1.1 Expansion of hiPSC-derived myogenic, neuronal and blood vascular cells
1.1.1 skeletal muscle cells
1.1.1.1 Transgene-based method
Transgene-based myogenic differentiation of hiPSC is based on our published protocol 56 outlined in steps 1-45. Characterisation of skeletal muscle cells is also detailed in steps 46 to 93 of the same protocol.
1. Thaw HIDEMs at 37°C until frozen clomps are no longer visible (up to 1 minute). Transfer cells to a 15 ml falcon tube containing HIDEM proliferation medium and spin at 300 rcf for 5 minutes.
2. Discard supernatant and resuspend cell pellet in HIDEM proliferation medium, plate 4-5x103/cm2 cells onto an uncoated surface and incubate cells at 37°C with 5% CO2 and 3-5% O2.
3. After 24 hours, replace the HIDEM proliferation medium. Continue culturing cells until approximately 80% confluent prior to using for artificial skeletal muscle generation.
PAUSE POINT: HIDEMs can be expanded onto uncoated surfaces using HIDEM proliferation medium and cryopreserved using HIDEM freezing medium 56.
1.1.1.2 Transgene-free method
Small molecule-mediated hiPSC differentiation into myogenic cells was performed using a published and commercially available method60, in which is detailed further insights into basic characterisation and testing of the differentiation capacity of the hiPSC myogenic derivatives prior to differentiation in 3D.
4. Thaw hiPSC-derived myogenic progenitors at 37°C for up to 2 minutes. Transfer cells to a 15ml falcon tube containing DMEM resuspension medium and spin at 300 rcf for 5 minutes.
5. Discard supernatant and resuspend cell pellet in Myoblast medium (Myocea), plate 4-5x103 cells per cm2 onto an uncoated surface and incubate cells at 37°C with 5% CO2 and 3-5% O2.
6. After 24 hours, replace the Myoblast medium (Myocea). Continue culturing until 70% confluent prior to use for artificial skeletal muscle generation.
CRITICAL STEP: hiPSC-derived myogenic progenitors take 2-3 days to recover from freezing. If the culture is too confluent or cells appear flattened and large (i.e., senescing), proceed with splitting the cells either in the same flask/dish (in situ splitting) or into additional flasks/dishes to facilitate recovery and expansion.
PAUSE POINT: hiPSC-derived myogenic progenitors can be expanded onto standard uncoated tissue culture plastic grade surfaces using MYOCEA myoblast medium and cryopreserved using HIDEM freezing medium 56,60.
1.1.2 motor neurons
Derivation of motor neurons was performed using published protocols39,40, steps from hiPSC to single cell NPCs or neurospheres are not detailed here. Characterisation of cell identity and differentiation potential of the neural progenitors according to published protocols39,40 is required prior to use in 3D muscle cultures. The following two sections provide a basic guide to thaw and prepare cells for use in 3D muscle cultures.
1.1.2.1 Neurosphere-based method
For complete derivation of neurospheres from hiPSCs, refer to steps 3-22 and 34-39 of Stacpoole et al.39
7. Thaw neurospheres at 37°C for up to 2 minutes. Transfer to a 15 ml conical tube containing pre-warmed CDM and spin at 250 rcf for 2 minutes.
8. Discard supernatant and resuspend cell pellet in 10-12 ml of fresh neurosphere proliferation medium, seed cells in a 10 cm dish and incubate at 37°C with 5% CO2 and 3-5% O2.
9. After 24 hours, replace neurosphere proliferation medium. Continue culturing neurospheres by changing proliferation medium every other day.
PAUSE POINT: This cell population can then be used in bi- or tetra-lineage muscles as detailed in 1.15.2.1 and1.15.2.2
1.1.2.2 NPCs (monolayer)
10. Coat a 3.5 cm dish with 1% (vol/vol) GFR Matrigel™ or Geltrex™ at 37°C for 30 or 60 minutes respectively.
11. Thaw NPCs at 37°C for up to 2 minutes. Transfer cells to a 15ml falcon tube containing NPC proliferation medium with ROCKi (1:300) and spin at 300 rcf for 5 minutes.
12. Discard supernatant and gently resuspend cell pellet in fresh NPC proliferation medium with 1:300 ROCKi and transfer cells to the coated wells. Seed at least 2 x 105 NPCs per cm2 and incubate at 37°C with 5% CO2 and 3-5% O2.
13. After 24 hours, change NPC proliferation medium. Continue culturing NPCs by changing NPC proliferation medium every other day.
PAUSE POINT: This cell population can then be used in bi-lineage or tetra-lineage muscles as detailed in 1.15.2.2 and 1.15.4.2.
1.1.3 Vascular endothelial cells (ECs) and pericytes (PCs)
ECs and PCs are derived from hiPSCs using a published protocol63. Key deviations from Orlova et al.63 include cell purification by FACS and labelling of PCs with a lentivirally delivered GFP transgene to facilitate detection. Cell sorting and isolation are performed as follows:
14. Starting from step 15 of the Orlova et al. protocol63, detach cells using Trypsin-EDTA.
15. Neutralise by resuspending in 20-30 ml of FACS buffer.
16. Hydrate the cell strainer with 2-3 ml of FACS buffer and immediately pass the cells through it.
17. Count cells and keep aside 2 × 105 cells resuspended in FACS buffer on ice for the unstained fraction to be used in calibrating the cell sorter.
18. Spin down the rest of the cells at 300 rcf for 5 minutes. Discard supernatant and incubate cell pellet with CD31 (Mouse Anti-Human CD31 antibody, TP1/15 – FITC) 1:150 for 1 hour at 4°C.
19. Resuspend cells in 10 ml of FACS sorting buffer, filter through a cell strainer and collect the cell suspension in a 15 ml tube.
