Stem Cell Biology

Neural Stem Cells Protocols

Plating Cells
Passage of Neurospheres
Cryopreservation and Re-establishment of Neurospheres
Preparation of Neurospheres for Cryostat Sectioning and Immunocytochemistry
The Artificial Cerebrospinal Fluid-For Hippocampal Slice Culture
Differentiation of ES Cells into Glial Cells and Neurons

Plating Cells

  • Calculate and adjust the viable cell concentration with a hematocytometer. Viable cells are determined by Trypan Blue dye (T8154) exclusion.
  • Add 0.5 ml of SFM containing the cells to each well of a 24 multi-well plate.
  • Incubate at 37 °C with 95% air and 5% CO2.

  

Passage of Neurospheres

  • Transfer the neurospheres and medium from all wells to a 15 ml conical tube and centrifuge for 5 minutes at 200 g.
  • Remove the supernatant and add 2.0 ml of TrypLE™ to the tube, use a Pasteur pipette to mix the neurospheres.
  • Place the tube in the water bath for 20 minutes at 37 °C.
  • Centrifuge for 5 minutes at 500 g. Remove the supernatant and re-suspend the cells in 0.5 ml of SFM. Triturate with a Pasteur pipette (60–70 times).
  • Determine the cell concentration and plate the desired concentration of cells to a new 24 multi-well plate. Incubate at 37 °C with 95% air and 5% CO2.

 

Cryopreservation and Re-establishment of Neurospheres

  • Pool the culture medium with the neurospheres from a 24 multi-well plate into a 15 ml conical tube. Centrifuge for 5 minutes at 200 g.
  • Remove the supernatant and re-suspend the cells in 10.0 ml SFM (without EGF and FGF) containing 15.0% DMSO (D2438).
  • Gently mix the neurospheres in the SFM. Aliquot 1.5 ml of the mixture into polypropylene cryovials. Transfer the tubes to a
    –80°C freezer overnight. The following morning, transfer the vials to a liquid nitrogen cryofreezer.
  • To re-establish the neurospheres in tissue culture, remove the cryovial from the freezer and let the vial warm up to room temperature on the bench.
  • Add the contents of the cryovial slowly to a 15 ml conical tube containing 10 ml of SFM.
  • Centrifuge for 5 minutes at 200 g and remove the supernatant. Gently mix the neurospheres with 4.0 ml of the SFM with a Pasteur pipette.
  • Fill 8 wells of a 24 multi-well plate with 0.5 ml of the mixture and incubate at 37 °C with 95% air and 5% CO2.

 back to top

Preparation of Neurospheres for Cryostat Sectioning and Immunocytochemistry

  • Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube. Allow the neurospheres to settle by gravity.
  • Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (D5773). Allow the neurospheres to settle in the tube.
  • Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS) (P7059). Allow the neurospheres to settle for at least 30 minutes before removal of the PBS. Repeat the rinsing procedure at least 3 times.
  • Immunocytochemistry may be performed on intact neurospheres at this stage. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1 M PBS). Transfer the tube to the fridge and allow the neurospheres to settle overnight.
  • Remove the 30% sucrose and add embedding medium for frozen tissue specimens for 1 hour (O.C.T. compound). Prepare the cryostat chuck with a layer of O.C.T. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
  • Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (A3648) coated glass microscope slides.
  • Continue with the desired immunocytochemical procedure (non-specific blocking, application of primary antibody etc.).

Protocols from Nature Protocols; Pacey K. L.; Stead S.; Gleave J. A.; Tomczyk K.; Doering L. C.

 

The Artificial Cerebrospinal Fluid-For Hippocampal Slice Culture
The artificial cerebrospinal fluid (aCSFin vivo) in the microdialysis probe was (in mM): NaCl 48 (S8776); KCl 3 (P9327) ; CaCl2 1.4 (C8106); MgCl2 0.8 (M1028); Na2HPO4 1.5 (S5136); NaH2PO4 0.2 (S5011), 2-[4-(2-Hydroxyethyl)-1-piperazine] ethanesulfonate monosodium salt (NaHEPES) 250 (H3784), pH 7.35. The concentration of NaCl (S8776) was decreased by 100 mM in the aCSFin vivo to maintain physiological osmolarity. Lactate aCSFin vivo contained 250 mM lactate (L7900) in place of 250 mM HEPES.

Protocol from Brain Research; Gilbert E. J.; Tang J. M.; Ludvig N.; Bergold P. J.

 

Differentiation of ES Cells into Glial Cells and Neurons

  • Day 1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells. Trypsinize (T4049, T3924, T4299, T4174) the cells as for normal passaging until the colonies lift off. Try to keep the loosely connected clumps of cells together by gentle handling. Then directly plate the cells 1:3 into bacterial grade Petri dishes in LIF free medium containing 1 microM all-trans retinoic acid (RA) (R2625).
  • Day 3: Collect cell aggregates and replate in tissue culture dishes (appr. 25 cell aggregates per 6 cm tissue culture dish) in ES cell culture medium without LIF and RA. Aspirate the medium carefully.
  • Day 8: Change half of the medium. From this day on at least 10% of the cells exhibits neuronal phenotype.

Nagy Lab, Samuel Lunenfeld Research Institute, Mount Sinai Hospital—online protocols

back to top