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.
2. Preparation of solutions
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.
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.
5. Slicing
6. Culture
NOTE: Perform this step in a laminar flow cabinet under sterile environment.
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.
A critical aspect to evaluate the quality and health of cultured slices is the presence and typical morphology of the expected neural cell types, neurons, and glial cells. The typical architecture of the human cortical lamination was observed in a slice at DIV4, revealed by neuronal immunolabeling (Figure 2D). In addition, the expected presence of microglia and astroglia (Figure 2B,C) was also observed. These results demonstrate that tissue architecture is not significantly affected either by the surgical procedure/sample processing or by the short-term period in vitro. In accordance with previous findings, it was shown that NeuN immunoreactivity was not altered between DIV0 and DIV432. Based on these results, the free-floating culture format, associated to the reduced thickness of the slice to 200 µm (compared to the widely used 300-400 µm when membrane inserts are used), contributed to better diffusion of oxygen and nutrients to inner cell layers in slices, which has been previously demonstrated to be critical to the health of cultured slices39,40.
Quantification of cell death in slices is also a valuable approach in ex vivo models of neuropathologies, such as slice cultures (reviewed by Lossi et al.41). In a previous study, we used the MTT assay to determine cell death levels along the period in culture32. In addition to viability, that same study also showed that cultured slices (up to DIV4) preserved the capacity to release neurotransmitters upon KCl-induced depolarization32. Here, those findings were expanded by investigating the neuronal response to KCl-induced depolarization on ERK phosphorylation, a central kinase involved in processes such as synaptic plasticity and memory42,43. Interestingly, a clear increase in ERK phosphorylation was seen in KCl-treated slices at DIV4 (Figure 3A,B).
Finally, the response of slices at DIV4 to a toxic challenge was evaluated with a known oxidative stress inducer, H2O2. The rationale was that the extent of cell death should be proportional to the level of cell viability in the cultured slices. As shown in Figure 3C, exposing the slices to 300 mM H2O2 for 24 h led to a robust decrease in MTT reduction. Taken together, the massive cell death observed in DIV5 after the H2O2 challenge and the KCl-induced depolarization results indicate that the preserved general health of slices at DIV4 responds adequately to a toxic stimulus such as oxidative stress.
Figure 1: Sample collection, transport, slicing, and culturing of cortical tissue from adult humans. The procedure starts at the surgical room with collection of cortical tissue from temporal lobectomy for the treatment of pharmacoresistant epilepsy (A,B). (C) Tissue fragment (n = 1) is immediately transferred to a tube containing ice-cold oxygenated transport medium (see below). (D) In the lab, meninges are removed using fine ophthalmic tweezers. Excess liquid is dried using filter paper, and the fragment is superglued (E) to the vibratome specimen disk with the white matter facing down and pial surface facing up. (F) Using a commercial shaving razor, the specimen is cut into 200 µm slices that are collected with a delicate paintbrush and transferred back to the Petri dish for further trimming of excess white matter and loose ends (not shown) prior to (G) plating and culturing in a free-floating format. (H) Slices cultures are kept viable for several days and can be used in a variety of experimental protocols. Please click here to view a larger version of this figure.
Figure 2: Morphological analysis of neural cells in slice cultures from adult human brain. Slices were fixed at the fourth day in vitro. (A,B,C) Representative steps of the sectioning procedure prior to immunohistochemistry. Tissue was digitally colored to improve visualization. (D) Normal distribution of neurons in cortical layers (Roman numerals). (E) Microglia and (F) astrocytes were also clearly observed (n = 1 slice per cell type labelling). All slices were obtained from tissue from a single donor. Scale bars = 100 μm. Please click here to view a larger version of this figure.
Figure 3: Functional and cell viability assays in adult human brain slice cultures. Neuronal activity was evaluated in slices at day in vitro 5 (DIV5) by KCl-induced depolarization and consequent increase in ERK phosphorylation. (A) A representative immunoblot result obtained with homogenates from one slice. Bands corresponding to phospho ERK (Perk) and total ERK (tERK) are indicated. (B) Quantification of pERK/tERK ratio in three independent slices from a single human donor. (C) Hydrogen peroxide (H2O2) toxicity was evaluated by the MTT assay in slices at DIV 5. Optical density values obtained were normalized by the mass of each slice. Slices were challenged with H2O2 at the indicated concentrations (No H2O2; 30 mM H2O2; 300 mM H2O2) for 24 h. Images of representative slices after incubation with MTT are shown above the bars. Results are presented as average ± standard error from data obtained from three independent slices from a single donor. Please click here to view a larger version of this figure.
