Culture and Phenotyping Protocol of Glial Cell, Gliospheres, and Neurospheres

Introduction of Culture and Phenotyping Protocol

The discovery of embryonic and adult stem cells has led to great interest and excitement about the therapeutic potential of these cells across medical disciplines. The two hallmark properties of stem cells are their ability to undergo self-renewal and their potentiality for differentiation into the multiple cell lineages specific from the organ from which they derive. Because of these properties, stem cells are essential for an organism to maintain tissue homeostasis and for tissue repair following injury. The features of self-renewal and pluripotentiality also make adult stem cells invaluable therapeutic tools for reversing the symptoms/pathology of neurodegenerative disease, promoting tissue repair after ischemia or stroke, or as a model system to understand how to inhibit the proliferative capacity of recently identified tumor-associated stem cells.

Figure 1. Germinal Matrix.

These cells may be isolated and cultured in vitro so that they can be manipulated and studied by researchers. Neural stem cells are present in two specialized niches in the adult mammalian brain, the dentate gyrus of the hippocampus and the germinal matrix, located in subventricular zone of the lateral ventricles. Once removed, they can be propagated as adherent cells in traditional two-dimensional cultures or embedded in extracellular matrices, or as floating spheres called neurospheres. Culturing neural stem cells in the form of neurospheres helps to select for and preserve stem cell-like properties of isolated cells, and neural stem cells grown as neurospheres can be maintained in this primitive state for many passages, creating a readily available, stable pool of stem cells for research. The cells can be used to ask defined questions about factors that alter stem cell properties, such as their capacity for self-renewal, or how experimental or genetic manipulations affect the neuronal or glial differentiation program.

In order for these types of experiments to be successful, researchers must be able to definitively identify the genetic and phenotypic changes resulting from the experimental manipulations. Immunocytochemistry is an invaluable tool for cell phenotyping. Neural stem cells and their progeny can be identified by the use of select protein biomarkers. These biomarkers are selectively expressed by stem, neuronal, or glial cells; or during specific stages in the differentiation process.

Methods of Culture and Phenotyping Protocol

Isolation of Neural Stem Cells

1. Adult-derived neurospheres are derived from tissue isolated from progenitor cells in the SVZ of 6- to 8-week-old mice.
2. Mince tissue using the blade of a scalpel for approximately 1 min, until only small pieces are visually evident.
3. Incubate tissue in 1 ml of 0.05% trypsin–EDTA for 5 min at 37°C to dissociate. Dissociation reaction is terminated by the addition of an equal volume of trypsin inhibitor—mix well to ensure trypsin is inactivated.
4. Centrifuge at 300Xg for 5 min and carefully aspirate the supernatant to remove all traces of trypsin.
5. Resuspend the cells in 250 μl of Neurosphere Growth Medium. Triturate cells using a P200 pipette tip to break up any remaining aggregated or clumped cells.
6. Add an additional 750 μl of Neurosphere Growth Medium and filter the cell suspension through a 40 μm mesh filter to remove tissue debris not digested by the trypsin.
7. Remove a 20 μl aliquot of the filtered cell suspension and mix with 80–180 μl Trypan Blue and count the number of viable cells using a hemocytometer (see Note 1).
8. Plate the filtered cell suspension (5000 cells/cm²) on uncoated tissue culture dishes in Growth Medium for 6–8 days. The cells are ready to be passaged when the majority of the neurospheres are approximately 150 μm in diameter (see Notes 2 and 3).

Passaging of Neurosphere Cultures

When the neurospheres are ready to be passaged, gently suspend the neurospheres, and transfer the media and suspended neurospheres into a sterile conical tube.

1. Wash the culture dish one time with Neurosphere Growth Medium and pool the wash with the media and cells in the conical tube.
2. Centrifuge at 100Xg for 5 min. Aspirate the supernatant and resuspend the cells in 1 ml of 0.05% trypsin–EDTA.
3. Incubate at 37°C for 2–3 min to dissociate the neurospheres. Inactivate trypsin by adding an equal volume of trypsin inhibitor.
4. Centrifuge at 300Xg for 5 min. Aspirate the supernatant and resuspend the cells in 250 μl of Neurosphere Growth Medium.
5. Triturate cells six to eight using a P200 pipette tip to make a single-cell suspension of cells.
6. Remove a 20 μl aliquot of the filtered cell suspension and mix with 80–180 μl Trypan Blue and count the number of viable cells using a hemocytometer (see Note 4).
7. Plate the cells in Neurosphere Growth Medium (5000 cells/cm²) on uncoated tissue culture and culture 6–8 days.

