Cancer stem cells (CSCs), also referred to as cancer initiating cells, represent a minority population of cells within a tumor. CSCs can self-renew and give rise to terminally differentiated cells. Many cancer disease models propose that the existence of CSCs contribute to cancer metastasis. While conventional cancer treatments often eliminate terminally differentiated cells within a tumor, CSCs have an inherent ability to be more resistant to such treatments. Therefore, the ability to properly isolate and culture these rare cells is a first step towards finding the right treatment. CSCs, when cultured in suspension, aggregate to form spheres known as tumorspheres.

This protocol outlines the steps needed to enrich for cancer stem cells under serum-free conditions using PRIME-XV Tumorsphere Serum-Free Medium. For more information on this medium, please refer to the product insert.

Efficient tumorsphere formation of MCF-7 cells under PRIME-XV Tumorsphere SFM (left) compared to control 10% serum-containing DMEM (right). Images taken at 10X magnification.

• PRIME-XV Tumorsphere SFM (FUJIFILM Biosciences, Catalog #91130)
• Heparin
• Hydrocortisone
• Ultra-low attachment vessel
• Phosphate Buffered Solution (PBS) (FUJIFILM Biosciences, Catalog #9236)
• TrypLE Express
• MEM NEAA (FUJIFILM Biosciences, Catalog # 9130)

The following cancer stem cell protocols are optimized for tumorsphere formation using HeLa, MCF-7 and A549 cells. 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.

• Preparing Complete Medium
• Thawing Cancer Stem Cells for Tumorsphere Formation
• Plating Cancer Stem Cells for Tumorsphere Formation
• Passaging Tumorspheres

Preparing Complete Medium

1. Thaw PRIME-XV Tumorsphere SFM at room temperature for a few hours or store overnight at 2-8°C. Pre-warm the amount of media needed at 37°C for no more than 30 minutes. Media should be pre-warmed before introducing cells.
2. The following supplements need to be added to PRIME-XV Tumorsphere SFM prior to use: 2U/mL of Heparin (Sigma Aldrich #H3149) and 0.5µg/mL Hydrocortisone (Sigma Aldrich #H0135). (Supplements can be added before or after pre-warming)

Thawing Cancer Stem Cells for Tumorsphere Formation

1. Rapidly thaw a frozen vial of cancer stem cells in a 37°C water batch by swirling the vial until the entire contents is liquid. This process should take less than 2 minutes.
2. Pipette the entire contents of the vial into a 15mL conical tube containing 5-10mL of pre-warmed PRIME-XV Tumorsphere SFM.
3. Centrifuge the cells for 5 minutes at 400xg. Aspirate supernatant.
4. Resuspend the cells in 5mL of pre-warmed fresh media.
5. Transfer contents into ultra-low attachment culture vessel.
6. Incubate cells at 37°C, 5% CO₂ incubator.

Plating Cancer Stem Cells for Tumorsphere Formation

1. Remove spent media from T-75 flask culture and gently rinse cells once with 10mL PBS for each T-75 flask.
2. Gently detach cells from culture dish with appropriate volume of room temperature TrypLE™ Express or equivalent solution to each T-75 flask. Tilt the flask in all directions to disperse the cell dissociation solution evenly over the cells.
3. Incubate the cells in 37°C, 5% CO₂ incubator. Monitoring periodically for cell detachment by observing the cells under the microscope. Cells will start to round and detach. Tap the side of the flask to aid the detachment of the cells and return culture to the incubator. Repeat the above process until at least 90% of cells are fully detached. This cell detachment process takes approximately 5-10 minutes.
4. Add 5mL of MEM NEAA medium to the flask. Disperse the cells by pipetting the media over the entire growing surface of the flask and transfer the contents to a 15mL conical tube. Take a cell count prior to centrifugation. It is important to not count in Tumorsphere medium as components may interfere with cell counting.
5. Centrifuge cells down at 400xg for 5 minutes. Aspirate off supernatant.
6. Gently resuspend cell pellet in appropriate volume of pre-warmed PRIME-XV Tumorsphere SFM and transfer to ultra-low adhesion plates. Recommended cells for plating can vary between 5 x 10³ and 6 x 10⁵ cells per well in a six-well plate. Density is dependent on cell type and it is recommended to test several densities to determine the optimum.
7. Incubate in 37°C, 5% CO₂ incubator. Sphere formation should occur within 3-10 days.

Passaging Tumorspheres (can also be used to prepare cells for flow cytometry)

1. Once spheres are formed (3-10 days), remove the entire contents of the of the well into a 15mL centrifuge tube.
2. Allow the cells to settle to the bottom (around 10-15 minutes) You can place the tube in an incubator if desired.
3. Remove as much media as possible without disturbing the cells at the bottom.
4. Add few milliliters of TrypLE or trypsin to break the spheres apart. Place the tubes in an incubator. The heat will aid in breaking the spheres apart.
5. Pipette up and down a few times to aid in breaking the spheres apart.
6. If trypsin is used, add an inhibitor (serum containing medium or soy bean inhibitor).
7. Centrifuge the cells for 5 minutes at 400xg. Aspirate supernatant.
8. Resuspend in MEM NEAA or PBS if running on flow cytometer.
9. Distribute cells into fresh ultra low attachment culture vessel at desired density or cells can also be used for FACS analysis.

* TrypLE is a registered trademark of Life Technologies.