早晚摇动盘子,使球体均匀分散。

 心脏分化和球体重新聚集的第 3 阶段

注意:球体的重新聚集增加了心脏分化过程中的纯度。作为阴性对照,不要在这一步重新聚合球体。为了确认重新聚集的效果,在程序 G 后通过流式细胞术分析 CTNT 阳性 iPS-CM 的纯度。在心脏分化的第 6 天,进入第 3 阶段以重新聚集球体。

注意:虽然建议在第 6 天执行此重新聚合步骤,但第 7 天也是可以接受的。准备第 3 阶段培养基(参见配方:表 8)并在 37°C 的水浴中预热。

将球体从 100 毫米 EZ-BindShut II 培养皿中收集到 50 毫升管中,并将其放入 37°C CO 2培养箱中 10 分钟以沉淀球体。

从收集的球体中取出上清液,加入 5 ml 室温 DPBS 清洗。

20°C 下以 50 × g离心3 分钟并去除上清液。

注意:对于阴性对照,跳过步骤 D6-D9 并继续执行步骤 D10。向球体中加入 1 ml Accutase,并在 37°C 的水浴中孵育 8 分钟,以将球体解离成单个细胞。在此孵育过程中,每 4 分钟将管涡旋 10 秒,剧烈混合细胞。

如果细胞聚集体在孵育 8 分钟后仍然存在,如上所述,强制额外孵育:使用移液器仅从单个分离的细胞中收集剩余的细胞聚集体,然后转移到新管中。然后,将双倍量(2 ml)的 Stage-3 培养基添加到单个解离的细胞中,并将 1 ml Accutase 添加到聚集体中。在 37°C 的水浴中再次孵育聚集体 8 分钟。在孵化过程中, 如上所述, 大力混合细胞。

将双倍量(2 ml)的 Stage-3 培养基加入 Accutase 中的单个解离细胞,20°C 下以 200 × g离心4 分钟,然后去除上清液。

5 ml Stage-3 培养基重悬收集的细胞沉淀,并计算直径范围为 8-27 μm 的细胞数。如果细胞聚集体仍然存在,请使用 70 μm 细胞过滤器将其除去。

Stage-3 培养基将细胞稀释至 1 × 10 5细胞/ml的密度,并将 3 × 10 5细胞接种到 35 毫米 EZSPHERE 903 型培养皿中。

注意:对于阴性对照,在第 5 步从一个 100 毫米 EZ-BindShut II 培养皿中获得的球体中添加 20 毫升第 3 阶段培养基,并将每个 3 毫升分配到 35 毫米 EZ-BindShut II 培养皿中。每 2 3 天更换一半体积的培养基。

 收获心肌细胞分化细胞

在心脏分化过程的第 10 14 天左右,收获获得的 hiPSC-CM 或阴性对照。

准备 4 ml 解离缓冲液(0.05% 胰蛋白酶-EDTA:AccuMax = 3:1)和 4 ml 淬灭缓冲液(DMEM:FBS 1:1 + 200 单位 ml -1 DNase I)。

在显微镜下观察心脏球体的跳动状态(见视频 1 2)。

注意:随着 EZSPHERE 培养皿在室温下变冷,心脏球体的跳动可能会衰减。因此,我们建议在显微镜观察期间使用台上培养箱将培养皿保持在 37°C 的温度。观察使用EVOS FL Auto Cell Imaging SystemEVOS台上培养箱,拍摄视频使用AGDRec AG-Desktop录像机。 视频 1. 心脏球体的代表性视频重新聚合 在 EZSPHERE 的微孔中。球体在第 6 天重新聚集并从第 8 天开始跳动。此视频是在第 14 天录制的。 视频 2.重新聚合过程后恢复的跳动心脏球体的视频。心脏球体从 EZSPHERE 培养皿转移到平皿中,然后在它们跳动时进行录像。   将球体收集到 15 毫升管中,静置 2-3 分钟,直到它们沉降。