20. Spin cells at 300 rcf for 5 minutes, discard supernatant and collect cell pellet for FACS.
21. Prepare 1 tube with 2 ml of EGM2 medium to collect the CD31- fraction (PCs) and 1 tube with 2 ml of EC-SFM proliferation medium to collect the CD31+ fraction (ECs).
22. Prepare 0.1% gelatine-coated flasks to plate the ECs and PCs and, once the coating is complete, seed the sorted cells into the flasks in their respective medium.
PAUSE POINT: This cell population can be used for bi-lineage, tri-lineage or tetra-lineage constructs; only the latter is reported in this protocol and detailed in 1.15.4.1 and 1.15.4.2.
The following points detail an optional strategy to label PCs to facilitate their detection in 3D cultures. A lentiviral GFP transgene is suggested here, but alternative labelling strategies are likely to be equally efficacious.
23. To lentivirally transduce the PCs with a PGK-GFP cassette, follow the next steps: plate 1 × 105 PCs in a 35-mm dish in EGM2 medium.
24. When cells are fully attached to the culture dish, thaw the PGK-GFP lentiviral vector on ice and calculate the volume necessary to transduce cells with a multiplicity of infection (MOI) of 1, 5 and 10. The following formula can be used for this purpose: μl of virus needed = number of cells to infect × desired MOI/titre of virus (in ml) × 1000.
25. Dilute the PGK-GFP lentiviral vector in 1 ml of EGM2 medium supplemented with 1 μl of polybrene.
26. Remove the medium from the 35-mm dish of PGK-GFP PCs and replace it with the 1 ml of viral suspension. Incubate cells for 12 hours at 37°C with 5% CO2 and 3-5% O2.
27. Remove medium and wash cells twice with PBS. Subsequently, add fresh EGM2 medium and incubate cells at 37 °C with 5% CO2 and 3-5% O2.
PAUSE POINT: This cell population can be used for bi-lineage, tri-lineage or tetra-lineage constructs; only the latter is reported in this protocol and detailed in 1.15.4.1 and 1.15.4.2
1.2 Making hiPSC-derived artificial skeletal muscles
1.2.1 Single lineage artificial muscle
1.2.1.1 Single lineage artificial muscle by transgene-based method
CRITICAL STEP: at least 24 hours prior to beginning the protocol, ensure that the Teflon spacers and PDMS post racks are cleaned and autoclaved and that an adequate amount of 2% agarose, thrombin, fibrinogen, Matrigel™ and aprotinin aliquots are available (see Calculation Sheet, Reagent Setup and Equipment Setup)
28. Expand HIDEMs (from step 3) to 80% confluence prior to making hydrogels.
PAUSE POINT: 106 cells per hydrogel are required, we suggest expanding HIDEMs in T175 flasks until the area is fully covered.
29. On the day, add fresh medium supplemented with ROCKi (1:300) to the HIDEMs and incubate for 2 hours at 37°C with 5% CO2 and 18% O2.
30. Place thrombin and Matrigel™ aliquots on ice to thaw. Fibrinogen aliquots can be kept at RT. Label one 1.5ml Eppendorf tube per condition/cell line and place on ice.
31. Prepare an appropriate amount of HIDEM proliferation medium prepared with heat inactivated FBS to resuspend cells to be embedded in hydrogels. Volume needed depends on the number of hydrogels. For a total of 20 constructs, we suggest preparing 5 ml.
32. After two hours of incubation with HIDEM proliferation medium and 1:300 ROCKi, discard medium, wash once with PBS and cover cells with trypsin-EDTA. Incubate for up to 5 minutes to detach cells.
33. Neutralise trypsin-EDTA using HIDEM proliferation medium and count cells.
34. Centrifuge the required number of cells at 300 rcf for 5 minutes. Discard supernatant and keep cell pellet on ice.
35. Add 1.5 ml of agarose to each well of a 24-well plate that will house a 3D artificial muscle, place the spacers into the wells and allow to cool.
CRITICAL STEP: ensure spacers are placed correctly to avoid spillage of cells and biomaterial mixture at a later stage.
36. Add minimal volume of HIDEM proliferation medium prepared with heat inactivated FBS to resuspend the cell pellet. We suggest adding 50-100 µl of medium depending on the number of hydrogels (see Calculation Sheet in Supplementary Information for details).
37. Aspirate the total volume using a P200, transfer content to a 1.5 or 2ml tube/vial and note the cell suspension volume.
CRITICAL STEP: be accurate when assessing the volume, ensure that there are no bubbles in the tip when aspirating the cell suspension. If a high number of hydrogels are generated, this step can be repeated to collect as many cells as possible from the tube but make a note of the volume of suspension transferred to the Eppendorf tube.
38. Add Matrigel™ to the cell suspension, homogenise the mixture and note the added volume (see Calculation Sheet in Supplementary Information for details).
39. For each condition/cell line, calculate the final master mix volume that will be in the Eppendorf tubes. Sum the volumes of cells calculated in step 36, Matrigel™ in step 38, and the volumes of fibrinogen and thrombin to be later added (see Calculation Sheet in Supplementary Information for details).
CRITICAL STEP: The final master mix should consist of the number of 3D muscle constructs needed plus one extra construct. Assume the volume of one construct will be lost.
40. Subtract the value calculated in step 39 from the total volume of the master mix and HIDEM proliferation medium prepared with heat inactivated FBS corresponding to the value obtained.
41. Take the required volume of fibrinogen using a P200 pipette (see Calculation Sheet in Supplementary Information for details), quickly dispense it and immediately after, resuspend the cell mixture with a P1000 pipette at least 10 times so that the fibrinogen to avoid forming clumps.
CRITICAL STEP: thaw fibrinogen at 37 °C for at least 3 minutes. This will ensure that the fibrinogen is less viscous for more accurate pipetting.
CRITICAL STEP: prepare both P200 and P1000 pipettes with tips attached prior to step 41, to be as quick as possible to prevent fibrinogen clumps forming, leading to defective constructs.
