May 15, 2020
Isolation of cancer stem cells by sphere formation assay
- Kwang Nickel1,
- Austin M. Maas1,
- Randall J Kimple2
- 1University of Wisconsin - Madison;
- 2University of Wisconsin - Madison, UW Carbone Cancer Center
Protocol Citation: Kwang Nickel, Austin M. Maas, Randall J Kimple 2020. Isolation of cancer stem cells by sphere formation assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bdt8i6rw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 18, 2020
Last Modified: May 15, 2020
Protocol Integer ID: 34400
Keywords: cancer stem cells,
Abstract
Cancer stem cells (CSCs) are a small subpopulation of tumor cells that are thought to be responsible for recurrence and metastasis of cancer due to their ability for self-renewal and differentiation into multiple cancer cell types.
CSCs are also known to play a key role in the development of metastases. Cancer cells undergoing Epithelial to Mesenchymal Transition (EMT) have also been shown to attain CSC phenotypes and acquire invasive and migratory properties. Therefore, understanding the characteristics of CSCs may help to develop better treatment strategies.
The sphere formation assay has been widely used to isolate CSCs using anchorage-independent sphere culture. CSCs can grow on ultra-low attachment plates that are coated with a layer to inhibit the attachment of cells. When cells are grown in serum-free and non-adherent conditions, CSCs can survive and clonally expand to form spheres, whereas differentiated tumor cells undergo apoptosis due to their anchorage dependence.
Materials
MATERIALS
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
Dulbeccos Modified Eagles Medium (DMEM)/F12Gibco - Thermo FischerCatalog #11320082
Human Epidermal Growth Factor (hEGF)Thermo Fisher ScientificCatalog #PHG0311
Basic Fibroblast Growth Factor (bFGF)Gibco - Thermo FischerCatalog #13256029
B-27™ Supplement (50X) minus vitamin AGibco - Thermo FischerCatalog #12587010
Costar® 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates Individually Wrapped SCorningCatalog #3473
Humidified, CO2 controlled tissue culture incubator, Cell counter (hemocytometer or automated cell counter), Tissue culture inverted microscope.
Prepare Sphere Formation Media (100 mL )
Base media is DMEM/F12
1 mL Penicillin and Streptomycin
2 mL B-27
20 µL bFGF
10 µL hEGF
96.97 mL of DMEM/F12.
Prepare a single cell suspension from either cell culture or primary tumor tissue. Count cells using a hemocytometer or an automated cell counter.
Note: In this example, a head and neck cancer cell line, UM-SCC47 
SCC071CA.pdf , is analyzed. The experimental approach can be used to study cells isolated from primary human cancers or with established cancer cell lines.
SCC071CA.pdf , is analyzed. The experimental approach can be used to study cells isolated from primary human cancers or with established cancer cell lines.Dilute cells to a final concentration of 2000 cells/ml in Sphere Formation Media.
Note that the final concentration of cells/ml should be optimized to your specific cell line or cancer type.
Add 250 µL of cell mix (from Step 3) to each well of a 24-well low attachement plate (final concentration of 500 cells/well).
Note that the optimal number of cells is dependent on tumor type. Use low-attachment plates (see materials) NOT standard tissue culture plates.
Place plates into humidified (95% relative humidity) incubator at 37 °C and 5% CO2.
Check plate daily and add fresh media to keep the wells approximately 20% filled based on evaporation.
Check spheres after 2 days and then daily by visualization under a microscope. We use a size of 50 µm to identify spheres. The time required for sphere formation can vary significantly based on the cell type used. We typically expect approximately 10 spheres per well. In this example, spheres were first identified 3-4 days after plating and a final count was made 7 days after plating the cells.
UM-SCC47 spheres.
Results
Results
- In this assay, the endpoint of interest is sphere formation efficiency (i.e., the percentage of plated cells that form a sphere). This is calculated using the following formula which is calculated for each well.
- Sphere formation efficiency (%) = (number of spheres)/(number of cells seeded) x 100.
A minimum of 3 biologic replicates should be performed and the average and standard deviation reported.