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  7. Culture of Neural Stem Cells in 96-Well Plates

Culture of Neural Stem Cells in 96-Well Plates

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Synchronous Differentiation

Pure population of cerebral cortical neuron

Axol-Bioscience-human-iPSC-image-p01.jpg

Following the protocol for systems A and B will result in the generation of a pure population of cerebral cortical neurons.

Using system A, iPSC-Derived Neural Stem Cells are expanded and then the synchronously differentiated into neurons, using Neural Differentiation-XF Medium.

Using system B, iPSC-Derived Neural Stem Cells are plated into the final assay format and then synchronously differentiated, using Neural Differentiation-XF Medium, from iPSC-Derived Neural Stem Cells to neurons.

Systems C and D – Spontaneous Differentiation

Spontaneous Differentiation

Mixed population of neuronal and glial cells

Axol-Bioscience-human-iPSC-image-p02.jpg

Following the protocol for systems C and D will result in the generation of a mixed population of cerebral cortical neurons and glial cells.

Using system C, iPSC-Derived Neural Stem Cells are expanded and then spontaneously differentiated, using Neural Maintenance-XF Medium, from iPSC-Derived Neural Stem Cells to neuronal and glial cells.

Using system D, iPSC-Derived Neural Stem Cells are plated into the final assay format and then spontaneously differentiated into neuronal and glial cells, using Neural Maintenance-XF Medium.

Caution!
Increasing the number of days the iPSC-Derived NSCs are in culture and the more expansions the NSCs undergo will increase the population of glial cells in the final culture. We recommend a maximum of 5 passages for healthy/control lines and a maximum of 3 passages for the disease/patient lines. Expansion must be conducted with caution and the cells should not be pushed to proliferate for too long before differentiation

Preparation of Coating Reagents and Coating the 96-Well Plate

SureBond-XF Coating Solution (required for all systems for final plating on plastic and cultures >50,000/cm2)

  • Upon receipt store SureBond-XF at 4°C.
  • Dilute the SureBond-XF stock solution (200x) 1:100 in Neural Plating-XF Medium to make a 2x working solution e.g. 60 μL in 6 mL.
  • Coat the surface of the 96-well plate with the SureBond-XF 2x working solution. We recommend coating 96-well plates at a volume of 100-150 μL per well however, please optimize for your experiments.

SureBond+ReadySet Coating Solution (required for all systems for final plating on glass and for low density cultures (<50,000/cm2)):

  • l Upon receipt store SureBond at or below -80°C and store ReadySet at 4°C.
  • Thaw the SureBond coating solution overnight at 4°C.
  • Coat the surface of the 96-well plate with the ReadySet at a volume of 150 μL per well.
  • Incubate at 37ºC for 45 minutes.
  • Wash the plate thoroughly four times using an equal volume of sterile distilled H2O (e.g. if 250 μL of ReadySet, use 250 μL sterile distilled H2O). During each wash rock the dish to ensure thorough washing.
  • Do not let the ReadySet dry out following washing, proceed straight to coating with SureBond.
  • Dilute the SureBond stock solution (50x) 1:50 in Neural Plating-XF Medium to make 1x working solution e.g. 120 μL in 6 mL.
  • Coat the surface of the 96-well plate with the SureBond 1x working solution. We recommend coating at a volume of 150 μL per well.
  • Incubate for 1 hour at 37ºC.

Important!
Do not let the SureBond or SureBond-XF coating dry out before seeding the cells.
DO NOT wash the vessel after coating with SureBond or SureBond-XF

Systems A and C

Plating Human iPSC-Derived Neural Stem Cells onto a 96-Well Plate

  • Pre-coat 96-well plate with SureBond-XF or SureBond+ReadySet.
  • Thaw an aliquot of Unlock, Neural Expansion-XF Medium and Neural Plating–XF Medium overnight at 4ºC before use and store at 4ºC.
  • Pre-warm all media and culture vessels to 37ºC before use.
  • Remove all spent medium from cell culture vessels.
  • Gently rinse the surface of the cell layer once with the Dulbecco’s-PBS (1x) (D-PBS, without calcium or magnesium, 2 mL D-PBS (1x) per 10 cm2 culture surface area).
  • Discard the D-PBS.
  • To detach the cells from a coating of SureBond use Unlock.
  • Add 1 mL per 10cm2 culture surface area of cold/room temperature Unlock. Evenly distribute it over the entire cell layer. Incubate the cells for 5 minutes at 37°C.
  • Use a P1000 pipette to transfer the cells drop-wise into a 15 mL sterile conical tube add four volumes of pre-warmed, 37°C, Neural Expansion-XF Medium. (For example if 1 mL of Unlock is used, then add 4 mL of the medium to stop the reaction). Gently pipette up and down a few times to disperse the medium.
  • Centrifuge cells at 200 x g for 5 minutes at room temperature.
  • Aspirate and discard the supernatant carefully. Gently resuspend the cell pellet in Neural Plating–XF Medium until they are in a single cell suspension.
  • Perform a cell count to ensure optimal seeding density.
  • Quickly remove the coating solution (SureBond-XF or SureBond+ReadySet) from the pre-coated wells before plating the resuspended cells. It is recommended that approximately 50 μL of the SureBond-XF or SureBond is left in the well to ensure that the wells do not dry out.
  • Plate the resuspended cells at 100 μL per well (totally approximately 150 μL in each well) drop-wise and evenly at no less than 50,000 cells/cm2 for healthy Human iPSC-Derived NSCs (ax0015, ax0016, and ax0018) and no less than 70,000 cells/cm2 for all other Human iPSC-Derived NSCs.
  • To ensure an even plating of Human iPSC-Derived NSCs, gently rock the culture vessel back and forth and side to side several times.
    l Incubate the cells at 37°C, 5% CO2.
  • The day after plating follow either system A or C:

