Department of Neurosurgery and Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI
BioTechniques, Vol. 55, No. 2, August 2013, pp. 83–88
Recent studies have found that the biological features of primary tumors are faithfully recapitulated when a patient's tumor is processed and then maintained as a 3-D spheroid in specialized cell culture media. However, a major drawback for maintenance and routine passage of primary tumors as spheroids has been the high cost of custom-formulated media compared to regular serum-supplemented media. Here we report the formulation of a cost-effective, serum-free medium in which high-grade primary brain tumor (glioblastoma) explants can be established and maintained as spheroids. Based on DMEM, this formulation requires only supplementation with several amino acids, vitamins, synthetic EGF, and bFGF, with most of the cost being associated with the growth factors. A simple addition of BSA (fraction V) obviated the need for numerous other components (or human serum) commonly used in the specialized commercial media formulations.
Maintenance of primary human tumors as in vitro monolayer (2-D) cultures in the presence of bovine, equine, or human serum supplemented media has been an established laboratory practice for decades. However, it has become apparent that these culture conditions irreversibly modify both genotype and phenotype, with passaged tumor cell lines bearing little resemblance to the tumor isolated from the patient (1). While cells derived from malignant brain tumors (glioblastoma) maintained in the presence of serum are tumorigenic in vivo, they tend to form well-circumscribed tumors in xenograft models that do not recapitulate the invasive (diffused) growth patterns observed in a clinical setting. In contrast, when tumors are established and maintained as 3-D spheroids using specialized serum-free media or media with minimum amounts of human serum, the biological features of the primary tumor is faithfully maintained, facilitating preclinical studies more relevant to the patient's tumor (1, 2). A major drawback for such maintenance and routine passage of tumors as spheroids has been the high cost associated with this specialized media in comparison to “regular” media which are most often supplemented with fetal bovine serum (FBS), in the average tissue culture laboratory. Here, we report the formulation of a cost-effective, serum-free medium for routine use, in which primary malignant glioblastoma tissues can be established and maintained as spheroids (neurospheres).
Most commercial formulations for spheroid culture are proprietary and expensive (e.g., Neurobasal medium, Life Technologies Inc., Grand Island, NY; NeuroCult medium, StemCell Technologies Inc., Vancouver, Canada; CellGro COMPLETE medium, Mediatech Inc., Manassas, VA). In addition, some formulations also contain human serum as a component necessary for initiation and propagation of spheroids from human neuron or glial tissue explants derived from human patients. However, media formulations developed during pioneering studies several decades ago utilized either DMEM alone, or in combination with F12 media supplemented with various growth factor/steroid/hormone combinations to culture neurons and glial cells in vitro (3-5).
This method describes a DMEM based formulation to establish and propagate spheroid (neurosphere) cultures from human glioblastoma resections. Lipid-fortified BSA (fraction V) was used as a substitute for several supplements present in commercial neurosphere culture media, including donor human serum; the resulting formulation is 1/6 the cost of commercially available neurosphere media.
We modified and supplemented the standard DME medium with components from the G-5 supplementation (5), but using off-the-shelf components and insulin-transferrin-selenium premixes from low-cost commercial sources (see Supplementary Protocol). Our lab-made formulation did not include any human or bovine serum and, in addition to the standard components of high-glucose DME medium, included three additional amino acids (Ala, Asn, and Pro), Zn2+, biotin, vitamin B12, hydrocortisone, the insulin-transferrin-selenium mixture, and the growth factors EGF and bFGF, which are needed for propagation and maintenance of glial cells and tumor cells of astrocytic lineage as neurospheres or spheroids (Table 1). Since human plasma contains albumin at 3–4 g/dL (6), our medium was supplemented with BSA preparations that maintain the fatty acid and growth factor traits of the original “fraction V” (cold-ethanol) preparations (Sigma Chemical Co., St. Louis, MO, or Life Technologies) at 4% (w/v) final concentration. Preliminary studies indicated that albumin was essential to the establishment and propagation of spheroids, serving as a carrier for cations, growth factors, and hormones, in addition to functioning as an osmoregulator. All other components listed in the proprietary B27-type (4) supplement that are used in commercial media formulations were not necessary for establishment and propagation of spheroid cultures from glioblastoma explants.
