Xeno-free Culture of Neural Stem Cells

实验概要

Neural  stem cells (NSCs) derived from human embryonic stem cells (hESCs) have  the potential to help provide understanding for human neurogenesis and  for potential cell therapy applications to treat Parkinson’s Disease or  spinal cord injuries. Standard methods of culturing NSCs raise concerns  about pathogen cross-transfer from nonhuman sources or contamination  with non-neural cells, limiting the efficiency and specificity of the  differentiation protocols. These concerns have led to the development of  xenofree conditions for maintaining and expanding NSCs, which are  described in this protocol.

主要试剂

CELLstart™ CTS™

Neurobasal® Medium

B-27® Supplement XenoFree

FGF-basic (AA 10-155), Recombinant Human

EGF, Recombinant Human

GlutaMAX™-I

TrypLE™ Select, 10X

Dulbecco’s Phosphate-Buffered Saline (D-PBS)

Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca² or Mg²

主要设备

15-mL conical tube

Microcentrifuge

实验材料

Neural stem cells

实验步骤

1. Preparing Media and Culture Vessels

1) Dilute  CELLstart™ CTS™ 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL  of CELLstart™ into 5 mL of D-PBS).   Note: CELLstart™ CTS™ should not be  frozen, vortexed, or exposed to vigorous agitation due to potential gel  formation.

2) Coat  the surface of the culture vessel with the working solution of  CELLstart™ CTS™ (2.5 mL for a T-25 flask or 60-mm dish, 1.5 mL for a  35-mm dish).

3) Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO₂ for 1 hour.

4) Use the dish immediately after incubation. Aspirate the CELLstart™ CTS™ solution immediately before use.

2. Thawing and Seeding NSCs

1) Remove  a vial of cells from liquid nitrogen and quickly thaw the vial in a  37°C water bath, being careful not to immerse the vial above the level  of the cap.

2) When  just a small crystal of ice remains, sterilize the outside of the vial  with 95% ethanol. Allow the ethanol to evaporate before opening the vial  in a cell culture hood.

3) Gently pipet the cell suspension up and down once, and place it into a 15-mL centrifuge tube.

4) Add 10 mL of warm culture medium to the tube dropwise to reduce osmotic shock.

5) Centrifuge the cell suspension at 200 × g for 5 minutes.

6) Remove  the supernatant, resuspend the pellet in 5 mL of culture medium, and  determine the total number of cells and percent viability.

7) Seed  the cells at a concentration of >90,000 cells/cm2 onto a dish or  flask that has been treated with CELLstart™ CTS™ solution. (Aspirate the  CELLstart™ CTS™ solution immediately before using the dish or flask.)

8) Incubate at 36–38°C in a humidified atmosphere (90%) of 5% CO₂ in air.

3. Culture and Propagation

1) Twenty-four hours after seeding the cells, replace the culture medium.

2) Replace  the spent medium every other day with an equal volume of fresh culture  medium. Note: If the medium turns yellow, change the medium daily.  Yellow medium will affect the NSC proliferation rate.

3) After 3–4 days, the culture will become semi-confluent.

4)  To split the cell culture 1:2, aspirate the medium and wash the cells  twice with 5 mL of D-PBS (without calcium and magnesium).

5) Add 1 mL of TrypLE™ Select to dissociate the cells, and incubate for 2 minutes at 37°C.

6) Add  4 mL of culture media to neutralize the TrypLE™ Select activity and  pipet up and down 2–3 times to get a uniform cell suspension. Check the  cells under a microscope.

7) Transfer the cell suspension to a 15-mL centrifuge tube.

8) Centrifuge the cells at 200 × g for 5 minutes.

9) Aspirate the supernatant and resuspend the cells in 10 mL of culture medium.

10)  Split the cell suspension into two fresh T-25 flasks that have been  treated with CELLstart™ CTS™ solution. Seed each flask with 5 mL of cell  suspension.

11) Incubate the flasks at 37°C in a humidified atmosphere (90%) of 5% CO₂ in air.

12) Grow the cells until semi-confluent, changing the medium once after 12 hours and every two days thereafter.

13) Passage the cells when the culture reaches ~80% confluence.