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MUSCLE Publication-derived

A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS

Source Massih et al., 2023 · Institute of Clinical Neurobiology, University Hospital Würzburg · 10.3389/fcell.2023.996952

👤 Bita Massih, Alexander Veh, Maren Schenke, Simon Mungwa, Bettina Seeger, Bhuvaneish T. Selvaraj, Siddharthan Chandran, Peter Reinhardt, Jared Sterneckert, Andreas Hermann, Michael Sendtner, Patrick Lüningschrör ⏱ 42 days 📋 10 phases 🧫 Human iPSC-derived motoneurons; Primary human skeletal myoblasts

Abstract

This protocol describes a 3D neuromuscular co-culture system combining iPSC-derived motoneurons and myoblast-derived skeletal muscle tissue to model human neuromuscular junction (NMJ) physiology and dysfunction. The system uses self-microfabricated silicone dishes with Velcro hooks to support 3D muscle tissue formation in a defined extracellular matrix, enabling functional NMJ formation and maturation for studying motoneuron diseases including ALS.

Cell source
Human iPSC-derived motoneurons; Primary human skeletal myoblasts
Application
Disease modeling

Protocol overview

82 steps across 10 phases

Human myoblast and fibroblast culture Prior to differentiation
  1. 1 Prepare myoblast growth medium
  2. 2 Culture primary human skeletal myoblasts
  3. 3 Culture primary human fibroblasts
iPSC differentiation into neuronal progenitor cells (NPCs) Days 1–21+
  1. 1 Expand iPSCs on Matrigel-coated dishes
  2. 2 Split iPSCs with ReLeSR reagent
  3. 3 Induce neuronal differentiation (Day 0)
  4. 4 Change to neuronal medium (Day 2)
  5. 5 Change to expansion medium (Day 4)
  6. 6 Select and dissociate embryoid bodies (Day 6 onwards)
  7. 7 Expand NPC cultures
MN differentiation according to Reinhardt et al. protocol Days 0–23
  1. 1 Culture NPCs on Matrigel-coated dishes in expansion medium
  2. 2 Initiate MN specification (Day 0)
  3. 3 Add Retinoic Acid (Day 2)
  4. 4 Change medium every other day (Days 0–9)
  5. 5 Switch to maturation medium (Day 9)
  6. 6 Increase GDNF/BDNF concentrations (Day 11)
  7. 7 Continue maturation culture
MN differentiation according to Kroehne et al. protocol Days 0–20+
  1. 1 Expand NPCs with smoothened agonist
  2. 2 Initiate NPC differentiation (Day 0)
  3. 3 Switch to MN maturation medium (Day 6)
  4. 4 Continue maturation for 14+ days
Self-manufactured 3D co-culture dishes (PDMS fabrication) Prior to co-culture assembly
  1. 1 Coat Petri dishes with liquid PDMS (first layer)
  2. 2 Add second PDMS layer and place templates
  3. 3 Remove air bubbles
  4. 4 Cure and anchor Velcro
  5. 5 Sterilize dishes
  6. 6 Store sterilized dishes
  7. 7 Prepare PDMS molds with Pluronic acid
Generation of 3D skeletal muscle tissue Days 0–14
  1. 1 Prepare hydrogel mixture
  2. 2 Fill PDMS channels with hydrogel
  3. 3 Incubate hydrogel in incubator
  4. 4 Prepare 3D differentiation medium
  5. 5 Add differentiation medium to hydrogel
  6. 6 Culture muscle tissue in incubator
  7. 7 Change medium every other day
3D neuromuscular co-cultures Days 0–42
  1. 1 Prepare hydrogel with muscle cells (co-culture protocol)
  2. 2 Add motoneurons to hydrogel
  3. 3 Prepare co-culture medium (Reinhardt protocol-derived MNs)
  4. 4 Prepare co-culture medium (Kroehne protocol-derived MNs)
  5. 5 Add co-culture medium to hydrogel + MNs
  6. 6 Increase growth factors after 2 days (Reinhardt protocol)
  7. 7 Increase growth factors after 2 days (Kroehne protocol)
  8. 8 Maintain co-cultures with medium changes every other day
Immunocytochemical staining and analysis Days 9–42 (at experimental endpoints)
  1. 1 Transfer tissues to Eppendorf tubes
  2. 2 Wash tissues with PBS
  3. 3 Fix tissues in paraformaldehyde
  4. 4 Wash fixed tissues
  5. 5 Permeabilize and block tissues
  6. 6 Wash tissues after blocking
  7. 7 Incubate with α-Bungarotoxin (for AChR labeling)
  8. 8 Wash after α-BTX incubation
  9. 9 Incubate with primary antibodies
  10. 10 Wash after primary antibodies
  11. 11 Incubate with secondary antibodies
  12. 12 Wash after secondary antibodies
  13. 13 Perform final PBS wash
  14. 14 Counterstain nuclei with DAPI
  15. 15 Quick PBS wash after DAPI
  16. 16 Mount tissues on glass slides
  17. 17 Allow mounting medium to cure
Functional assays: Calcium imaging and muscle contraction Days 14–42
  1. 1 Prepare ACh stimulation stock solution
  2. 2 Prepare glutamate stimulation stock solution
  3. 3 Prepare blocker solutions (BTX, TTX, BoTN)
  4. 4 Record baseline videos before stimulation
  5. 5 Apply ACh or glutamate directly to culture dish
  6. 6 Continue video recording during stimulation
  7. 7 Analyze muscle displacement using ImageJ
  8. 8 Quantify maximal muscle displacement
  9. 9 Perform calcium imaging with GCaMP6-expressing muscle
  10. 10 Analyze calcium transients using ImageJ
  11. 11 Blind assessment for ALS co-cultures
Molecular characterization: RT-qPCR Days 0–23 (at differentiation milestones)
  1. 1 Collect cells at differentiation milestones
  2. 2 Isolate total RNA
  3. 3 Treat RNA with DNase I
  4. 4 Quantify RNA concentration
  5. 5 Synthesize cDNA from 100 ng total RNA
  6. 6 Prepare negative control (no reverse transcriptase)
  7. 7 Dilute cDNA 1:5 with water
  8. 8 Prepare qPCR reactions
  9. 9 Perform qPCR on LightCycler instrument
  10. 10 Analyze qPCR data using ΔΔCt method
  11. 11 Assess differentiation efficiency using gene expression

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Massih et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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