1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences
Azari, H., Millette, S., Ansari, S., Rahman, M., Deleyrolle, L. P., Reynolds, B. A. Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay. J. Vis. Exp. (56), e3633, doi:10.3791/3633 (2011).
Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion.
1. Collection of Primary GBM Tissue
2. Dissociation of Primary Tumor into Single-Cell Suspension
3. Cell Count and Plating
4. Passaging and expansion of GBM derived spheres:
5. Representative Results:
After plating the single cells harvested from GBM tumor tissue, tumor stem-like cells proliferate and generate small clusters of cells composed of few cells in 3-4 days (see the video and Figure 1). As these clusters grow, they acquire a more spherical shape so that by 7-8 days, proper phase bright spheres with an average diameter of 150-200 microns form (See the video and Figure 2). At higher magnification, healthy spheres usually demonstrate microspikes at their periphery. Having large sphere like clusters in 1-2 days after culture initiation is due to existence of non-dissociated clumps at the binging of the culture and should not be mistaken as true spheres. The amount of debris in primary tumor sphere culture varies depending on the initial source of tissue and whether or not it includes any surrounding brain tissue. Proper tissue preparation techniques including enzymatic and mechanical dissociation, and subsequent filtration of the sample with sufficient amount of medium can result in less debris in culture.
Figure 1. Primary GBM sphere culture 4 days after plating. Tumor stem-like cells proliferate and generate small clusters of cells in 3-4 days. Original Magnification; 20x.
Figure 2. Passage one GBM sphere culture 8 days after plating. Original Magnification; 20x.
To isolate neural stem and progenitor cells from normal adult and fetal brains1, 2, 3, 4 and also tumor stem-like cells from cancer tissues such as lung5, prostate6, breast7 and brain8, 9 the neurosphere assay has been frequently used as the method of choice. Using this simple and reproducible assay, one can generate an indefinite number of cells from resected tumor tissue that show similar characteristics as somatic stem cells; ex vivo multipotency, the ability to create new tumors upon implantation, and self-renewal. These cells could be used to study the basic cancer cell biology including cell-to-cell interactions, and differentiation, migration, invasion and cell death. In addition, isolated tumor stem-like cells provide an invaluable tool to study how tumors form, progress, and relapse and also to unravel the underlying deriving cellular mechanisms that eventually could provide insights to therapeutic options.
No conflicts of interest declared.
This work was supported by Grants from the Florida Centre for Brain Tumor Research; Preston A. Wells Jr. Center for Brain Tumor Therapy.
|NeuroCult NSC Basal Medium (Human)||Medium||Stem Cell Technologies||05750|
|NeuroCult NSC Proliferation Supplements (Human)||Medium supplement||Stem Cell Technologies||05753|
|%0.05 trypsin-EDTA||Reagent||GIBCO, by Life Technologies||25300-062|
|*MEM||Reagent||GIBCO, by Life Technologies||41500-018||HEM component|
|*Distilled water||Reagent||GIBCO, by Life Technologies||15230-147|
|**DNase I||Reagent||Roche Group||104159|
|**Soybean trypsin inhibitor||Reagent||Sigma-Aldrich||T6522|
|Pen/Strep||Reagent||GIBCO, by Life Technologies||15140-122|
|No. 10 scalpel blade||Surgical tool||BD Biosciences||371610|
|Petri Dish||Culture ware||BD Biosciences||353003|
|Small forceps||Surgical tools||Fine Science Tools||11050-10|
|Cell strainer||Sieve||BD Biosciences||352340|
|T25 flask||Culture ware||Nalge Nunc international||136196|
|T80 flask||Culture ware||Nalge Nunc international||178905|
|15 ml tubes||Culture ware||BD Biosciences||352096|
|50 ml tubes||Culture ware||BD Biosciences||352070|
|EGF||Growth factor||R&D Systems||2028-EG|
|b-FGF||Growth factor||R&D Systems||3139-FB|
|Heparin||Growth factor||Sigma-Aldrich||H4784||Reconstituted in PBS|
|* To make HEM, mix 1x10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4°C.|
|** To prepare trypsin inhibitor solution, first make 10 ml of DNase I solution (100 mg DNase dissolved in 100 ml of HEM) and then add 0.14 g of trypsin inhibitor to DNase solution and finally make the volume up to 1 Liter using HEM. Keep aliquots of the final products in -20°C freezer.|