co-culture of primary neurons and microglia for studying microglia-neuron interactions

 Protocolo de cocultivo primario de microglía y neurona para estudios de interacción con el SNC

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)1,2,3. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development4,5,6&#160;and are maintained throughout the organism&#39;s life span through self-renewal, which involves proliferation and apoptosis7. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance8,9,10.
Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS11,12 and to promote inflammatory responses in the event of infections or tissue injury12,13,14, as well as during neurodegenerative diseases9,15, such as&#160;multiple sclerosis (MS)16,17. Microglia also express receptors to various neurotransmitters and neuropeptides18,19,20, which suggests they may also respond to and regulate neuronal activity21,22. Indeed, microglia and neurons interact in various forms of bidirectional communication8,23 such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells23,24.
For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia25,26,27. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state28. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry29 and influence neuronal signaling30,31,32. As disruptions of these interactions induce hyperexcitability of neurons30 and&#160;microglia reactivity33,34, dysregulated microglia-neuronal interactions&#160;are implicated&#160;as a contributing factor to&#160;neurological diseases33,35. Indeed, psychotic23,26 and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions33. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.
Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death37, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections38 or neuronal injury and repair39. Overall, in vitro microglia-neuronal co-culture systems&#160;enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

Autor destacado: Modelo de cocultivo in vitro para el estudio de las interacciones microglía-neuronas en enfermedades

Generación y cocultivo de microglía primaria murina y neuronas corticales

Our research aims to investigate the interactions between neurons and microglia during disease conditions such as multiple sclerosis. We aim to recapitulate microglia neuronal interactions in vitro to better understand their functional importance in the CNS. We previously demonstrated that microglia phagocytosis protects neurons from oxidized phosphatidylcholine-mediated cell death with this co-culture.While other co-culture systems are also employed, this mixed culture system enables direct and close contact between microglia and neurons. In addition, it can remain healthy from 12 days to potentially three weeks, allowing for long-term studies of neuronal microglia interactions. Our protocol offers a simple and accessible method to co-culture microglia neurons that are easily manipulatable and can be further used in downstream assays to determine cellular functional changes.

Cocultivo de neuronas primarias y microglía para el estudio de las interacciones microglía-neurona

co-culture-primary-neurons-microglia-for-studying-microglia-neuron

Research

JoVE Journal

Neuroscience

Co-Culture of Primary Neurons and Microglia for Studying Microglia-Neuron Interactions

47 Views. To begin, take neurons grown in T25 flasks for co-culture. Replace the media with five milliliters of versene solution containing trypsin and Dnase I.Add 0.5 milliliters of fetal bovine serum to neutralize the trypsin and Dnase I.Transfer cells to a 15 milliliter conical tube. Wash the flask with five milliliters of complete neurobasal media once to maximize the collected neurons.Then, centrifuge the cell suspension at 300 G for five minutes at four degrees celsius. After discarding th...