CRITICAL STEP: when releasing the fibrinogen into the Eppendorf tube, do not touch the cell suspension with the pipette tip otherwise clump could form blocking the tip.
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42. Remove the spacers from the agarose and place the PDMS post rack into the moulds.
CRITICAL STEP: make sure both PDMS posts are positioned correctly into the moulds before proceeding with the next steps.
43. Homogenise the content of the Eppendorf tube. Take 113 µl of the mixture and add it to a single thrombin aliquot (see Calculation Sheet in Supplementary Information for details). Quickly mix by pipetting up and down 1-2 times and decant content between the PDMS posts filling the gap.
CRITICAL STEP: make sure that the mixture is quickly mixed with thrombin for a maximum of two times to prevent formation of fibrin in the tube or tip. Make sure no bubbles are formed.
CRITICAL STEP: do not push the pipette until the end as bubbles might be released into the mixture which might result in potential premature snapping of the muscle constructs.
44. Cover the plate and place inside the incubator at 37 °C with 5% CO2 and 18% O2 for 2 hours.
45. Prepare a solution with normal HIDEM proliferation medium supplemented with aprotinin (1:1000) to prevent fibrin degradation. Add 1.5 ml per well of a new 24-well plate.
46. Add 1 ml DMEM per well on top of the hydrogels and incubate at 37 °C with 5% CO2 and 18% O2 for 10 minutes. This facilitates dislodging the hydrogels from the 2% agarose moulds.
47. Transfer the PDMS post racks with hydrogels to the 24-well plate containing the prewarmed medium prepared in step 45. Incubate constructs at 37 °C with 5% CO2 and 18% O2 for 48 hours.
48. Change medium with HIDEM proliferation medium supplemented with 1 µM 4-OH tamoxifen and aprotinin (1:1000) (first tamoxifen pulse).
49. After 24 hours, change medium with HIDEM differentiation medium supplemented with 1 µM 4-OH tamoxifen and aprotinin (1:1000) (second tamoxifen pulse).
50. Keep the hydrogels in culture until day 9, change medium every other day with HIDEM differentiation medium supplemented with aprotinin (1:1000).
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51. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.1.2 Single lineage artificial muscle by transgene-free method
CRITICAL STEP: at least 24 hours prior to beginning the protocol, make sure that the Teflon spacers and PDMS post racks are cleaned and autoclaved and that an adequate amount of 2% agarose, thrombin, fibrinogen, Matrigel™ and aprotinin aliquots are available (see Calculation Sheet, Reagent Setup and Equipment Setup)
52. Expand the hiPSC-derived myogenic progenitors (from step 6) to 75% confluence prior to making hydrogels.
PAUSE POINT: 106 cells per hydrogel are required, we suggest expanding the myogenic progenitors in T175 flasks until they reach ~80% confluency.
53. On the day, pre-treat the hiPSC-derived myogenic progenitors with 1:300 ROCKi for 2 hours at 37 °C with 5% CO2 and 18% O2.
54. Place thrombin and Matrigel™ aliquots on ice to thaw. Fibrinogen aliquots can be kept at RT. Label one 1.5ml Eppendorf tube per condition/cell line and place them on ice.
55. Prepare an appropriate amount of heat inactivated MYOCEA myoblast medium to resuspend cells to be embedded in hydrogels. Volume needed depends on the number of hydrogels to be made. For example, 5 ml of inactivated MYOCEA myoblast medium is sufficient to make 20 constructs.
CRITICAL STEP: as MYOCEA myoblast medium contains horse serum, it needs to be inactivated to prevent interference with the polymerising factors, which would lead to an impaired hydrogel structure. Inactivation can be achieved by incubating a MYOCEA myoblast medium-containing 1.5 ml Eppendorf tube at 56 °C for 30 minutes.
56. Detach cells by adding 2 ml of trypsin-EDTA in each T175 flasks and incubating at 37 °C with 5% CO2 and 3-5% O2 for up to 5 minutes.
57. Neutralise the trypsin-EDTA with DMEM resuspension medium and count cells.
58. Follow steps 34-44 to generate the transgene-free artificial muscles.
59. Prepare a solution with normal MYOCEA myoblast medium and aprotinin (1:1000). Add 1.5ml per well of a new 24-well plate.
60. Transfer the PDMS post racks with hydrogels to the 24-well plate containing prewarmed MYOCEA myoblast medium and incubate 37 °C with 5% CO2 and 18% O2 for 48 hours.
61. After 2 days, switch to MYOCEA fusion medium supplemented with aprotinin (1:1000) to begin terminal differentiation.
62. Keep hydrogels in culture for at least 7 days, change medium every other day with MYOCEA fusion medium supplemented with aprotinin (1:1000).
63. To generate constructs containing PAX7+ cells, follow steps 53-60 and use early passage hiPSC-derived myogenic progenitors (cells at day 2 of the MYOCEA myoblast stage60).
64. Transfer hydrogels to prewarmed MYOCEA myoblast medium supplemented with aprotinin (1:1000).
65. Change MYOCEA myoblast medium with aprotinin (1:1000) every other day for the subsequent 5 days.
66. On day 6, switch to MYOCEA fusion medium and aprotinin (1:1000) to begin terminal differentiation.
67. Keep the hydrogels in culture until day 14 adding fresh MYOCEA fusion medium supplemented with aprotinin (1:1000) every other day.
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68. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.2 hiPSC-derived bi-lineage muscles containing skeletal myofibres and motor neurons
1.2.2.1 Bi-lineage artificial muscle using neurospheres
69. Generate the desired number of single lineage artificial muscles as described in steps 28-47.
70. Prepare a mixture of 113 μl of MegaCell DMEM and 14 μl of thrombin. Keep on ice.
71. Hold the PDMS post in hand and deposit 5 μl of fibrinogen on the artificial muscle.
CRTICAL STEP: warm up fibrinogen by hand or in a bead bath for 3 minutes to reduce its viscosity to pipette more accurately.
