Millifluidic culture improves human midbrain organoid vitality and differentiation Emanuel Berger University of Luxembourg
3D midbrain organoids for modelling Parkinson s disease Healthy control Midbrain Parkinson s patient Parkinson s disease core symptoms Demands to the human midbrain organoid model: Spatial 3D organisation of heterogeneous midbrain-residing cells Physiological functions of midbrain-specific cells (e.g. dopamine release ) Stable system for extended cultivation and manipulation Patient-specificity Reproducibility Dopaminergic neurons Applications: Disease modelling, Drug testing, Regenerative medicine Double et al. 211
3D midbrain organoids Generation Human neuroepithelial stem cells: - Resemble cells of the neural plate border - Are pre-patterned towards mid-/hindbrain identity Protocol developed by A.S. Monzel et al. (Stem Cell Reports. 217) in dependence on Lancaster et al. 213
3D midbrain organoids Features Midbrain organoids exhibit neuronal, astroglial and oligodendrocyte differentiation Midbrain specific, due to mda marker expression Midbrain organoids exhibit spatial asymmetry of mda neurons Midbrain organoids are functional Synaptic connections Myelination Electrophysiological activity Monzel AS. et al. Stem Cell Reports. 217
3D midbrain organoids Limitations Dead core Shaking Solely shaking does not ensure proper supply High effort of frequent manual media change Susceptible for culture perturbations Need for fluidic culture system Agarose sections
Millifluidic culture system Cell culture chamber 6-Chamber Tray Quasi Vivo Peristaltic pump E Berger et al. Lab on a chip (accepted)
Reduced dead core size under millifluidic conditions 34769 WT T12.9 WT K7 WT A r e a r a tio (d e a d c o r e / to ta l o r g a n o id ) 34769 WT T12.9 WT K7 WT o r g a n o id a r e a [m m 2 ] Shaking Fluidic Shaking Fluidic.2 n.s. n.s. n.s. * 2. n.s. n.s. n.s. n.s. 1.5.1 1..5.. K 7 W T T 1 2.9 W T 3 4 7 6 9 W T a ll lin e s K 7 W T T 1 2.9 W T 3 4 7 6 9 W T a ll lin e s E Berger et al. Lab on a chip (accepted)
Increased oxygenation under millifluidic conditions Shaking Fluidic E Berger et al. Lab on a chip (accepted)
Accelerated dopaminergic differentiation K7 WT T12.9 WT 34769 WT T H /F O X A 2 b y n u c le a r p e r im e te r T H + F O X A 2 + b y n u c le a r p e rim e te r [p ix e l r a tio ] [p ix e l ra tio, % o f C T R L ] Shaking Fluidic TH/FOXA2/ TUJ1/Hoechst TH TH/FOXA2/ TUJ1/Hoechst TH 1 n.s. n.s. * n.s. 1-1 1-2 1-3 1-4 1-5 K 7 W T T 1 2.9 W T 3 4 7 6 9 W T a ll lin e s 8 6 4 2 E Berger et al. Lab on a chip (accepted)
Accelerated dopaminergic differentiation K7 WT T12.9 WT 34769 WT T H + F O X A 2 + [% o f s in g le c e lls ] T H + /F O X A 2 + [% o f s in g le c e lls ] Shaking Fluidic 1 8 6 4 2 K 7 W T T 1 2.9 W T 3 4 7 6 9 W T 5 4 3 2 1 E Berger et al. Lab on a chip (accepted)
Accelerated dopaminergic differentiation K7 WT T12.9 WT 34769 WT S O X 2 + b y n u c le a r m a s k S O X 2 b y n u c le a r m a s k [p ix e l ra tio, % o f C T R L ] [p ix e l r a tio ] Shaking Fluidic SOX2/Hoechst SOX2 SOX2/Hoechst SOX2 1.5 * n.s. ** * 1..5. K 7 W T T 1 2.9 W T 3 4 7 6 9 W T a ll lin e s 1 8 6 4 2 E Berger et al. Lab on a chip (accepted)
Metabolic maturation under millifluidic conditions g lu c o s e c o n s u m p tio n la c ta te p r o d u c tio n [m o l/c e ll* d a y ] [m o l/c e ll* d a y ] g lu ta m in e c o n s u m p tio n g lu ta m a te p r o d u c tio n [m o l/c e ll* d a y ] [m o l/c e ll* d a y ] 2. 1-1 3 *** ** *** -2. 1-1 2-4. 1-1 2 1.5 1-1 3-6. 1-1 2 1. 1-1 3-8. 1-1 2-1. 1-1 1-1.2 1-1 1 * ** *** 5. 1-1 4 D 2 3 D 3 c o m b in e d D 2 3 D 3 c o m b in e d 2. 1-1 1 ** * *** 1.5 1-1 1-1. 1-1 2-2. 1-1 2 1. 1-1 1-3. 1-1 2 5. 1-1 2-4. 1-1 2-5. 1-1 2-6. 1-1 2 *** *** *** D 2 3 D 3 c o m b in e d D 2 3 D 3 c o m b in e d E Berger et al. Lab on a chip (accepted)
Summary and outlook Millifluidics improve midbrain organoid cultures in terms of Dead core area Organoid oxygenation Dopaminergic differentiation efficiency Metabolic maturation Practical solution for advanced organoid cultures Quasi Vivo Quasi Brain Further improvement strategies Vascularization Physiological cell density Microglia incorporation disease modeling
Acknowledgements LCSB DVB Jens Schwamborn Silvia Bolognin Sarah Nickels Kathrin Hemmer Jonas Walter Javier Jarazo Thea van Wuellen Marie Fossépré Isabel Rosety Philippe Lucarelli Lisa Smits Anna Monzel Gemma Gomez Giro Sonia Sabate Soler Jennifer Modamio Developmental and Cellular Biology Group Collaborators o Chiara Magliaro and Arti Ahluwalia (Università di Pisa) o Silvia Bolognin, Paul Anthony & Anna Monzel (LCSB) o Nicole Paczia & Carole Linster (LCSB Metabolomics core facility)
In Vitro Models for the Study of Neurological Diseases Q & A Session Sponsor