The overall goal of this procedure is to develop routine neuros sphere, culturing of high grade astrocytomas and large scale expansion of tumorogenic cells for preclinical studies. This is accomplished by first acutely dissociating fresh tumor specimens and separating the single cells from the red blood cells and debris. In the second step, the tumor cells are cultured in neuros sphere, medium supplemented with growth factors until neurospheres are observed.
Neurospheres may be cultured for in vivo experiments and molecular characterization or formal and fixed and embedded in paraffin for immunological characterization. Ultimately, the long-term self-renewal orthotopic tumor formation and molecular profiling of the neuros sphere cultures can be evaluated. Main advantage over existing methods like 10%FBS long-term serum cultures is that this technique preserves the molecular and genetic characteristics of the original tumor.
This method makes possible the establishment of a live tumor bank for the generation of patient specific glioblastoma cell cultures and animal models for clinically relevant research. The implications of this model for therapeutic testing for patients with malignant glioma is obvious. The model most closely represents the primary tumor and allows therapeutic testing in a preclinical environment.
Though this method can provide insight into high grade gliomas, it can also be applied to other human and animal tumors. Generally, individuals new to this method will struggle cause it requires proper sterile techniques and care and multiple steps to favor long-term cell viability. We first had the idea for the 3D histology method because of the superior subcellular localization of labeled proteins and the sections in relation to the more common methods of labeling and imaging the whole neurospheres or dissociating the cells.
Visual demonstration of this histology method is critical as the fixing, clearing, processing and embedding steps are difficult to learn. Care is needed to assure that all the neurospheres are adequately exposed to the reagents and processed evenly, giving the best available pellet for morphology and immunohisto chemistry. Starting with 200 to 500 milligrams of tumor sample, use a scalpel blade to mince the tissue.
Transfer the pieces to a 15 milliliter tube containing 10 milliliters of medium, and then invert the suspension several times to mix the tissue solution. Let the tumor pieces sediment by gravity and then remove the medium. Continue to wash the debris from the tumor pieces until the medium is no longer turid.
Next, remove the medium. Add two milliliters of enzymatic tissue dissociation solution per 0.5 grams of mixed tumor sample and gently mix the solution by inversion. Incubate the tissue solution at 37 degrees Celsius in a tissue culture incubator under rotation.
After 30 minutes, tri rate the suspension with a two milliliter pipette. When the tumor is digested to a mostly single cell suspension, stop the enzymes with two volumes of stop solution and mix the cell suspension with a five milliliter serological pipette. Now filter out any undigested material through a 40 micrometer cell strainer and pellet the cells for five minutes at 800 times G and room temperature.
Then after three washes in 10 milliliters of media, resus, suspend the final cell pellet in five milliliters of media. Slowly layer this cell suspension over five milliliters of lympho light M, and then separate the cell populations for 20 minutes at 1300 times G and room temperature. Now collect the interface layer containing the nucleated cells into a 15 milliliter tube containing 10 milliliters of media.
Then resuspend the cells in neuros sphere medium supplemented with growth factors, and plate them at less than one times 10 to the fifth cells per milliliter. In irregular T 25 tissue culture flask at 37 degrees Celsius and 5%carbon dioxide. In a humidified tissue culture incubator.
Transfer the floating neurospheres that form over one to three weeks to fresh flask to separate them from the attached cells and debris. To analyze the neurospheres by immunohisto chemistry first pellet the neurospheres, then taking care not to disturb the pellet. Remove the supernatant and add 10 milliliters of DPBS to the tube.
Then remove the DPBS, re suspend the pellet in 10%neutral buffered formin and incubate the cells for 20 minutes at room temperature. After pelleting the steroids again, re suspend them in a series of ethanol incubations. After the second 95%ethanol incubation resuspend the PHE with absolute ethanol until the cells are bright, white and condensed.
Next, make a small cone out of lens paper. Place it into a small funnel within a beaker and wet the lens paper with absolute alcohol. Loosen the pellet slightly with fresh 100%ethanol and then pour off excess alcohol.
Pour the spheres through the lens paper cone, making sure they go to the tip and rinse the tube with additional absolute ethanol. Pour any remaining steroid through the lens paper cone and then remove the cone from the funnel. Then fold the lens paper into a square, making sure the steroids are as securely enclosed as possible, and transfer the package neurospheres to a cassette.
Now place the cassette into an automatic tissue processor and run the processor. Then remove the cassette and place it on the heated part of a paraffin embedding system. After confirming the surface is free of fragments, dust, or other debris, siphon all the paraffin from the forceps in warming wells.
Then warm a clean pair of paraffin free forceps and add a small amount of paraffin to a warmed base mold. Now remove the packet from the cassette and place it on the heated part of the embedding system. Carefully open the paper until the sphe are visible.
Use the prewarm forceps to gather as much of the material as possible, and then transfer the spheroids into the liquid paraffin in the base mold. Carefully break up any clumps and move the steroid away from the corners into a uniform central layer. Move the base mold to the cooling area to secure the steroid in place.
Finally, add the cassette and fill it slowly with paraffin. Once the cassette has completely chilled, the neuros sphere paraffin block can be sectioned for immunohistochemistry as described in the table. The efficiency of this protocol for establishing long-term neuros sphere cultures from 88 newly diagnosed and 37 recurrent tumors total was 41.6%Similar for both newly diagnosed tumors and recurrent tumors.
For some of the glioblastoma samples, neurospheres formed within the first few days while for others longer culturing time was required. The efficiency of the neuros sphere formation was not exclusively dependent on the level of necrosis in the tissue as exemplified by the results from a newly diagnosed tumor with a high cell density and a recurrent and necrotic tumor both processed as just demonstrated and yielding neurospheres testing the tumorogenic potential of each neuros sphere. Culture in immunocompromised mice is a crucial validation of this approach for the enrichment of cancer stem cells.
In this experiment, two different glioblastoma neuros sphere cultures were implanted into the brains of immunocompromised mice. The xenograft tumors presented morphological characteristics of glioblastomas such as invasion into the brain, parenchyma and necrosis. This in this experiment, cells which did not form neurospheres were cultured in neuros sphere medium supplemented with 2%FBS, the tumor formation of these alternative media cells.
Compared to glioblastoma, neurospheres cultures xenograft into nude mouse recipients was evaluated. No differences in survival. Tumor growth dynamics or morphology were observed between the two pre-plant culture methods.
While neural stem cell markers, including SOX two are downregulated in most primary glioblastoma cells cultured in 10%FBS glioblastoma cells grown in neurospheres medium, supplemented with 2%FBS retain SOX two expression after processing as just demonstrated. And this experiment, neurospheres were labeled with h and e antiox two antibody for demonstrating nuclear localization and epidermal growth factor antibody for illustrating cell membrane localization. As the resulting images illustrate the demonstrated method optimizes the analysis of protein expression and post-translational modifications while maintaining the 3D architecture of the neurospheres Once mastered, this tissue dissociation technique can be done in approximately two hours if performed properly While attempting procedure, it is important to remember that the time interval between surgery and association, as well as the heterogeneous molecular composition of high grade gliomas can affect outcome Following this procedure.
Other methods like intracranial implantation can be performed in order to recapitulate the original tumor in animal models. This technique is revolutionizing neuro-oncology, allowing preclinical testing in a clinically relevant environment. After watching this video, you should have a good understanding of how to develop and maintain long-term neurospheres, as well as how to produce formal and fixed paraffin embedded neurospheres for molecular analysis.
Don't forget that working with human tissue can be extremely hazardous, and precautions such as personal protection, compatible and potentially hazardous agents should always be used while performing this procedure.
