Protocol for Neural Progenitor Cell Expansion

Neural progenitor cells (NPCs) are multipotent stem cells with the capability to differentiate into neurons and glial cells (oligodendrocytes and astrocytes). Therefore, successful in vitro neural progenitor cell expansion offers significant therapeutic potential for cell therapy applications.

This protocol outlines the steps needed to expand and maintain human-, mouse-, and rat-derived NPCs using PRIME-XV® NPC Expansion Xeno-free, Serum-Free Medium. PRIME-XV NPC Expansion XSFM is manufactured in cGMP conditions and comes completely formulated and ready for use. For more information on this medium, please refer to the product insert.

NPC, DAPI staining, Nestin

click to enlarge

Human neural progenitor cells plated on PRIME-XV Human Fibronectin-coated plate and cultured in PRIME-XV NPC Expansion XSFM retained Nestin expression. Nuclei counterstained with DAPI (blue).

Materials

Protocols

The following neural progenitor cell expansion protocols can be found below:

These procedures are general guidelines for culturing human neural progenitor cells isolated from fetal tissue or embryonic stem cells, as well as for murine neural progenitor cells isolated from fetuses or post-natal brains. Procedures for optimal growth conditions should be determined for each application and cell type as appropriate. Safe laboratory procedures should be followed and protective clothing should be worn when handing this medium. The acute and chronic effects of over-exposure to this medium are unknown.

Coating Culture Vessels

Note : For neural progenitor cell expansion in monolayer, culture vessels have to be pre-coated with substrate for cell attachment. Either PRIME-XV Human Fibronectin or MatrIS F should be used as coating substrates. For consistent performance, usage of Poly-L-Ornithine is also recommended to be used in conjunction with PRIME-XV Human Fibronectin or PRIME-XV MatrIS F. Specific concentrations and types of substrates should be optimized for each end-user application.

 

1. Dissolve Poly-L-Ornithine in sterile PBS to make a 15 mg/mL stock (1000X). Aliquot and store at ≤-20˚C in a manual defrost freezer for up to 6 months. Avoid repeated freeze-thaw cycles.

2. Dilute the 1000X Poly-L-Ornithine stock 1000-fold in sterile PBS to make a 15 µg/mL (1X) solution. Prepare fresh as needed.

3. Add the (1X) Poly-L-Ornithine solution to culture vessel at a ratio of 0.15 mL/cm2. Incubate 3 hours to overnight at 37˚C and 5% CO2.

4. Discard the Poly-L-Ornithine solution. Wash each vessel 3 times with equal amount of PBS.

5. Add PBS to each vessel at a ratio of 0.15 mL/cm2. Incubate overnight at 37˚C and 5% CO2.

6. Allow all the vial of attachment substrate, either PRIME-XV Human Fibronectin or PRIME-XV MatrIS F, to warm to room temperature without agitation. Make a 10 µg/mL solution pipetting the attachment substrate into sterile PBS and gently inverting the tubes. Prepare fresh as needed.

7. Discard the PBS from each Poly-L-Ornithine coated dish. Wash once with PBS.

8. Add the 10µL/mL PRIME-XV Human Fibronectin or PRIME-XV MatrIS F solution to each dish at a ratio of 0.15 mL/cm2. Incubate at 37˚C and 5% CO2 for 3 hours to overnight.

9. Discard the solution. Wash each vessel with PBS once before use.

neural progenitor cell, NPC,  preservation, frozen, thaw

click to enlarge

Thawing Neural Progenitor Cells

1. Thaw PRIME-XV NPC Expansion XSFM at room temperature. Pre-warm at 37˚C the amount of medium needed for one procedure (repeated warming of medium may reduce product performance). Store the remaining medium at 2-8˚C. Thawed medium can be kept at 2-8°C for up to 7 days.

2. Pre-coat tissue culture vessel using the above protocol to make "Coating culture vessels."

Note: For neurosphere suspension cultures, use uncoated low or ultra-low attachment surface culture vessels.

3. Rapidly thaw a frozen vial of NPCs in a 37˚C water bath while swirling the vial until all its content is liquid. The vial should still feel cold to the touch. The process typically takes less than 2 minutes.

4. Pipette the content of the entire vial into a 15 mL conical tube. Add 5 to 10 mL pre-warmed PRIME-XV NPC Expansion XSFM drop-wise (3-5 drops per second) while swirling.