去除上清液并加入 5 ml DPBS 清洗球体。

将管以 50 × g离心3 分钟并去除上清液,注意不要丢失球体。

向收集的球体中加入 1 ml 解离缓冲液,在 37°C 的水浴中孵育 8 分钟,将球体解离成单个细胞。在孵化过程中, 如上所述, 大力混合细胞。

如果孵育 8 分钟后细胞聚集体仍然存在,请执行额外的孵育步骤。使用移液器仅从单个分离的细胞中收集剩余的细胞聚集体,然后转移到新管中。然后,向解离的细胞中加入等体积(1 ml)的淬灭缓冲液,并在聚集体中加入 1 ml 的解离缓冲液。在 37°C 的水浴中再次孵育聚集体 8 分钟。在孵化过程中, 如上所述, 大力混合细胞。

向解离的细胞中加入等体积 (1 ml) 的淬灭缓冲液。

将管以 200 × g离心5 分钟,并通过小心移液去除上清液。不要使用抽吸器来防止吸入与一些破碎的细胞衍生 DNA 缠绕在一起的细胞聚集体。

对于细胞计数分析,用 DPBS 重悬收获的细胞。对于下一步的培养,用含有 10% FBS DMEM 重悬细胞。

计算直径在 8-30 μm 内的细胞数。

 心脏中胚层的流式细胞术分析。

使用第 6 天收获的细胞(步骤 D8)。

准备两管(#1 #2)并将0.5 × 10 6细胞分别转移到每个管中,用于细胞染色及其同种控制。

准备 20 ml 流式细胞术缓冲液 [HBSS (-), 3% FBS, 0.03 mM EDTA]

向细胞中加入 2 ml 流式细胞术缓冲液。

将细胞在20-25°C 下以 300 × g离心5 分钟并吸出上清液。

重复步骤 F4-F5

100 μl 流式细胞术缓冲液中重悬 0.5 × 10 6细胞。

APC-KDR PE-PDGFRα 抗体添加到管 #1 中进行细胞染色,并将 APC PE-同型控制添加到管 #2 中进行同型控制(表 1)。

在室温下在黑暗中孵育每管 45 分钟。

在每个管中加入 2 ml 流式细胞术缓冲液。

将细胞在20-25°C 下以 300 × g离心5 分钟,然后吸出 DPBS

重复步骤 F10-F11

300 μl 流式细胞术缓冲液中重悬细胞。

使用流式细胞仪 (BD FACS Verse) 测量细胞并使用 FlowJo 软件分析数据 ( 3)

  3. iPSC 衍生的心脏中胚层在第 6 天的代表性流式细胞仪配置文件。使用流式细胞仪 (BD FACS Verse) 获取事件并使用 FlowJo 软件进行分析。首先,主要群体是通过 SSC FSC 点图 (A) 中的门控选择的。显示了每种抗体的直方图:APC-KDR (B) PE-PDGFRα (C)。该图显示了同种型对照(空心直方图)与染色样品(红色直方图)。第 6 天出现的主要群体是 KDR PDGFRα 阳性(5.7% 的细胞为 KDR高,71.7% 的细胞为 KDR低,86.7%的细胞为PDGFRα + )。该结果表明第 6 天存在的主要群体被认为是心脏中胚层样细胞。 hiPSC 衍生的心肌细胞 (hiPSC-CM) 的流式细胞术分析

