Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Live adult brain tissues were obtained from patients undergoing resective neurosurgery for the treatment of pharmacoresistant temporal lobe epilepsy (Figure 1A). All procedures were approved by the Ethics Committee from the Clinics Hospital at the Ribeirão Preto Medical School (17578/2015), and patients (or their legal responsible person) agreed and signed the informed consent terms. Collection of the tissue was done by the neurosurgery team at the Epilepsy Surgery Center (CIREP – Clinics Hospital at the Ribeirão Preto Medical School, University of São Paulo, Brazil).

1. Sterilization of materials

NOTE: All material and solutions must be sterilized prior to use.

1. Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 °C.
2. Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.
3. Sterilize media and solutions by autoclavation or filtration through 0.22 µm pore membranes.

2. Preparation of solutions

1. Prepare 15-20 mL of transport solution: 50% v/v Hanks' balanced salt solution (HBSS) pH 7.4, 50% v/v basal medium for maintenance of post-natal and adult brain neurons (Table of Materials), supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 3 mg/mL glucose, and 33 µg/mL gentamicin.
   NOTE: Transport solution must be refrigerated and oxygenated (bubbling with carbogen gas) for at least 20 min prior to sample collection.
2. Prepare 300 mL of slicing solution (HBSS supplemented with 10 mM HEPES and 3 mg/mL glucose) and cool it down in a freezer to the point of initial crystal formation.
3. Prepare 20 mL of culture medium: basal medium for maintenance of post-natal and adult brain neurons (Table of Materials) supplemented with 1% L-glutamine derivative (Table of Materials), 2% supplement for neural culture (Table of Materials), 1% penicillin/streptomycin, and 0.25 µg/mL amphotericin B.

3. Setting up the slicing apparatus

NOTE: This protocol is ideally performed with the assistance of a colleague due to the logistics of sample collection in the surgical room.

1. In a bucket of salt-added ice, let the slicing solution rest under carbogen mixture bubbling (95% O₂, 5% CO₂) for at least 20 min prior to use.
2. Prepare a block of 3% agarose (approximately 2 cm x 2 cm x 2 cm) and superglue it to the vibratome specimen disk in order to create additional mechanical support to the tissue sample during slicing (Figure 1E).
3. Set the vibratome for slicing: section thickness of 200 µm, frequency of vibration of 100 Hz, and speed of slicing between 0.5-1.0 mm/s.
4. 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.

4. Sample collection

NOTE: In this protocol, human neocortical tissue was collected in the surgical room and transported to the laboratory.

CAUTION: When dealing with human samples, follow the appropriate safety protocols established by the Institution.

1. Set up the transport apparatus (Figure 1C) that consists of: a portable gas cylinder with carbogen mixture connected to a pressure/flux valve that controls the gas output connected to a silicon tubing that connects gas output to the transport vessel; a transport vessel, usually a 50 mL conical centrifuge tube with perforated lid for gas input, containing the transport solution; and ice for sample cooling during transport.
2. Collect and transport the specimen (Figure 1B) immediately to the lab. Submerse the specimen in cold transport solution (constantly bubbled with carbogen mixture).

5. Slicing

1. Transfer the specimen to a Petri dish (100 mm x 20 mm) containing slicing solution and, with fine surgical tools, carefully remove as much as possible of the remaining meninges in the sample (Figure 1D).
2. Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design, and with a no. 24 scalpel blade, trim a flat surface to be the base glued to the specimen disk.
3. Using a disposable plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry excess solution using filter paper (dry by capillarity and avoid touching the tissue fragment with paper).
4. Using superglue, attach the tissue to the vibratome specimen disk until it is firmly adhered to the disk and in contact with the agarose block (Figure 1E).
5. Place the vibratome specimen disk (with tissue properly attached) in the vibratome buffer tray filled with slicing solution that must be bubbling during the whole process.
6. Lock the knife holder in place with the razor blade firmly fixed.
7. The slicing solution must cover both the specimen and the blade, only then start slicing (Figure 1F).
8. Cut the specimen into 200 µm slices.
   NOTE: Although some vibratomes cut the specimens automatically, the close observation and minor adjustments in slicing speed during the process may help producing better slices. Discard initial irregular slices.
9. Transfer the slices from the buffer tray to a Petri dish with slicing solution and trim loose edges and excess white mater to a proportion of around 70% cortex/30% white matter.