5. Centrifuge the cells for 5 minutes at 200xg at room temperature.

6. Resuspend the pellet in a minimum volume of pre-warmed PRIME-XV NPC Expansion XSFM, count the cells and determine the total viable cell number.

7. Add thawed cell suspension to a pre-coated culture dish (see “Protocol: Culturing Culture Vessels”) at a seeding density of 2-5x104 viable cells/cm2 in an appropriate volume of medium for the culture vessel.

8. Incubate the cells in 37˚C, 5% CO2 humidified incubator.

9. 24 hours post-thaw, remove the spent medium from the vessel and add pre-warmed PRIME-XV NPC Expansion XSFM.

10. Remove and discard spent medium and re-feed the cells with pre-warmed medium every 2 days.

11. Subculture when the cells reach 80-90% confluency.

neural progenitor cell, NPC,  preservation, frozen, thaw

click to enlarge

Expansion and Subculture Human Neural Progenitor Cells

Monolayer adherent cultures

1. Pre-warm PRIME-XV NPC Expansion XSFM and a cell dissociation reagent (i.e. Accutase®) at 37°C.

2. Aspirate the medium from the culture flask.

3. Rinse the cells with sterile PBS without calcium and magnesium.

4. Add ~ 0.1 mL/cm2 of pre-warmed Accutase® to the flask to make the solution cover the entire cell culture surface. Incubate at 37°C for 2-5 minutes; check the flask after 2 minutes, the cell layer should start to detach from the culture dish surface.

5. Once the cells are detached, very gently pipette them up and down to create a single cell suspension. Minimize cells exposure to air by avoiding air bubbles.

6. Add 10 mL of pre-warmed PRIME-XV NPC Expansion XSFM and mix gently; transfer the cell suspension to a 15 mL conical tube.

7. Centrifuge the cell suspension at 200xg for 5 minutes.

8. Aspirate the medium and gently resuspend the cell pellet in minimum volume of pre-warmed PRIME-XV NPC Expansion XSFM. Count the cells to determine the number of total viable cells.

9. Seed the cells in a pre-coated culture vessel at a density of 2-5x104 viable cells/cm2 and incubate at 37°C and 5% CO2 humidified incubator.

10. Remove and discard spent medium and re-feed the cells with pre-warmed medium every 2 days.

NPC, neural progenitor cell, expansion, culture, suspension

click to enlarge

Harvesting Neurosphere Suspension Cultures

1. Add thawed cell suspension (from “Protocol: Recovery of Cryopreserved Human NPC”) to a low or ultra low attachment surface culture vessel at a seeding density of >2x105 cells/cm2. Note: the initial seeding density will impact the number of neurospheres. Low cell viability (Less than 90%) will result in increased number of dead cells and debris in the culture suspension.

2. Without removing neurospheres, gently change the neurosphere culture medium every 2 to 3 days with pre-warmed PRIME-XV NPC Expansion XSFM. The cells within the neurospheres should look bright and almost transparent under the microscope. Neurospheres should be harvested after 3 to 4 days of culture for easy dissociation.

3. When the neurospheres reach 100-200 µm, in size, aspirate suspension culture and transfer to a 15 mL conical tube. Let the neurospheres settle down by gravity and carefully aspirate most of the culture medium. Alternatively, centrifuge at 100xg for 2 minutes and remove supernatant without exposing neurospheres to air.

4. Add 5 mL of sterile PBS without calcium and magnesium and let the neurospheres settle down or centrifuge as in previous step.

5. Add 1 mL of pre-warmed Accutase® and incubate for 15 minutes in a water bath at 37˚C.

6. After incubation, pipette neurospheres up and down 10 to 20 times to create single cell suspension. Add 5-10 mL of PRIME-XV NPC Expansion XSFM.

7. Remove a sample to count cells to determine cell density and viability, and centrifuge at 200xg for 5 minutes.

8. Resuspend the cells in pre-warmed PRIME-XV NPC Expansion XSFM at the desired density and incubate in a 37˚C and 5% CO2 humidified incubator.

neurosphere, monolayer, NPC, expansion, culture, NSC

click to enlarge

 

neurosphere, monolayer, NPC, expansion, culture, NSC

click to enlarge

PRIME-XV NPC Expansion XSFM supports rat neural progenitor cell expansion in monolayer culture (top) and neurosphere culture (bottom). Images taken at 10X magnification.

Accutase® is a registered trademark of ICT inc.
Parafilm® is a registered trademark of Bemis Company, Inc.