The overall goal of this Indirect Coculture Assay, is to investigate the impact of astrocytes on the development of neurons. Our laboratory is interested in the role of neuron-glia interactions. It is believed that astrocytes contribute to the formation of the synapses, the connecting structures of the central nervous system.
In order to study their role, we have designed an in vitro model where we can combine primary astrocytes and primary embryonic neurons, in separate compartments. The compartments are connected by a permeable membrane, which allows for the analysis of exchanges between both cell types. Using this approach, we have been able to monitor synapse formation over periods of up to four weeks.
In addition, it is possible to investigate the individual roles of astrocytes on the one hand, and neurons on the other hand, using this assay. And finally, we can also investigate in more detail the secretome of our cell compartments which contains molecules that mediate the reciprocal influence of both cell compartments in that system. This method can provide insights into the interactions between neurons and astrocytes.
Also, it can be applied to other model organisms, such as the cells from rat. Generally individuals new to this method would struggle because experience is required to obtain the optimal maturation operation of hippocampal tissue after dissection. To dissect the cortices, remove the skin from the skull along the midline starting from the neck up towards the level of the eyes.
Next, hold the head by the rostral end with a pair of forceps and make an incision along the midline of the skull. Then, clip off the left and the right halves of the skull to expose the brain. After that, lift the brain up from the skull and transfer it to a 10 cm petri dish filled with HBSS.
Proceed in the same manner with remaining specimens until all the brains are collected in the dish. To separate the cortices, pinch off the hindrind using a pair of forceps. Make a midline incision between the two hemispheres.
Carefully fix the brain and cut off the midbrain and the olfactory bulb using a second pair of forceps, which results in two cortical halves. Following this, fix one cortical half with one pair of forceps and peel the meninges off by detaching it from the lateral edge. And subsequently pulling it from the cortical surface using a second pair of forceps.
Orient the cortical half with its surface facing down. Carefully dissect and remove the crescent shaped hippocampus. Then transfer the cortices into a 15 ml conical tube.
Add one ml of denem and 0.1%wv papain to a 2ml reaction tube. Incubate the suspension in a water bath at 37 degrees Celsius until the solution is clear. Add L-cysteine and DNAse to the mixture and shake gently.
Afterward filter the mixture to obtain one ml of sterile solution, then add it to the cortical tissue and incubate for 30 minutes to one hour at 37 degrees Celsius. To terminate the digestion reaction, add one ml of astrocyte medium and carefully titrate the digested tissue. Afterwards, add five ml of astrocyte medium to the cell suspension.
Once the tissue has been dissociated into a single cell suspension, centrifuge it for five minutes. After that, aspirate the supernatant carefully from the resulting pellet and re-suspend the cells in one ml of astrocyte medium. Add the cell suspension to the T75 flasks replenished with nine ml of astrocyte medium.
After seven days of culture, check if the cells have formed a confluent monolayer. Ensure that the temperature is adjusted to 37 degress Celsius and the filter of the flask is sealed with laboratory film to prevent evaporation of the carbon dioxide. In order to get rid of the progenitor cells and obtain a pure astrocyte culture, place the T75 flasks on an orbital shaker and shake the cultures overnight at 250 rotations per minute.
The next day aspirate the medium and add 10 ml of fresh culture medium replenished with 20 micromolar RSE to eliminate residual dividing cells. In this procedure place as many inserts as needed in the 24 well plate. Coat each insert with 10 micrograms per ml of poly d'lysine and incubate for one hour.
After one hour wash the inserts twice with PBS. In the meantime, aspirate the astrocyte medium and wash the culture once with 10 ml of PBS to ensure that residual serum is removed. Next, add three ml of 0.05%trypsin edta to the flasks and incubate the cells for trypsinization for approximately 10 minutes.
Afterward, gently re-suspend the cells in seven ml of astrocyte medium. Transfer the cell suspension to a 15 ml tube and centrifuge for five minutes at 216 x g. Then carefully aspirate the supernatant and re-suspend the cell pellet in one ml of astrocyte medium.
Count the cells using a counting chamber. Subsequently, fill the 24 well plate with 500 microliters of astrocyte medium per well. Aspirate the PBS and transfer 25, 000 cells and 500 microliters of astrocyte medium into each individual insert and incubate the culture at 37 degrees Celsius.
To dissect the hippocampi, prepare three 10 cm dishes filled with preparation medium and one 2 ml tube with one ml of preparation medium. Dissect the hippocampi from the cortex noyadees and collect them in a two ml tube filled with preparation medium. It is critical for the hippocampi to be isolated without an adjacent tissue from other regions of the CNS.
Therefore, after removing the hippocampus the foreign contaminating tissues have to be additionally removed. After dissection, transfer the tube to a sterile laminar flow bench. Remove the preparation medium carefully and digest the hippocampal tissue of one ml of digestion solution containing papain.
After 15 minutes of digestion, carefully withdraw the digestion solution by gentle suction with the pipette. Due to the neuron's high sensitivity, it is critical to titrate the hippocampal carefully in order to avoid bubbles and to obtain the optimal titration intensity. Wash the hippocampi three times with neuron medium by adding and carefully aspirating one ml of fresh culture medium per wash cycle.
After the final washing step, carefully titrate the tissue in one ml of neuron medium. Then count the cells using a counting chamber. Plate 35, 000 cells in 500 microliters of neuron medium per well of the 24 well plate and incubate the neurons at 37 degrees Celsius for one hour.
Now take the prepared inserts, which have been seeded with confluent astrocyte monolayers out of the incubator. Exchange the medium by aspirating the astrocyte medium and replacing it with 500 microliters of fresh neuron medium. Then place the inserts with astrocytes carefully into the wells that contain the neuron cultures using sterile forceps.
Subsequently, place the resulting indirect neuron astrocyte coculture back into the incubator until the experiments. This image shows the monolayers formed by astrocytes on the cell culture insert membrane. The astrocytes are immunostained for GFAP and MMP2.
The scheme here illustrates the indirect coculture setup. Although two cultures are physically separated, they share the same medium. Two secreted molecular mediators of neuron glia interactions are revealed in the co cultivation medium using western blot with multiple isoforms of TNC, an outright growth regulator and MMP2, an extra cellular matrix modifier.
This image shows that the primary neurons develop highly interconnected networks by day 14 of cultivation. The co localization of the presynaptic marker bassoon with the postsynaptic PSD95 scaffolding documents the structurally completed synaptic formation. In conclusion, using our assay system we have shown that it possible to combine primary astrocytes and primary neurons in the coculture model.
Both surtypes are separated but share the same medium. Hence, we can also investigate the secretome of both surtypes in our model. In this model synapses develop and maturate.
And furthermore, we can also show the emergence of additional specific structures such as perineuronal nets. Thus, our model system is well suited to investigate the influence of astrocytes on synapse formation, plasticity and function.
