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

Artur Fernandes; Niele  Dias Mendes; Glaucia  Maria Almeida; Giovanna  Orlovski Nogueira; Carla de Moraes Machado; Jose de Anchieta de Castro Horta-Junior; João  Alberto Assirati Junior; Norberto Garcia-Cairasco; Luciano Neder; Adriano Sebollela

doi:10.3791/59845

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Method Article

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

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DOI:

10.3791/59845

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November 5th, 2019

Artur Fernandes¹^,² , Niele Dias Mendes¹^,³ , Glaucia Maria Almeida¹ , Giovanna Orlovski Nogueira¹ , Carla de Moraes Machado⁴ , Jose de Anchieta de Castro Horta-Junior⁴ , João Alberto Assirati Junior⁵ , Norberto Garcia-Cairasco² , Luciano Neder³ , Adriano Sebollela¹

¹Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, ²Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, ³Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School, University of São Paulo, ⁴Department of Anatomy, Institute of Biosciences, São Paulo State University, ⁵Clinical Hospital at the Ribeirão Preto Medical School, University of São Paulo

Summary

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Abstract

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.

Introduction

The use of human samples in research is unequivocally a great option to study human brain pathologies, and modern techniques have opened new ways for robust and ethical experimentation using patient-derived tissue. Methods like organotypic/slice cultures prepared from adult human brain have been increasingly used in paradigms such as optogenetics¹, electrophysiology²^,³^,⁴^,⁵, plasticity⁶^,⁷^,⁸^,⁹....