72. Collect at least 3 of the neurospheres described in step 8 using a 20 μl pipette and add within the fibrinogen drop.
CRITICAL STEP: when collecting the neurospheres, aspirate as little neurosphere proliferation medium as possible to avoid negative interactions with fibrinogen.
73. Immediately add 7 μl of the MegaCell DMEM and thrombin solution (from step 70) on top of the neurospheres and fibrinogen mixture. Wait 3 minutes to allow formation of fibrin.
74. Transfer hydrogels to a 24-well plate containing 1.5 ml of neurosphere bi-lineage differentiation medium supplemented with 1 μM of 4-OH tamoxifen, 5 μg/ml heparin and aprotinin (1:1000) per well and place in the incubator at 37 °C with 5% CO2 and 18% O2.
75. After 24 hours, perform a full medium change with fresh neurosphere bi-lineage differentiation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000).
76. Change neurosphere bi-lineage differentiation medium supplemented with aprotinin (1:1000) every other day until day 6.
77. From day 6, change medium with neurosphere bi-lineage differentiation medium supplemented with 1 μM purmorphamine (PM) and aprotinin (1:1000) every other day until day 14.
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78. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.2.2 Bi-lineage artificial muscle using NPCs (as single cells, not in neurospheres)
79. Thaw and culture the HIDEMs and hiPSC-derived NPCs as described in steps 1-3 and 10-13, respectively, until they reach 90-100% confluence.
80. To prepare reagents for making the bi-lineage complex artificial muscles, follow the steps 30-31.
81. Pre-treat both NPCs and HIDEMs with ROCKi (1:300) for 2 hours prior to making the bi-lineage complex muscle constructs.
82. Collect the required cell numbers of HIDEMs by following step 32.
PAUSE POINT: A mix of 70% HIDEMs and 30% NPCs (e.g., 7 × 105 and 3 × 105 per hydrogel respectively) is required.
83. Incubate NPCs with 0.5 mM EDTA at 37°C for 5 minutes and then remove the EDTA solution. Add N2B27 medium.
84. Detach cells from the bottom of the dish by gently pipetting up and down the N2B27 medium. When cells have detached, collect in a 15ml falcon tube and count NPCs.
85. Upon mixing the appropriate number of the two cell types, spin cell mixture at 300 rcf for 5 minutes.
86. Discard supernatant and add a minimal volume of NPC bi-lineage proliferation medium containing heat inactivated FBS to resuspend the cell mixture. We suggest adding 50-100 µl of heat inactivated NPC bi-lineage proliferation medium depending on the number of hydrogels (see Calculation Sheet in Supplementary Information for details).
87. Note cell mixture volume, then aspirate the contents using a P200 pipette and transfer to the respective Eppendorf tubes.
88. Follow steps 38-44 to generate the bi-lineage constructs using NPC bi-lineage proliferation medium with heat inactivated FBS.
89. Transfer hydrogels into prewarmed NPC bi-lineage proliferation medium supplemented with aprotinin (1:1000) and incubate the hydrogels at 37 °C with 5% CO2 and 18% O2 for 48 hours.
90. Change medium with NPC bi-lineage proliferation medium supplemented with 1 µM 4-OH tamoxifen, 0.1 µM y-secretase inhibitor and aprotinin (1:1000) (first tamoxifen pulse).
91. After 24 hours, change medium with NPC bi-lineage differentiation medium with 1 µM 4-OH tamoxifen, 0.1 µM y-secretase inhibitor and aprotinin (1:1000) (second tamoxifen pulse).
92. Hydrogels are kept in culture performing daily changes of NPC bi-lineage differentiation medium containing 0.1 µM y-secretase inhibitor and aprotinin (1:1000).
93. On day 5, perform an NPC bi-lineage differentiation medium change with supplementation of 0.1 nM agrin and aprotinin (1:1000). Perform the same NPC bi-lineage differentiation medium change using 0.5 nM and 1 nM of agrin on day 6 and 7, respectively.
94. From day 8, change medium with NPC bi-lineage differentiation medium supplemented with aprotinin (1:1000) and 1 nM of agrin daily until day 14 (or beyond this date for longer cultures).
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95. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.3 hiPSC-derived tri-lineage artificial muscles containing myofibres and blood vascular endothelial cells and pericytes
96. Culture HIDEMs (steps 1-3), ECs (step 21) and PCs (step 27) in T175 flasks containing HIDEMs proliferation, EC-SFM and EGM2 medium, respectively, until they reach desired confluence (see section 1.14 ).
97. To prepare reagents for making the tri-lineage complex artificial muscles, follow steps 30-31.
98. Pre-treat ECs, PCs and HIDEMs with ROCKi (1:300) 2 hours prior to making the tri-lineage complex muscle constructs.
99. Obtain the required cell numbers of HIDEMs, ECs and PCs by following steps 32-34. Make sure to use the appropriate medium for each cell type for inactivation of trypsin.
PAUSE POINT: A mix of 70% HIDEMs (7 × 105) and 30% vascular cells (6% ECs and 24% PCs (6 × 104 ECs and 2.4 × 105 PCs)) per hydrogel is required.
100. Spin cells at 300 rcf for 5 minutes, discard supernatant and resuspend in appropriate volume of tri-lineage proliferation medium with heat inactivated FBS.
101. Follow steps 38-44 to generate the tri-lineage constructs using tri-lineage proliferation medium with heat inactivated FBS.
102. Transfer the PDMS post rack with hydrogels into prewarmed tri-lineage proliferation medium containing aprotinin (1:1000) and incubate the hydrogels at 37 °C with 5% CO2 and 18% O2 for 48 hours.
103. Change medium with tri-lineage proliferation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000) (first tamoxifen pulse).