In brief, a tumor tissue section (0.25–0.5 g wet weight, the size of a pencil-tip-eraser) was resuspended immediately after surgical resection in 10 mL ice-cold sterile PBS (or saline) in a 60 mm or 100 mm diameter tissue culture dish, and then sliced into approximately 1–2 mm3 fragments by using a cross-cut pattern with a sterile scalpel. The tissue fragments, along with the PBS or saline, were aspirated into a 15 mL conical tube and allowed to settle for 1 min. The spent PBS (or saline) containing blood and serum components was aspirated off, and the tissue fragments were washed while in the tube in the same manner as above, with 10 mL aliquots of cold PBS until the wash was visibly clear of blood (approximately 2–3 additional washes are needed depending on the vascularity of the tumor). Then, tissue fragments were aspirated and transferred onto a fresh 100 mm tissue culture dish, and all residual PBS was aspirated off. Five mL of AccuMax (Innovative Cell Technologies, San Diego, CA), a cell-dissociation enzyme formulation of crustacean origin that is more mild than trypsin, was added to the plate, and the plate placed on a rocker platform for 30 min to 1 h (but no longer) at room temperature until at least half of the tissue fragments had sloughed off as cell clumps consisting of 2–10 cells when visualized under a low-power (40×) inverted microscope. Next, the mixture was placed back in a 15 mL conical tube and residual undigested tissue fragments were allowed to settle for 1 min. The supernatant was aspirated into a new 15 mL conical tube and the cell clumps then allowed to “gravity-settle” for 10 min. The supernatant from this step was discarded; although some tumor cells are lost at this step, almost 90% of the erythrocytes that contaminate primary tumor cell preparations can be eliminated from the tissue digest. Ten mL of our “home-made serum free” (hereafter referred to as HMSF medium) medium formulated as described above (see Supplementary Protocol) was added to the tube. The suspension was then aspirated into a 100 mm tissue culture dish and placed in a 37°C CO2 tissue culture incubator (5% CO2-air). After 24 h, the culture was aspirated into a 15 mL conical tube and centrifuged in a swing-bucket rotor at 100× g, in 1 min steps until most of the glial cells were pelleted while residual erythrocytes remained in the supernatant (the stepwise centrifugations are carried out until the “whitish” cell pellet just starts to become covered with a “reddish layer” of erythrocytes). Again, supernatant was discarded, and the cell pellet resuspended in a fresh 10 mL aliquot of HMSF medium and placed in a new 100 mm tissue culture dish. This process was repeated every 72 h, until tumor spheroids were clearly visible under low-power microscopy (1–2 weeks based on tumor grade, with highly aggressive tumors giving rise to spheroids within a week). Media changes were continued once every three days during long-term propagation of the spheroids.
Once the spheroids reached a diameter of roughly 100 µm (approximately 100 cells/spheroid), they were spun down at 100× g for 5 min, washed once in 10 mL PBS, and then resuspended in either 2.5 mL of Accutase (another mild trypsin substitute of crustacean origin; Innovative Cell technologies) or 2.5 mL of Accumax. This suspension was incubated at room temperature for up to 10 min with occasional gentle mixing until most of the spheroids had dispersed into a fine suspension of cell clumps consisting of 2 to 10 cells. Ten mL HMSF medium was added to the mixture and centrifuged as before. The pellet was resuspended in 10 mL fresh HMSF medium, roughly enumerated using a hemocytometer or with a Coulter counter, and the equivalent of 1 × 106 cells (in 10 mL) plated per 100 mm diameter tissue culture plate for passaging the cells as spheroids (see Supplementary Protocol for alternate non-enzymatic fragmentation of spheroids for routine passage).
To date, we have established and maintained spheroid cultures from eight primary glioblastoma explants using our HMSF media (with one failure, where the tumor explant grew as attached cells in both HMSF and commercial neurosphere media). It is important to note that even established glioma cell lines such as U87MG and U251MG generate spheroids in HMSF within 1–2 weeks (Figure 1).