6. Culture

NOTE: Perform this step in a laminar flow cabinet under sterile environment.

1. Add 600 µL of culture medium per well (in a 24 well plate) and incubate for at least 20 min at 36 °C and 5% CO₂ prior to plating the slices.
2. Plate one slice per well using a paintbrush (Figure 1G).
3. If there are any unused wells in the plate, fill them with 400 µL of sterile water.
4. Incubate the plate at 36 °C, 5% CO₂.
   NOTE: First medium replacement must be done in between 8-16 h after plating depending on the size of the slice.
5. Supplement 10 mL of the previously prepared culture medium with 50 ng/mL brain derived neurotrophic factor (BDNF).
   NOTE: During the first 8-16 h, the slices are incubated in 600 µL of medium to avoid nutrient deprivation and acidification, since medium consumption in this phase is accelerated. From the next step, the volume of medium per well is adjusted to 400 µL.
6. Remove 333 µL of the conditioned medium from each well and add 133 µL of fresh BDNF-supplemented medium.
7. Repeat the process of medium replacement every 24 h by replacing one-third of the conditioned medium with fresh BDNF-supplemented medium.

7. Evaluating health, morphology, and function in cultured slices

NOTE: To induce cell death for the purpose of illustration in the representative results, some slices were submitted to a 24 h treatment with the oxidative stress inducer H₂O₂. Steps 7.1.2 and 7.1.3 describe use of H₂O₂ to induce cell death.