104. After 24 hours, change the medium with tri-lineage differentiation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000) (second tamoxifen pulse).
105. Keep the hydrogels in culture until day 17 performing a full medium change with tri-lineage differentiation medium supplemented with aprotinin (1:1000) every other day.
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106. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.4 Tetra-lineage artificial muscles containing myofibres, motor neurons and vascular cells
1.2.4.1 Option A - Neurospheres:
107. Generate a tri-lineage construct following steps 96-101.
108. After 1 hour of polymerisation, decant 3-6 neurospheres on top of each hydrogel using 10 μl of fibrinogen.
109. Transfer the PDMS post racks with hydrogels into prewarmed neurosphere tetra-lineage proliferation medium containing aprotinin (1:1000) and keep at 37 °C with 5% CO2 and 18% O2 for 48 hours.
110. Change medium with neurosphere tetra-lineage proliferation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000) (first tamoxifen pulse).
111. After 24 hours, change medium with neurosphere tetra-lineage differentiation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000) (second tamoxifen pulse).
112. Perform medium changes every 2 days with neurosphere tetra-lineage differentiation medium and aprotinin (1:1000) until day 7.
113. From day 8, perform full medium changes with fresh neurosphere tetra-lineage differentiation medium supplemented with 1 μM of PM and aprotinin (1:1000) every other day.
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114. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.2.4.2 Option B - Neural Progenitor Cells (NPCs):
115. Culture HIDEMs (steps 1-3), ECs (step 21) and PCs (step 27) in T175 flasks containing HIDEMs proliferation, EC-SFM and EGM2 medium respectively until they reach appropriate confluence.
116. Culture NPCs as described in steps 10-13 until the full confluence of at least one 6-well plate is reached.
117. To prepare reagents for making the tetra-lineage artificial muscles, follow steps 28-30.
118. Pre-treat ECs, PCs, HIDEMs and NPCs with ROCKi (1:300) for 2 hours prior to making the tetra-lineage constructs.
119. Obtain the required numbers of HIDEMs, ECs and PCs by following steps 32-34. Make sure to use the appropriate medium for each cell type for inactivation of trypsin.
120. Count the number of NPCs.
121. Mix the 4 different cell types in the required ratio and spin the cell mixture at 300 rcf for 5 minutes.
PAUSE POINT: 70% myogenic cells (7 × 105), 15% vascular cells (10% ECs and 5% PCs (1 × 105 ECs and 5 × 104 PCs)) and 15% NPCs (1.5 × 105).
122. Discard supernatant and resuspend cells in a minimal volume of NPC tetra-lineage proliferation medium containing heat inactivated FBS. We suggest adding 50-100 µl of heat inactivated NPC tetra-proliferation medium depending on the number of hydrogels (see Calculation Sheet in Supplementary Information for details).
123. Follow steps 38-44 to generate hydrogels using NPC tetra-lineage proliferation medium with heat inactivated FBS.
124. Transfer the PDMS post racks with hydrogels into prewarmed NPC tetra-lineage proliferation medium containing aprotinin (1:1000) 2 hours after embedding the cells into fibrin hydrogels and incubate at 37 °C with 5% CO2 and 18% O2 for 48 hours.
125. Change medium with NPC tetra-proliferation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000) (first tamoxifen pulse).
126. After 24 hours, change medium with NPC tetra-lineage differentiation medium supplemented with 1 μM of 4-OH tamoxifen and aprotinin (1:1000).
127. Keep the gels in culture until day 24 and add replace NPC tetra-differentiation medium supplemented with aprotinin (1:1000) every other day.
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128. Upon completion of differentiation, proceed to functional assays: steps 185-202 for electrical stimulation contraction or steps 203-223 for calcium transients. Alternatively, fix the gels in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Or proceed directly to steps 161-171 for mRNA expression analysis and steps 172184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
1.3 Characterisation of 3D artificial muscle
Please note that this section is organised based on technical workflows and not according to the tiers mentioned in figure 3, which we advise the reader to follow.
1.3.1 Morphological characterisation
1.3.1.1 Immunofluorescence
129. Remove the fixed hydrogels from the PDMS posts using the tweezers and transfer to a conical tube containing the blocking buffer. The artificial muscles can also be stored in PBS at 4°C for up to 1 week.
CRITICAL STEP: carefully run the tweezers along one post and remove one end of the construct. Follow the same procedure for the other end of the construct. Do not touch the central portion of the hydrogels with tweezers as this could damage the construct.
130. Blocking and permeabilisation: incubate fixed artificial muscles in blocking buffer for 6 hours at 4°C to block non-specific binding.
131. Remove the blocking buffer and incubate with primary antibody/antibodies in permeabilisation buffer at 4 °C overnight.
132. Remove the primary antibody and wash the samples six times for 60 minutes each in TBS 1X at RT.
133. Incubate samples with secondary antibody/antibodies and Hoechst in permeabilisation buffer at 4 °C overnight on a plate shaker.
134. Remove the secondary antibody and wash samples six times for 60 minutes each in TBS 1X at RT on a plate shaker.
CRITICAL STEP: antibodies conjugated with fluorophores are light sensitive, so step 134 on must be performed while protecting the samples from light (e.g., by covering samples with aluminium foil).
135. Once the immunofluorescence is completed, either proceed with confocal (steps 136-145) or light-sheet microscopy (steps 146-160).
PAUSE POINT: immunohistochemistry can also be performed on sectioned constructs to further characterise and image tissue architecture. For further details see Maffioletti et al., 2018 1.
1.3.1.2 Confocal and light sheet microscopy
1.3.1.2.1 Confocal microscopy
136. For confocal imaging, mount the immunolabelled hydrogels (129-135) on microscope glass slides with a concave depression using 2-3 drops of fluorescence mounting medium.
137. Gently place a coverslip on top of the sample and let dry for 20 minutes.
CRITICAL STEP: make sure the constructs are positioned in the middle of the concave depression, covered with fluorescence mounting medium while avoiding bubbles.