System A – Synchronous Differentiation After Expansion

  • 24 hours after plating, replace 100 μL of spent medium with fresh, pre-warmed, 37°C, Neural Expansion-XF Medium WITHOUT EGF or FGF2.
  • After a further 24 hours, replace 125 μL of the medium with 125 μL of fresh, pre-warmed, 37°C, Neural Differentiation-XF Medium.
  • Replace half the volume of medium with fresh pre-warmed, 37°C, Neural Differentiation-XF Medium every 3 days.
  • Detection of a pure population of neurons is visual. Neurite formation will begin to occur and you will see an increase in neuronal cell types. Check the culture each day to observe neuronal development. When the cells are 60% confluent before starting differentiation, a pure neuronal population will take at least 3 days to develop. If the culture is >60% confluent it may take longer (up to 10 days). If the culture is fully confluent, a pure population of neurons will be difficult to obtain.
  • Once the culture has reached a pure population of neurons, replace half the volume of medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.
  • 24 hours after the half medium change to Neural Maintenance-XF Medium, replace half the volume of medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.
  • Every 3 days replace half the volume of medium with the same volume of Neural Maintenance-XF Medium

System C – Spontaneous Differentiation After Expansion

  • 24 hours after plating, replace 100 μL of spent medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.
  • After a further 24 hours, replace 75 μL of the medium with 75 μL with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.

MAINTENANCE

  • To maintain a healthy neuronal culture, replace half the volume of medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium every 3 days.
  • It is important that the medium is pre-warmed and that the medium changes are conducted gently by pipetting to the side of the culture dish.
  • Terminally differentiated neurons can now be maintained in culture until ready for experimental assays.

Systems B and D

Thawing and Plating Human iPSC-Derived Neural Stem Cells onto a 96-Well Plate

The day before thawing Human iPSC-Derived NSCs:

  • Thaw Neural Plating–XF Medium overnight at 4ºC.
  • Pre-coat 96-well plate with SureBond-XF or SureBond+ReadySet.

On the day of thawing Human iPSC-Derived NSCs:

  • Pre-warm all media and culture vessels to 37ºC before use.
  • To thaw cells – transfer the cells from storage by transporting the cells buried in dry ice. Remove the cells from dry ice and transfer the cells to a 37°C water bath.
  • Quickly thaw the vial of cells in a 37°C water bath. Do not completely submerge the vial (only up to 2/3rd of the vial).
    Remove the vial before the last bit of ice has melted, after ~1-2 minutes.
  • Do not shake the vial during thawing.
  • Take the vial of cells to a biological safety cabinet, spraying the vial and hood thoroughly with 70% ethanol and wiping with an autoclaved paper towel before placing the vial in the hood.
  • Once thawed, use a P1000 pipette to transfer the cells drop-wise into a 15 mL sterile conical tube containing 10 mL of pre-warmed, 37°C, Neural Plating–XF Medium. Gently wash the vial with 1 mL of Neural Plating–XF Medium.
  • Transfer this to the 15 mL sterile conical tube containing the cells Centrifuge cells at 200 x g for 5 minutes at room temperature.
  • Aspirate and discard the supernatant carefully. Gently resuspend the cell pellet in Neural Plating–XF Medium until they are in a single cell suspension.
  • Perform a cell count to ensure optimal seeding density.
  • Quickly remove the coating solution (SureBond-XF or SureBond+ReadySet) from the pre-coated wells before plating the resuspended cells. It is recommended that approximately 30 μL of the SureBond-XF or SureBond is left in the well to ensure that the wells do not dry out.
  • Plate the resuspended cells at 120 μL per well (totally approximately 150 μL in each well) drop-wise and evenly at no less than 50,000 cells/cm2 for healthy Human iPSC-Derived NSCs (ax0015, ax0016, ax0018 and ax0019) and no less than 70,000 cells/cm2 for all other Human iPSC-Derived NSCs.
  • To ensure an even plating of Human iPSC-Derived NSCs, gently rock the culture vessel back and forth and side to side several times.
  • Incubate the cells at 37°C, 5%, CO2.
  • The day after plating follow either system B or D:

Important!
Do not mix the cells vigorously. Avoid generating bubbles.

Differentiation

System B – Synchronous Differentiation

  • 24 hours after plating, replace 100 μL of spent medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium. This will allow the cells to recover after thawing.
  • After a further 24 hours replace 125 μL of the medium with 125 μL of fresh, pre-warmed, 37°C, Neural Differentiation-XF Medium.
  • Replace half the volume of medium with fresh pre-warmed, 37°C, Neural Differentiation-XF Medium every 4 days.
  • Detection of a pure population of neurons is visual. Check the culture each day to observe neuronal development. A pure population will take at least 3 days to develop at 60% confluency, if the culture is very dense it may take longer.
  • If the culture is too confluent a pure population will be difficult to obtain.
  • l Once the culture has reached a pure population of neurons, replace half the volume of medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.

Differentiation

System D – Spontaneous Differentiation

  • 24 hours after plating, replace 100 μL of spent medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium. This will allow the cells to recover after thawing.
  • After a further 24 hours, replace 75 μL of the medium with 75 μL fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium.

Maintenance

  • To maintain a healthy neuronal culture, replace half the volume of medium with fresh, pre-warmed, 37°C, Neural Maintenance-XF Medium every 3 days.
  • It is important that the medium is pre-warmed and that the medium changes are conducted gently by pipetting to the side of the culture dish.
  • Terminally differentiated neurons can now be maintained in culture until ready for experimental assays