Neurospheres from passages 1–3 (P1-P3) are often irregularly shaped and disorganized clusters, with debris still present from the isolation procedure. After P3, healthy neurosphere should be shiny, smooth with a uniform outline when viewed through a tissue culture microscope. When Neurospheres reach this state, they are ready for downstream studies.

The plating density of the neural stem cells in later passages is highly dependent on the question of the experiment. If the experimental outcome is to determine the clonality of isolated or treated cells, the cells must be plated at very low densities to endure that the resulting neurospheres are the result of single cell expansion and not the result to aggregation of multiple cells and smaller neurospheres. The most stringent plating method calls for cells to be plated in 96-well plates at very low plating densities (1–10 cells/μl with 200μl/well). The researcher can then identify which wells contain only a single cell and monitor just those wells for neurosphere formation. If the experiment is designed to measure other outcomes such as the influence of growth factors or pharmacologic treatments on neuronal development, then less stringent plating conditions are required (see Note 5).

Differentiation of Neurospheres

Coat Chamber Slides or Coverslips with Poly-L-Ornithine/Laminin

1. Dilute the poly-L-ornithine stock solution 1:100 in cell culture grade distilled water to make 10 μg/ml working solution.
2. Cover the surface of the chamber dish or coverslip with the 10 μg/ml poly-L-ornithine (typically about 1 ml for one well of a 2-well chamber slide or about 125–250 μl/cm²) and incubate for 1 h at 37°C.
3. Rinse the culture dish or coverslips twice with sterile water.
4. Cover the surface of the culture dish or coverslip with the laminin (10 μg/ml) and incubate for 2 h at 37°C.
5. Rinse the culture dish or coverslips twice with PBS (see Note 6).

Differentiation of Intact Neurospheres

The neurospheres are ready for plating when they reach approximately 150 nm in diameter (between day 6–8 of culture).

1. Collect the media and neurospheres from the culture dish and transfer to a conical tube and centrifuge at 100Xg for 5 min. Intact neurospheres should be handled very gently in order to avoid dissociation.
2. Aspirate the Growth Medium and gently resuspend the neurosphere in Differentiation Medium. Transfer the neurospheres suspension to a sterile 60- or 100-mm2 dish and select individual neurospheres using a 1 ml pipette.
3. Transfer an appropriate number of neurospheres to the tissue culture dishes containing the precoated coverslips or chamber slides. Neurospheres should be plated sparsely so that there is adequate space for the neurospheres attach and disperse on the coated surface without any merging of differentiating neurosphere populations. The differentiation process typically takes 6–8 days, by which time the differentiated cells should form a monolayer on the culture surface.
4. Fix the cells and analyze the resulting population of differentiated cells by immunocytochemistry.

Differentiation of Dissociated Neurospheres

When neurospheres are ready for harvesting, precoat glass coverslips or chamber slides and collect neurospheres as described above.

1. Aspirate the Neurosphere Growth Medium and resuspend the cells in 1 ml of 0.05% trypsin–EDTA. Incubate at room temperature for 2–3 min. Add an equal volume of trypsin inhibitor and mix well to terminate the digestion. Do not overtrypsinize cells as this can lead to decreased viability of cells after plating.
2. Centrifuge at 300Xg for 5 min and remove the supernatant. Immediately resuspend the cells in 250 μl of differentiation medium. Triturate the cells using a P200 pipette tip until only single cells are observed when the suspension is checked under the microscope.
3. Count the cells using a hemocytometer.
4. Dilute the appropriate volume of cell suspension in Differentiation Medium and plate in tissue culture dishes containing precoated coverslips or in precoated chamber slides. There is a great deal of variability in the required plating density of dissociated cells. It is best to try a range of densities from 1X10⁴ to 5X10⁵ cell/cm² to determine which density works best for each experiment.
5. Differentiation should be complete after 4–7 days. Fix the cells and analyze the differentiated cells by immunocytochemistry.

The resulted cell population will reflect the potentiality of the stem cell culture and can be used to test the effects of genetic or therapeutic manipulation on neural stem cell fate. Alternatively, culture conditions can be adjusted to selectively push neural stem cells into different lineages, as outlined below.

Culture Conditions to Preferentially Differentiate Neural Stem Cells into Neurons

1. Follow the preceding steps to harvest and plate either intact or dissociated neurospheres as described above.
2. In place of the generic Neurosphere Differentiation Medium, instead resuspend cells in Neuronal Differentiation Medium.
3. Culture the cells for 5–7 days. The medium can be replenished every 2 days by removing 1/2 to 3/4 of the medium and replacing it with an equal volume of fresh Neuronal Differentiation Medium (see Note 7).
4. On Day 5–7, add 500 μMdibutyral cAMP may be added to the differentiation medium and cell cultured for an additional 3 days to further select for neuronal cells.

