We present a protocol to prepare free-floating slice cultures from brain tissue collected from living human donors submitted to resective brain surgery. This culture is amenable to perform biochemical and cell biology assays or immunohistochemistry. It is expected to contribute to the elucidation of the mechanism underlying neurodegeneration in associated brain diseases.
The main advantage of this technique is that it's a simpler and cost effective alternative to the classic slice culture protocol using membrane inserts. Although the overall protocol is not complex, some steps such as removal of meninges, gluing the tissue to the vibratome specimen disc, and resection for immunohistochemistry are best understood when demonstrated visually. Helping to demonstrate the procedure will be Niele Mendes and Glaucia Almedia who are grad students, and Giovanna Nogueira, another grad student.
To begin this procedure, add salt to a bucket of ice. Transfer the slicing solution to this bucket and let it rest under carbogen mixture bubbling for at least 20 minutes prior to use. Next, prepare a block of 3%agarose that is about two centimeters by two centimeters by two centimeters.
Super glue the agarose block to the vibratome specimen disc in order to create additional mechanical support to the tissue sample during slicing. On the vibratome, set the section thickness to 200 micrometers, the vibration frequency to 100 hertz, and choose a slicing speed between 0.5 and 1.0 millimeters per second. Then lock the vibratome buffer tray to the vibratome base and add ice to keep it refrigerated prior to receiving the slicing solution and the sample and throughout the slicing procedure.
First, set up a transport apparatus that consists of a portable gas cylinder containing a carbogen mixture connected to a pressure flux valve that controls the gas output connected to silicone tubing that connects that gas output to the transport vessel and the transport vessel which contains the transport solution and ice for sample cooling during transport. Collect and transport the specimen and transport as outlined in the text protocol. Transfer the specimen to a Petri dish containing slicing solution.
Using fine surgical tools, carefully remove as much of the remaining meninges as possible. Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design and use a number 24 scalpel blade to trim a flat surface to be the base that will be glued to the specimen disc. Using a disposal plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry off excess solution with filter paper.
Next, use super glue to attach the tissue to the vibratome specimen dish until it is firmly adhered to the dish and in contact with the agarose block. Place the vibratome specimen disc into the vibratome buffer tray. Lock the knife holder in place with the razor blade firmly fixed.
Add slicing solution and ensure that it is covering both the specimen and the blade. Then begin slicing the specimen into 200 micrometer slices. Transfer the slices from the buffer tray to a Petri dish containing slicing solution and trim any loose edges and excess white matter to a proportion of approximately 70%cortex and 30%white matter.
In a laminar flow cabinet, add 600 microliters of culture medium to each well of a 24-well plate and incubate at 36 degrees Celsius with 5%carbon dioxide for at least 20 minutes prior to plating the slices. After this, use a paintbrush to plate one slice in each well. If there are any unused wells in the plate, fill them with 400 microliters of sterile water.
Incubate at 36 degrees Celsius with 5%carbon dioxide. Supplement 10 milliliters of the previously prepared culture medium with brain derived neurotrophic factor at a concentration of 50 nanograms per milliliter. After eight to 16 hours, remove 333 microliters of the conditioned medium from each well and add 133 microliters of fresh BDNF supplemented medium.
Replace one-third of the conditioned medium with fresh BDNF supplemented medium every 24 hours. First, transfer the slices from the wells containing culture medium to a new 24-well plate containing PBS. Remove the PBS from each well and replace it with one milliliter of 4%paraformaldehyde.
Incubate overnight at four degrees Celsius. The next day, carefully remove the paraformaldehyde from the wells and replace it with one milliliter of 30%sucrose solution. Incubate at four degrees Celsius for 48 hours.
After 48 hours, set the freezing microtome to negative 40 degrees Celsius. Prepare a sucrose base on the microtome stage where the slices should be placed. After it is frozen completely, cut some of the frozen sucrose to produce a flat surface on which the slice will be placed.
Next, place each slice over a stretched plastic film and use a paintbrush to flatten the tissue. In a single move, transfer the stretched slice to the frozen sucrose base. After the slice has rested for five to 10 minutes to freeze properly, cut the slice into 30 micrometer sections.
Transfer the 30 micrometer sections to a Petri dish containing PBS and proceed to a histology protocol adequate for the experimental design. When determining the quality and health of cultured slices, a critical aspect to evaluate is the presence and typical morphology of the expected neural cell types, neurons and glial cells. The typical architecture of the human cortical lamination is observed in a slice at day in vitro four revealed by neuronal immunolabeling.
In addition, the expected presence of microglia and astroglia is also observed. These results demonstrate that tissue architecture is not significantly affected either by the surgical procedure, the sample processing, or by the short-term period in vitro. The neuronal response to potassium chloride-induced depolarization have also been quantified in cultured slices by following ERK phosphorylation.
Interestingly, a clear increase in ERK phosphorylation is seen in potassium chloride-treated slices at day in vitro four. Finally, the response of slices at day in vitro four to a toxic challenge is evaluated with a known oxidative stress inducer hydrogen peroxide. Exposing the slices to 300 millimolar hydrogen peroxide for 24 hours led to a robust decrease in MTT reduction.
Taken together, the massive cell death observed in day in vitro five slices after the hydrogen peroxide challenge and the potassium chloride-induced depolarization results indicate the preserved general health of slices at day in vitro four which respond to a toxic stimulus such as oxidative stress. This protocol is mainly devoted to studies based on assays lasting than one week such as the investigation of molecular mechanisms of neurotoxicity and neuroprotection by candidate drugs. Although this protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment for micro resistant temporal lobe epilepsy, it's likely that tissue collected from other brain regions or conditions could also be sources to produce free-floating slice cultures.
Slice cultures from adult human brain may be key in advancing our understanding of human neuropathologies due to their unique cellular circuitry and molecular machinery lacking in slices produced from rodent brains.
