實驗概要
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.
主要試劑
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
主要設備
15-mL conical tube
Microcentrifuge
實驗材料
Neural stem cells
實驗步驟
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.