PAUSE POINT: to seal the coverslip to the glass slide, nail polish can be applied to the sides of the coverslip and left to dry for 20 minutes at RT.
138. Place the slide onto the stage of the confocal microscope and use the following settings: 63× objective, scanning parameters: 1,024x1,024, speed of 600, zoom factor 1.0, frame average of 3 and z-stack spacing of 1.5 µm. Set the fluorescent filters as required and their intensity, which should not exceed a wavelength of 30 nm.
139. For 3D reconstruction of nuclei, use the same settings as in the step 138 but use a zoom factor of 1.5 and z-stack spacing of 0.5 µm.
140. As the 63× is an immersion objective, dispense two drops of the immersion oil on top of the coverslip prior to imaging.
141. Lower the objective until it contacts the immersion oil. Use the ocular to further lower the objective until the sample is in focus.
142. Select the field to image and set the start and end of the Z position moving the objective up and down across the sample. This will represent the portion of artificial muscle over which the z-stack will be collected.
PAUSE POINT: we advise choosing the start/end of the Z position based on the fluorescent channel of major interest (e.g., Lamin A/C to reconstruct myonuclei)
143. For each fluorescent channel, use the LUT tool to adjust the gain and the offset to improve signal intensity and background noise respectively.
144. Initiate imaging. Upon completion of this process, use the Intensity Max Projection tool to combine stack to generate a single image to be exported as a TIFF file.
145. For 3D nuclear reconstruction, export the files in LIF format and proceed to step 224.
1.3.1.2.2 Light sheet microscopy method
The steps listed are the same as recommended by the manufacturer, with minor changes to better fit 3D artificial skeletal muscles. It is worth noting that the 3D artificial skeletal muscle is quite opaque and so the maximum depth that can be imaged is 1-2 myotubes from the exterior of the construct.
146. Prepare the light sheet fluorescence microscope by setting up the objective (5×), the filter cubes and perfusion chamber (filled with PBS to the top of the chamber windows) onto the chamber mount. Make sure the relevant filter cubes are installed.
147. Assemble the syringe into the microscope’s sample holder disk with syringe adapter ring.
148. Following immunolabelling (steps 129-135), using forceps, place the construct on a 10 cm dish.
CRITICAL STEP: ensure that there is no excess of PBS but that the construct is not completely dry.
149. Dip the tip of the needle into a droplet of superglue and attach to the furthest end of the muscle construct, having a couple of millimetres of overlap between the needle and construct so that both are in tandem.
CRITICAL STEP: this step must be performed rapidly as superglue sets quickly.
150. Leave to set for 30 seconds and then attach the needle to the syringe.
151. Slowly mount the sample holder disk onto the microscope stage and into the upper opening.
152. Using the Zen software specimen locate sample tool, find and centre the specimen along the 3 axes.
153. In the acquisition tab, setup the light path by defining the necessary tracks.
154. Activate multi-view checkbox in multidimensional input field, use the now-available z-stack tool window to select the upper and lower limit of imaging using the “set first” and “set last” buttons, the interval (recommended 1 μm) and the number of rotations (recommended 3).
155. Start imaging of the constructs by clicking start experiment.
156. Once done, remove the holder disk from stage and the needle from the syringe.
157. Place sample gently onto 10 cm dish and hold needle at 30°, then quickly slide the blade on the needle to detach the superglued muscle construct from the needle.
158. Using forceps, place the construct back into PBS-filled conical tube and keep away from light.
159. The acquired image is exported in CZI file extension and imported in Arivis Vision4D software.
160. Reconstruct the 3D muscle and export images and videos.
PAUSE POINT: the same sample can be imaged by both light sheet and confocal microscopy. It is advisable to not excessively manipulate the artificial muscle to preserve its structure and to perform steps 146-160 quickly to avoid photobleaching of the fluorophores.
1.3.2 Molecular characterisation
1.3.2.1 mRNA expression analysis
161. Wash constructs 3 times with PBS, then remove hydrogels from the PDMS posts as described in step 129 and transfer to Eppendorf tubes containing 500 μl of Trizol per sample.
PAUSE POINT: if RNA extraction is not performed immediately, dry constructs can be stored in vials/tubes at -80°C for up to 6 months.
162. Homogenise for approximately 30 seconds on ice, then incubate at RT for 5 minutes.
163. Add 100 μl of chloroform per sample and shake intermittently for 3 minutes at RT.
164. Centrifuge at 12,000 rcf (or more) for 15 minutes at 4°C.
165. Transfer aqueous phase containing the RNA without touching the interphase to a new Eppendorf tube.
166. Add 250 μl of isopropanol to the aqueous phase. Incubate for 10 minutes and centrifuge at 12,000 rcf for 10 minutes at 4°C
167. Discard supernatant and add 1 ml of chilled 70% ethanol, vortex and centrifuge at 7,000 rcf for 5 minutes at 4°C.
168. Discard supernatant, air dry the RNA pellet for few minutes at RT and resuspend in 20 μl of nuclease free water and measure the RNA yield and purity using a Nanodrop.
169. Use 1 μg of RNA to retro-transcribe to cDNA with the ImProm-II™ Reverse Transcription System kit.
170. Perform a PCR for a housekeeping gene (e.g. GADPH, TBP) to evaluate the efficacy of the retro-transcription reaction.
171. Perform Quantitative Real Time-PCR to assess gene expression (e.g., for myogenic factors) with the GoTaq® qPCR Master Mix kit following manufacturer’s directions using a BioRad CFX96 machine.
1.3.2.2 Western Blot
172. Add ice-cold lysis buffer to cell pellets (50 μl/ sample) or 3D muscle constructs (200 μl/ sample).
173. Homogenise samples for 30 seconds using the Ultra-Turrax homogeniser (power level 5, 20,500 rpm) on ice.
174. Incubate samples on ice for 30 minutes, mixing very well every 5-10 minutes and then centrifuge at 12,000 rcf for 10 minutes at 4°C.