2-Propanol | Merck | 1096341000 | |
Acrylamide/Bis-Acrylamide, 30% solution | Sigma Aldrich | A3449 | |
Agarose | Sigma Aldrich | A9539 | |
Ammonium persulfate | Sigma | A3678-25G | |
Amphotericin B | Gibco | 15290-018 | |
Antibody anti-ERK 2 (rabbit) | Santa Cruz Biotecnology | sc-154 | Dilution 1:1,000 in BSA 2.5% |
Antibody anti-pERK (mouse) | Santa Cruz Biotecnology | sc-7383 | Dilution 1:1,000 in BSA 2.5% |
B27 | Gibco | 17504-044 | |
BDNF | Sigma Aldrich | SRP3014 | |
Bovine Serum Albumin | Sigma Aldrich | A7906 | |
Bradford 1x Dye Reagent | BioRad | 500-0205 | |
EDTA | Sigma | T3924 | Used in RIPA buffer |
Glucose | Merck | 108337 | |
Glutamax | Gibco | 35050-061 | |
Hank's Balanced Salts | Sigma Aldrich | H1387-10X1L | |
Hepes | Sigma Aldrich | H4034 | |
Hydrochloric acid | Merck | 1003171000 | |
Hydrogen Peroxide (H2O2) | Vetec | 194 | |
Mouse IgG, HRP-linked whole Ab (anti-mouse) | GE | NA931-1ML | |
NaCl | Merck | 1064041000 | Used in RIPA buffer |
Neurobasal A | Gibco | 10888-022 | |
Non-fat dry milk (Molico) | Nestlé | Used for membrane blocking | |
PBS Buffer pH 7,2 | Laborclin | 590338 | |
Penicilin/Streptomicin | Sigma Aldrich | P4333 | |
Potassium Chloride | Merck | 1049361000 | |
Prime Western Blotting Detection Reagent | GE | RPN2232 | |
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) | GE | NA934-1ML | |
SDS | Sigma | L5750 | Used in RIPA buffer |
TEMED | GE | 17-1312-01 | |
Thiazolyl Blue Tetrazolium Bromide (MTT) | Sigma Aldrich | M5655 | |
Tris | Sigma | T-1378 | Used in RIPA buffer |
Triton x-100 | Sigma | X100 | Used in RIPA buffer |
Ultrapure Water | Millipore | Sterile water, derived from MiliQ water purification system | |
Equipment and Material | |||
24-well plates | Corning | CL S3526 | Flat Bottom with Lid |
Amersham Potran Premium (nitrocellulose membrane) | GE | 29047575 | |
Carbogen Mixture | White Martins | 95% O2, 5% CO2 | |
CO2 incubator | New Brunswick Scientific | CO-24 | Incubation of slices 5% CO2, 36ºC |
Microplate Reader | Molecular Devices | ||
Microtubes | Greiner | 001608 | 1,5mL microtube |
Motorized pestle | Kimble Chase | ||
Plastic spoon | Size of a dessert spoon | ||
Razor Blade | Bic | Chrome Platinum, used in slicing with vibratome | |
Scalpel Blade | Becton Dickinson (BD) | Number 24 | Used for slicing of tissue; recommended same size or smaller |
Superglue (Loctite Super Bonder) | Henkel | Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6'-di-terc-butil-2,2'-metilenodi-p-cresol; homopolymer | |
Vibratome | Leica | 14047235612 – VT1000S | |
Name of Material/ Equipment for Immunohistochemistry | |||
Antibody anti-NeuN (mouse) | Millipore | MAB377 | Dilution 1:1,000 in Phosphate Buffer |
Antibody anti-GFAP (mouse) | Merck | MAB360 | Dilution 1:1,000 in Phosphate Buffer |
Antibody anti-Iba1 (rabbit) | Abcam | EPR16588 – ab178846 | Dilution 1:2,000 in Phosphate Buffer |
Biotinylated anti-mouse IgG Antibody (H+L) | Vector | BA-9200 | |
DAB | Sigma Aldrich | D-9015 | |
Entellan | Merck | 107960 | |
Ethanol | Merck | 1.00983.1000 | |
Gelatin | Synth | 00G1002.02.AE | Used for coating slides |
Microtome | Leica | SM2010R | Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40ºC |
Normal Donkey Serum | Jackson Immuno Research | 017-000-121 | |
Paraformaldehyde | Sigma Aldrich | 158127 | |
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) | GE | NA934-1ML | |
Slides (Star Frost) | Knittel Glaser | Gelatin coated slides | |
Sucrose | Vetec | 60REAVET017050 | |
Vectastain ABC HRP Kit (Peroxidase, Standard) | Vector | PK-4000, Kit Standard | |
Xylene | Synth | 01X1001.01.BJ |
Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.
Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.
Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.