使用 1 × 10 6在步骤 E10 中获得的分化或阴性对照细胞。

注意:如果细胞是在培养基中收获的,请用 DPBS 清洗。将细胞在20-25°C 下以 300 × g离心5 分钟,然后吸出上清液。

加入 200 μl 4% 磷酸多聚甲醛缓冲溶液,室温孵育 20 分钟以固定细胞。

向细胞中加入 2 ml DPBS,并在 20-25°C 下以 300 × g离心5 分钟。

取出含有多聚甲醛溶液的上清液,将其丢弃到适当的废液瓶中。

2 ml DPBS 中重悬细胞沉淀。细胞可以在这一步储存长达 7 天。

将细胞在20-25 °C 下以 300 x g离心5 分钟,并吸出上清液。

将细胞沉淀重悬在 200 μl(每 100 μl 0.5 × 10 6 个细胞)的流式细胞术透化/洗涤缓冲液 I 中,并在室温下孵育 30 分钟。

100 μl0.5 × 10 6 个细胞)的重悬细胞转移到 96 孔圆底板的一个孔中作为染色样品。将剩余的 100 μl0.5 × 10 6细胞)重悬细胞转移到另一个 96 孔圆底板的一个孔中作为同种型对照。

向样品中加入 2.5 μl PE 抗心脏肌钙蛋白 T 抗体,向同型对照中加入 2.5 μl PE 同型对照抗体(表 1),并在室温下在黑暗中孵育 30 分钟。

向每个孔中加入 100 μl 流式细胞术透化/洗涤缓冲液 I,并在室温下在黑暗中轻轻摇动板 5 分钟。

将板在20-25°C 下以 300 × g离心5 分钟,并通过倾析或移液去除上清液。

小心地将 200 μl 流式细胞术透化/洗涤缓冲液 I 添加到每个板中,不要破坏细胞沉淀。

将板在20-25°C 下以 300 × g离心5 分钟,并通过倾析或移液去除每个板的上清液。

重复步骤 G13-G14

在每个板中用 200 μl DPBS 重悬细胞沉淀并混合均匀。

使用流式细胞仪 (BD FACSVerse) 测量每个板中的细胞并使用 FlowJo 软件分析数据 ( 4)

  4. hiPSC-CM 的代表性流式细胞仪配置文件。使用流式细胞仪 (BD FACS Verse) 获取事件并使用 FlowJo 软件进行分析。首先,主要群体是通过 SSC FSC 点图 (A) 中的门控选择的。直方图分别显示了无重新聚集的阴性对照(左)和重新聚集的球体(右)(B)的心脏肌钙蛋白 T 阳性 hiPSC-CM 群体。该图显示了同种型对照(开放直方图)与染色样品(红色直方图)。直方图显示,与阴性对照相比,重新聚集的球体中心脏肌钙蛋白-T hiPSC-MS 的纯度增加。 数据分析 使用 ImageJ 软件测量球体大小

5 显示了球体尺寸分析的流程。使用 ImageJ 软件打开球体图像。

如果距离以像素显示,请使用 ImageJ 中的设置比例命令设置已知距离。例如,450 像素 = 1,000 μm

将图像类型转换为 8 位。

(选项)如果下一步“双极化”不起作用,则使用“高斯模糊(Sigma 2.0命令过滤图像。

使用“阈值”命令对图像进行双极化。

(选项)使用“分水岭”命令分割重叠的球体。

要测量每个球体的表面积,请使用“分析粒子”和参数“尺寸:706.5 (Φ30 μm)” – 无穷大、圆度:”0.50-1.00显示:轮廓;选中显示结果边缘排除包括孔