175. Collect supernatant (containing proteins) and use to determine sample concentration. Protein suspensions can be stored at -80°C indefinitely.
176. Prepare a standard curve using bovine serum albumin (BSA) 8 mg/ml to 0.5 mg/ml and quantify samples using the colourimetric reaction DC Protein Assay and the iMark microplate reader.
177. Heat samples at 98°C for 5 minutes in reducing loading Laemmli buffer.
PAUSE POINT: if samples are stored at -80°C, thaw on ice before use.
178. Load a molecular weight ladder and 30 μg of protein per sample on the 4%-15% precast gel and run at 100 V for 2 - 3 hours.
179. Transfer the proteins onto the polyvinylidene difluoride (PVDF) membrane using a semi-dry transfer system (iBlot gel transfer system) at 20 V for 7 minutes.
180. Stain the membrane using Ponceau S to confirm correct protein transfer.
181. Rinse the membrane 3 times using TBST buffer then block using milk-TBST blocking buffer for 60 minutes at RT.
182. Incubate PVDF membrane in milk-TBST blocking buffer containing primary antibodies of choice at respective concentration on a shaker overnight. Wash 3 times using TBST buffer for 10 minutes each at room temperature.
183. Incubate PVDF membrane in milk-TBST blocking buffer containing HRP-conjugated secondary antibody at respective concentration for 1 hour at RT on a shaker. Then wash 3 times using TBST buffer for 10 minutes each at RT.
184. Visualise and develop the signal by incubating the membrane with ECL reagents, according to manufacturer’s instructions, and image using the chemiluminescence detector (ChemiDoc).
1.3.3 Functional characterisation
1.3.3.1 Contractility assay
185. Assemble the imaging set up: the plate holder, the lamp and the inverted Basler camera and connect to computer.
186. On the computer, create a new folder (with subfolders) to save the recordings, and start the Pylon program and select the Basler ace camera.
187. Select “Windows” and then “Recording setting” and set the acquisition speed to 25 fps (records a frame every 40 milliseconds), the recording time to 20 seconds and select the desired output folder.
188. Prepare the electric stimulator and connect it to the autoclaved Pacing electrodes.
189. Add 500 μl of warm differentiation medium to each well to reach a total volume of 2 ml per well.
190. Place on the plate-holder and remove lid. Position the light above the skeletal muscle construct and position the camera under it.
191. Gently mount the carbon electrodes on the well containing the muscle construct. Dip the two electrodes into the medium. Use Blu Tack® to fix the electrodes to the plate.
CRITICAL STEP: Avoid touching the constructs with the electrodes or the electrodes touching each other.
192. Switch on the electrical stimulator.
CRITICAL STEP: Make sure that the pulse is off at this stage.
193. On the pylon software, select the camera icon and record the first video of the hydrogel without an electrical stimulus; this is the baseline.
194. Set up the desired voltage, frequency on the electrical stimulator to achieve the required current.
PAUSE POINT: start with a stimulation of 2 Hz and 5 V. Increase voltage or frequency if the aim is to further challenge the muscle.
195. Select the new destination folder, turn the pulse switch to the on state and start recording the video immediately. Alternatively, start recording before turning on the stimulation to obtain the baseline and contraction from the same video.
196. Turn off the pulse switch and the stimulator.
197. Gently remove the electrodes and fix the constructs with PFA 4% for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform normal confocal imaging and continue with steps 129-135 for immunofluorescence. Alternatively, proceed directly to steps 161-171 for mRNA expression analysis or steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
Analysis:
198. To analyse the video (e.g. supplementary video 1), install the MUSCLEMOTION plugin on ImageJ.
199. Drag the folder with the pictures to FIJI and in select “Use virtual stack” to mount the video.
200. Save the entire video.
201. Click on the MUSCLEMOTION icon. In the “analysis parameters wizard” insert the frame rate of the recording (25 fps in step: 187) and the speed to analyse the video should be set to 1 frame. Select “yes, but keep it simple” to decrease the noise in the output and detecting the reference frame and “No” for analysis of the transient time.
PAUSE POINT: Refer to the MUSCLEMOTION user manual for further information regarding the plugin and settings.
202. Select the folder to save the analysis and click on the video to analyse.
1.3.3.2 Whole construct calcium transients
203. Incubate artificial muscles anchored to the PDMS post racks in Fluo-4 Loading Buffer for 30 minutes at 37°C.
204. Wash the constructs using DMEM for 5 min.
205. Transfer constructs and posts on glass bottom dishes.
CRITICAL STEP: For high quality imaging, make sure constructs adhere to the bottom of the wells. If the construct does not touch the bottom of the plate, fill the gap with medium.
206. Set the long-term time-lapse wide-field system Nikon microscope to fluorescence excitation of 488 nm with a 10X lens. Set image acquisition every 0.333 sec for a total of 90 sec.
CRITICAL STEP: Ensure that the CO2 and temperature settings in the chamber are correct.
207. Place the plate on the stage and gently mount the carbon electrodes on the well containing the muscle construct as described in step 188 and 191.
208. Start the time lapse acquisition. The first 10 seconds are acquired as baseline, after setting up the desired voltage and frequency, switch on the electrical stimulator.
209. At the end of recording constructs can be fixed or used for RNA/protein extraction.
Analysis:
210. Export the image series into Fiji / ImageJ
211. Click on Image and select the Stacks option. Click on Plot Z-axis profile. Save the data as xls.
1.3.3.3 Single cell calcium transients
212. With the 3D artificial muscles retained on the PDMS post racks, incubate in Fluo-4 Loading Buffer for 30 minutes at 37°C.