1. Cell viability
   NOTE: A simple, straightforward method to evaluate neurotoxic/neuroprotection in challenged slice cultures is the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay³⁶, which measures the percentage of metabolic active cells under normal conditions compared to treated samples.
   1. In the laminar flow cabinet, replace one-third of the medium with fresh medium.
   2. From a 30% H₂O₂ stock solution, add a volume of H₂O₂ per well to reach the intended final concentration (30 mM or 300 mM).
      NOTE: H₂O₂ was added after the daily change of the medium to guarantee the proper supply of fresh nutrients prior to the challenge.
   3. Incubate the plate for 24 h at 36 °C, 5% CO₂.
      NOTE: After H₂O₂ treatment (or other toxic stimulus of interest), the following steps describe cell viability determination by the MTT assay.
   4. Add 40 µL of MTT solution to a final concentration of 0.5 mg/mL to each well in the plate.
   5. Incubate the plate at 36 °C, 5% CO₂ for 3 h.
   6. Wash the slices with phosphate-buffered saline (PBS) and transfer to a microtube.
   7. Carefully remove any remaining solution by pipetting.
   8. Weigh the microtubes containing the slices to determine the mass of each slice (this is key for normalizing the absorbance readings obtained).
      NOTE: If needed, samples can be frozen (-20 °C) at this stage for later processing.
   9. Homogenize the slices in 200 µL of isopropanol/HCl using a motorized pestle.
   10. Centrifuge at room temperature (RT) for 2 min at 2600 x g.
   11. Collect the supernatant and measure the absorbance at 540 nm.
2. KCl-induced neuronal depolarization
   NOTE: Phosphorylation of the mitogen activated protein kinase (MAPK) signaling cascade protein ERK, followed by western blotting, can be used for the quantification of the neuronal response to KCl-induced depolarization³⁷ .
   1. At the flow cabinet, replace the culture medium by 300 µL of HBSS previously equilibrated to 36 °C.
   2. Replace the HBSS with 300 µL of fresh HBSS previously equilibrated to 36 °C.
   3. Incubate the plate at 36 °C, 5% CO₂ for 15 min.
   4. Replace the HBSS with either the fresh HBSS or with 80 mM KCl depolarizing solution (both at 36 °C) and incubate at 36 °C, 5% CO₂ for 15 min.
   5. Transfer the slices from the plate to microtubes. At this step, slices can be stored at -20 °C for later processing.
   6. Prepare tissue extracts in 150 µL of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% nonionic surfactant; 0.1% sodium dodecyl sulfate; pH 7.5). Centrifuge extracts at 4 °C for 10 min at 16000 x g, collect supernatant, and determine total protein concentration using the Bradford method.
   7. Load 30 µg of total protein onto a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
   8. After electrophoresis, transfer the gel content to a nitrocellulose membrane.
   9. After blocking the membrane with 5% non-fat dry milk in TBS plus 0.1% Tween, incubate with mouse anti-pERK (1:1,000) for 16 h at 4 °C. After washing, incubate for 2 h at RT with rabbit anti-ERK1/2 (1:1,000).
   10. Incubate with the appropriate HRP-conjugated secondary antibody at RT for 1 h.
   11. Reveal with the preferred HRP substrate.
3. Morphological evaluation
   NOTE: In addition to cell survival and functional evaluations, it is important to analyze tissue morphology. Be aware that resectioning the cultured slice is an important step to producing as much high-quality material as possible for morphological analysis.
   1. Fixing, cryoprotecting and resectioning the slices
      1. Transfer the slices from the wells with culture medium to a new 24 well plate containing PBS.
      2. Remove the PBS and add 1 mL of 4% paraformaldehyde (PFA). It is important that slices be kept flat prior to adding PFA. Incubate overnight at 4 °C.
      3. Carefully remove the PFA solution and add 1 mL of 30% sucrose solution. Incubate for 48 h at 4 °C.
      4. Set the freezing microtome to -40 °C.
      5. Prepare a sucrose base on the microtome stage where the slices should be placed (Figure 2A). Let it freeze completely and carefully cut some of the frozen sucrose to produce a flat surface on which the slice will be placed.
      6. Place each slice over a stretched plastic film and use a paintbrush to flat the tissue.
      7. Transfer the stretched slice to the frozen sucrose base in one single move.
         NOTE: It is not possible to move the slice once it is over the frozen sucrose base. Perform this transfer step carefully.
      8. Let the slice rest for 5-10 min for proper freezing.
      9. Cut the slice into 30 µm sections.
      10. Transfer the 30 µm sections to a Petri dish containing PBS.
      11. Proceed to the histology protocol more adequate to the experimental design.
          NOTE: The 30 µm sections can be readily used for free-floating immunohistochemistry, mounted onto microscopy slides for further histology or stored in antifreeze solution at -20 °C.
   2. Immunohistochemistry
      NOTE: Immunohistochemistry and immunofluorescence standard protocols vary among labs. For a detailed version of the protocol used here, refer to Horta et al.³⁸. Primary antibodies used for immunostainings presented in Figure 2 include neuronal nuclei (NeuN), healthy mature neuron marker, glial fibrillary acidic protein (GFAP), astorcytes marker, ionized calcium binding adapter (Iba-1), and microglia marker.
      1. Incubate the slices in blocking solution (e.g., 2% normal donkey serum in PBS) for 40 min.
      2. Incubate overnight with primary antibody under mild agitation at 4 °C.
      3. After washing with PBS, incubate for 120 min with biotinylated secondary antibody under mild agitation at RT.
      4. After washing with PBS, incubate at RT for 120 min with avidin-peroxidase conjugate (Table of Materials).
      5. Reveal with DAB + 0.04% nickel ammonium.
      6. Mount the stained sections on gelatin coated microscopy slides and let them air dry. Dehydrate in ethanol, diaphanize in xylene, finish with mounting medium (Table of Materials), cover with a coverslip, and image.