Culture Conditions to Preferentially Differentiate Neural Stem Cells into Astrocytes

1. Follow the preceding steps to harvest and plate either intact or dissociated neurospheres as described above.
2. In place of the generic Differentiation Medium, instead resuspend cells in Astrocyte Differentiation Medium.
3. Culture the cells for 5–7 days. The medium can be replenished every 2 days.
4. On day 6–7, shake tissue culture dish overnight at 150 rpm in a 37°C incubator remove nonadherent glial cells (oligodendrocytes) to further purify culture for astrocytes (see Note 8). Cells may be split and subjected to additional rounds of shaking when confluent to continue to select for astrocytic lineage.

Culture Conditions to Preferentially Differentiate Neural Stem Cells into Oligodendrocytes

1. Follow the preceding steps to harvest and plate either intact or dissociated neurospheres as described above.
2. In place of the generic Differentiation Medium, instead resuspend cells in Oligodendrocyte Differentiation Medium.
3. Culture the cells for 5–7 days. The medium can be replenished every 2–3 days.

Immunocytochemical Analysis of Intact, Undifferentiated Neurospheres

The neurospheres can be fixed and immunolabeled as intact neurospheres. This may be desired if the goal is to identify pathways associated with maintenance of, or the differentiation from, the stem cell phenotype. Immunocytochemical analysis of the intact neurosphere would allow the investigation of how the expression of proteins of interest changes during the differentiation program, or to identify the presence of representative markers of apoptosis or cell cycle regulation following experimental manipulation of the neurosphere cultures. Identifying the patterns of protein expression in intact neurospheres is advantageous since dissociation and plating of the neural stem cells initiates differentiation and alters gene expression. In addition, a model containing the majority of cells present in the brain allows one to track cytopathological changes to specific phenotypes, and to observe the interactions between different cell types.

Fixation and Permeabilization of Intact Neurospheres

1. Thaw the Matrigel bottle at 4°C overnight to prevent polymerization. The next day, prechill neurobasal medium and 1.5 ml Eppendorf tubes. On ice, dilute Matrigel 1:50 with cold neurobasal medium. Aliquot the undiluted Matrigel into chilled Eppendorf tubes and store at -20°C.
2. Cover the surface of the chamber dish or coverslip with the diluted Matrigel and incubate for 2 h at 37°C.
3. Collect the media and neurospheres from the culture dish and transfer to a conical tube and centrifuge at 100Xg for 5 min. Intact neurospheres should be handled very gently in order to avoid dissociation.
4. Aspirate the old growth medium and gently resuspend the neurospheres in fresh growth medium. Transfer the neurosphere suspension to a sterile 60- or 100-mm2 dish and select individual neurospheres using a 1 ml pipette.
5. Transfer an appropriate number of neurospheres to the Matrigel-coated tissue culture dishes containing the precoated coverslips or chamber slides.
6. Incubate the neurospheres at 37°C for 20 min to allow the neurospheres to attach to the Matrigel.
7. Fix in 4% paraformaldehyde for 15 min, and wash 1 time with PBS for 20 min at room temperature.
8. Proceed with immunolabeling protocol discussed above.

Notes of Culture and Phenotyping Protocol

1. The amount of Trypan Blue added will depend on the cell density of the cell suspension.
2. Avoid moving the neurospheres-containing dishes too much to prevent mechanical aggregation of neurospheres.
3. The neurospheres should not be allowed to grow larger than 150 nm to preserve the viability and undifferentiated state of the inner cells.
4. If there is not a single-cell suspension, pass the cells through the P200 pipette tip several more times. It is important the cells are plated as individual cells to help ensure the resultant neurospheres are the result of clonal expansion and do not arise from multiple cells.
5. Cells grow better if media is changed 1/2 a time.
6. Coverslips or culture dishes may be prepared in advance. Keep the coated surfaces covered with PBS, wrapped tightly with Parafilm, at RT for up to a week.
7. Neurons are not all that adherent. Be careful not to dislodge them during media changes.
8. These the floating cells can be taken and replated in Oligodendrocyte Diff Medium to concurrently generate an oligodendrocyte culture.
9. It is best to use freshly diluted 4% PFA.
10. If cells are easily dislodged/washed off, they may be washed just one time with a greater volume of PBS.
11. Fixed cells may be stored for up to 3 weeks in PBS at 4°C before staining. Do not allow slides to dry.
12. If methanol or acetone are used as a fixatives, a permeabilization step is not needed.
13. Include a secondary only control to ensure observed signal is specific to primary antibody. If colabeling, also label slides with singly with each primary antibody.
14. Can be 10–25 ml depending on the starting weight of the tumor sample.
15. The cell suspension may be filtered through a 40 μm mesh filter to remove debris.

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