213. Wash the constructs using recording buffer.
214. Take the 3D artificial muscles out of medium and carefully disassemble them from the PDMS posts using fine tip forceps, place them in a 10 cm dish and submerge samples in recording buffer.
CRITICAL STEP: remove 3D artificial muscles very gently and place carefully to avoid snapping them. For the best imaging quality, make sure most of the construct body is sticking to the bottom of the plate.
215. Set the Zeiss 880 confocal LSM to fluorescence excitation of 488 nm and emission collected at >530 nm and prepare to acquire as a time series at 33 frames per second (fps).
CRITICAL STEP: make sure that the CO2 and temperature settings in the chamber are correct.
216. Place the plate under the stage, choose the field with most aligned myotubes and perform imaging using a 10× objective.
217. Add caffeine to the recoding buffer at a final concentration of 10 mM and immediately mark the time/captured frame on the microscope software.
218. Repeat step 217 when fluorescence excitation subsides, for as many times as necessary.
219. Fix the constructs in 4% PFA at 4°C for 3 hours to reconstruct the nuclei in 3D or 8 hours to perform regular confocal imaging and continue with steps 129-135 for immunofluorescence. Alternatively, proceed to steps 161-171 for mRNA expression analysis and steps 172-184 for western blotting. Constructs to be used for mRNA expression analysis and western blotting can be snap frozen in a conical microtube in liquid nitrogen and stored in -80 °C.
Analysis:
220. Export time series from microscope and open in Fiji software (also known as ImageJ).
221. Select areas (each representing a myotube and covering a maximum of 10% of its area to avoid interfering signals from other cells) using the “Lasso” tool.
222. From the “Analyse” menu, choose “Set measurements” and select “Mean grey value”.
223. From the “Image” menu, select “Stacks”, “Plot z axis profile” and save as an xls file.
1.4 Disease Modelling: a stepwise guide to study nuclear morphology and model laminopathy-associated nuclear abnormalities using 3D artificial muscles
One of the most characteristic pathological hallmarks of laminopathies is nuclear shape abnormality. This aspect can be modelled utilising the 3D artificial muscle platform described in this protocol by reconstructing the nuclei in three-dimensions2. For both 3D artificial muscles and 2D classic monolayer cultures, the imaging and the three-dimensional nuclear reconstruction were performed in the same way for a more robust comparison. For more details see also Supplementary Information.
224. Import the raw (LIF) files in Imaris® 8.4.1, obtained in step 145, by clicking “Image” in the upper menu (the images will automatically appear in the “Arena” window). Images can also be arranged in folders named “Assays” or “Groups” also found in the upper menu.
225. Select the image to reconstruct, which will show under the “Surpass” window in a “3D view”. To reconstruct the nuclei, click on to “Add new surfaces” on the left-hand menu. The “Surface” window will show in the below panel. If the “Display Adjustment” window does not appear automatically, it can be opened by clicking Edit>Show Display Adjustment. This function will allow the operator to tick and untick the channels to be visualised.
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226. Algorithm: choose “Default” as a Favourite Creation Parameters and proceed by clicking on the blue arrow at the bottom of the panel.
PAUSE POINT: the operator can also generate and store Creation Parameters that can be applied to any number of grouped images.
227. Source channel: select the channel to reconstruct (e.g., the red channel indicating lamin A/C), tick the box “Smooth” and choose the “Surface Detail” value that better sets up the smoothness of the resulting area (e.g., 0.200-0.300 µm). Tick “Absolute Intensity” box as the parameter to base the thresholding on and proceed by clicking on the blue arrow at the bottom of the panel.
228. Threshold: this is based on the intensity of the immunofluorescence in the analysed image. The lower the value the more objects are detected and reconstructed within the image. By ticking the box “Enable”, it is possible to split the touching objects. Click on the blue arrow at the bottom of the panel.
CRITICAL STEP: it is advisable to set the threshold at around a value that allows the detection of low-intensity objects while preserving the natural size of the objects with higher fluorescence intensity.
229. Classify surfaces: this function allows the removal of objects that can interfere with the final results and analysis. To achieve this, several filters can be selected (e.g., number of voxels, area, volume, etc.). Use this function to remove small unspecific and partially reconstructed objects that are not relevant. Click on the green arrow on the bottom of the panel to execute the creation steps.
CRITICAL STEP: ensure most unspecific objects are removed. To achieve this goal, untick/tick the “Volume” box on the upper panel to better understand what is the specific and the unspecific signal detected.
230. Major axis length analysis: upon 3D reconstruction of the nuclei, select “Add new measurement point” in the left panel menu (where the “Add surfaces” tool is).
231. Choose “Settings” in the below panel to set the point/line shape, size and labels. Select “Pairs” for “Line mode” and tick “Distance” for “Line Labels”.
232. Click on “Edit” and choose “Surface of object” as the intersection site when adding the points. This action is performed by shift-clicking the left mouse button to add the point on to the desired object. Observe the image and use the “Navigate” function on the top right corner menu under the item “Pointer” to assess which myonucleus to measure.
CRITICIAL STEP: use the “Display adjustment” tool to assess whether a nucleus is located within or outside a myotube.
233. Choose “Select” from the top right menu to pinpoint one extremity of the myonucleus by shift-clicking the mouse left button. After selecting “Navigate” and turning the image, add the second extremity to the same nucleus. The distance between the two points will display automatically.
CRITICAL POINT: make sure the extremities are positioned in the most appropriate way by selecting “Surface”> “Colour”> “Transparency”. This function will allow a better visualisation of the line throughout the objects.
234. After measuring the desired objects in the image, select “Statistics”, click on “Detailed” tab, “Specific Values” and export the excel files including all the nuclear long axis values to the desired directory. The resulting spreadsheet will include all relevant information such as the type of object measured (e.g., surface), the outputted value, the unit of measurement as well as the object ID to enable tracking of analysed items. Examples of possible additional measures other than the major axis length, include nuclear surface area and volume.