(可选)在原始图像中叠加轮廓以确保正确识别球体。

使用“Lookup Tables > Green”命令更改大纲的颜色。

使用“Lookup Table > Invert LUT”命令反转轮廓的颜色。

使用“图像计算器(图像 1:原始图像,图像 2:轮廓)命令在原始图像上叠加轮廓。

Excel 分析由每个球体的表面积组成的数据集。

  5. 在数据分析 A 中使用 ImageJ 分析球体大小的流程  食谱 细胞培养试剂和培养基制备试剂(表 2

  2. 试剂制备试剂制备方法库存集中度L-抗坏血酸在 4°C 下用 5 mg/ml 的预冷超纯水溶解 L-抗坏血酸,并使用涡旋混合器混合直至完全溶解。使用 22 μm 注射器过滤器进行消毒。将溶液分装成 1 ml 体积并在 -20°C 下冷冻。制备培养基时,将溶液在 4°C 或冰上避光解冻。5 毫克/毫升硫代甘油 (MTG) MTG 分装至 1 ml 体积并在 -20°C 下冷冻。在 3 个月内使用解冻的 MTG。为防止 MTG 氧化,尽量减少库存管盖的打开和关闭。在培养基制备当天,用 1 ml StemPro-34 稀释 13 μl MTG100%(1.3%(v/v) 使用中)转铁蛋白用无菌超纯水以 30 mg/ml 的浓度溶解转铁蛋白。将转铁蛋白溶液等分至 1 ml,并在 -20°C 下冷冻。30 毫克/毫升4 mM 盐酸 (HCl)0.1% BSA 缓冲液在通风室中,将 30 μl 6.0 N HCl 转移到 50 ml 灭菌超纯水中。使用 22 μm 注射器过滤器进行消毒。将 1 ml 30% BSA 添加到 30 ml HCl 溶液中。4 毫米盐酸0.1% BSADPBS0.1% BSA 缓冲液将 10 μl 30% BSA 添加到 3 ml DPBS 中。0.1% BSA 细胞因子和小分子化合物(表 3

  3. 细胞因子和小分子化合物的储备制备生长因子/小分子化合物缓冲器库存集中度BMP44 mM 盐酸,0.1% BSA10 微克/毫升bFGFPBS0.1% BSA10 微克/毫升血管内皮生长因子PBS0.1% BSA5微克/毫升激活素APBS0.1% BSA10 微克/毫升IWP-4二甲基亚砜1.2 毫米 使用适当的缓冲液以表 2 中所示的库存浓度溶解细胞因子或小分子化合物。如果需要,将每种溶液分装并在 -20°C 下冷冻。 改良的 StemPro-34(表 4

  4. 改良 StemPro-34 的制备组合物最终浓度体积StemPro-34——48.5 毫升笔/链球菌 (100×)1×500 微升L-谷氨酰胺 (100×)1×500 微升转铁蛋白(30 毫克/毫升)150 微克/毫升250 微升抗坏血酸 (5 毫克/毫升)50 微克/毫升500 微升1.3% (v/v) MTG0.0039% (v/v)150 微升 球体形成介质(表 5

  5. 球体形成介质的制备组合物最终浓度体积改良的 StemPro-34——50毫升BMP4 (10 微克/毫升)1 纳克/毫升5 微升Y-27632 (10 mM)10微米50 微升 Stage-1 培养基 (2×) ( 6)

  6. Stage-1 培养基 (2×) 的制备组合物最终浓度体积改良的 StemPro-34——50毫升BMP4 (10 微克/毫升)20 纳克/毫升100 微升bFGF (10 微克/毫升)10 纳克/毫升50 微升激活素 A10 微克/毫升)12 纳克/毫升60 微升 Stage-2 培养基(表 7

  7. Stage-2 培养基的制备组合物最终浓度体积改良的 StemPro-34——50毫升VEGF5微克/毫升)10 纳克/毫升100 微升IWP-4 (1.2 毫米)2.5微米104 微升 第 3 阶段培养基(表 8

  8. Stage-3 培养基的制备组合物最终浓度体积改良的 StemPro-34——50毫升VEGF5微克/毫升)10 纳克/毫升100 微升bFGF (10 微克/毫升)5 纳克/毫升25 微升 致谢 这项工作的一部分得到了日本医学研究与开发机构 (AMED) 实现再生医学网络计划的支持。该协议改编自先前发表的作品(Miwa等人,2020 年)。

 

利益争夺

不存在利益冲突或竞争利益。

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引用:Miwa, T., Idiris, A. and Kumagai, H. (2021). High-throughput 3D Spheroid Formation and Effective Cardiomyocyte Differentiation from Human iPS Cells Using the Microfabric Vessels EZSPHERETM. Bio-protocol 11(21): e4203. DOI: 10.21769/BioProtoc.4203.
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