FluidFM® for Neuroscience
Pattern, stimulate, inject into, and analyze single neurons

The nervous system is a highly complex system built-up from diverse, highly specialized cell types that carry out various functions in the brain and throughout the entire body. The fundamental building blocks of the brain are the 80-100 billion neurons that are organized in complex neuronal structures. 

Neurodegenerative diseases are a group of diseases that is characterized by deterioration of cells in the nervous system and brain. On a cellular level, diseases of the central nervous system (CNS) are often associated with mechanisms of biological ageing (senescence) of the different brain cell types such as astrocytes, microglia, oligodendrocytes and neurons. While cellular senescence has a pivotal role in normal biological development of cells and tissues, when it becomes chronic it causes alterations in cellular functioning. As such, different mechanisms of cellular senescence have been implicated to play a role in the development of various neurodegenerative diseases such as Alzheimer’s disease, Ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and more. [1]

These disorders cause progressive loss of cognitive and/or motor functions and can affect every aspect of a person’s life, from sleep to mobility and balance, breathing, memory and cognitive abilities. Current estimates suggest that 1 in 6 of the world’s population suffers from a neurological disease. [2] With estimated societal costs of dementia alone to amount up to $2 trillion by 2030 [3], these disorders pose a major challenge for societies with rapidly aging populations.

In a healthy brain, extensive cell-cell interactions of neurons with neighboring cells like other neurons as well as with glial cells are essential. Therefore, to fully comprehend the complexity of the brain’s functioning in health and disease, it has always been the aim in neuroscience to look at the single cell level and to understand the processes involved in single neuron interactions. 

In neuronal networks, the neuronal cell body or soma forms connections with other neurons through outgrowth of dendrites and axons. These neuronal outgrowths interact with other nerve cells through connections called synapses. To communicate, neurotransmitters and ions travel through these connections with high speed to transmit physiological and electrical signals. In the development of the brain, these types of neural circuits are highly dynamic and continuously change and refine to support learning and memory in a process called neuronal plasticity. 

To successfully develop new therapeutic strategies to halt or reverse neurodegeneration, it is paramount to first better understand molecular and signaling mechanism in the brain that lead to decreased neuron connectivity and neuronal deterioration.

Due to the high complexity of the brain, research still does not fully comprehend the underlying pathophysiology of many neurological diseases. One of the hallmarks of neurodegenerative diseases is the accumulation and aggregation of misfolded proteins that lead to cellular dysfunction, loss of synaptic connections, and ultimately brain damage [1]. 

In Alzheimer’s disease, the leading hypotheses are on accumulation and misfolding of tau-protein and beta-amyloid. Accumulation of beta-amyloid supposedly forms plaques between neurons and interferes with neuron-neuron interactions. The tau-protein hypothesis proposes that it forms neurofibrillary tangles inside neurons and blocks their transport system. 

In the recent years, the application of cryo-EM techniques helped to understand the role of such prion-like mechanisms in disease progression. Unfortunately, a clear understanding on how protein aggregates spread within the brain remain poorly understood. To date, the relation between tau and beta amyloid aggregation and what other factors drive the development and progression of Alzheimer’s disease, are still unclear. 

The lack of translational understanding of how cellular and molecular drivers relate to causative mechanisms of neurodegeneration poses a major challenge in drug development and is believed to be one of the main reasons for the failure of many therapeutics in the clinic. 

Also because of this, the scientific community has started to investigate other factors that are observed during neurodegeneration. As such, the fields of neuro-immunology and neuroinflammation have gained increasing interest. Especially because of the growing understanding of the importance of these topics in the autoimmune disease multiple sclerosis. 

In neuroimmunology, another type of cells called microglia, a type of glial cells, plays an important role as they are responsible for the homeostasis in the brain. Microglia account for 10-15% of cells found in the brain and act as the active immune defense in the central nervous system (CNS). 

In all these research topics, research techniques such as FluidFM that allow observation and manipulation of single neurons in a controlled neuronal network can help to answer important questions. 

For example, FluidFM enables injection of CRISPR reagents, plasmids, or any type of cargo into both primary and iPSC-derived neurons maintaining high viability. As such, specific neurons in a network can be stimulated by directly injecting a drug or neurotransmitter into a single neuron, which can provide highly valuable information on neuronal signaling and functioning in health and disease states.  

In addition, our FluidFM OMNIUM system can also very accurately select and place neurons or any type of relevant cell type on custom surfaces. Individual cells of different types can be brought in proximity to generate highly defined mixed-cell CNS models. Inclusion of neurons, glial- or neuromuscular cells in one defined model opens-up a wide array of possibilities to study important cellular mechanisms involved in neuroimmunology or neuromuscular diseases.  

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In neuronal cell biology, transfection of reagents, experimental drugs, nucleic acids for knock-down of target proteins or plasmids are essential to answer fundamental research questions. Neurons are highly sensitive cells, for which it is challenging to get any type of cargo into the cytosol or nucleus with the usually harsh traditional transfection methods. In addition, genetic engineering of neurons is particularly difficult, as neurons are typically non-dividing cells. Due to this, the nuclear envelope never opens, making it cumbersome to get any type of gene editing reagents into the nucleus where it needs to be. Secondly, in non-dividing cells the genomic DNA is less accessible, making it harder to access for gene editing reagents such as CRISPR. 

Commonly used transfection methods with neurons are: 

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• Electroporation: a method that exposes cells to a high voltage pulse. The electric pulse alters the properties of the neural membrane and allows charged materials like plasmids to flow into the cytosol. The downside of this is that only low transfection efficiencies can be achieved (<20%), and it only works effectively with undifferentiated, post-mitotic cells. 
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• Nucleofection is a variation of electroporation that uses a series of electrical pulses to enable entry into the nucleus. Due to this, transfection efficiency can be significantly higher than for regular electroporation. 
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• Chemical transfection, a method that relies on the formation of calcium-phosphate/DNA complexes followed by precipitation onto the neuronal membrane, which then presumably are taken-up through endocytosis. The effectiveness of this method is generally low, and it is mostly used in applications that should resemble physiologically normal behavior of cells (e.g. live imaging).  
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• Lipid-mediated transfection or lipofection are technically simple transfection methods that show low toxicity to cells. Cargo is encapsulated in unilamellar liposomes and deliver the cargo into the cell in an endocytosis-type of mechanism. Efficiencies of lipofection can vary greatly among cell types, but generally works very poorly for neurons (1-5%). 
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• Virus-based transfection with AAV-, lentiviral-, and herpes simplex derived viral vectors show very high transduction efficiency in different types of neurons. The downside of these viral vector-based transfection is that it requires a lot of preparative work and a lab that is suited to work with viruses. Also, there is always the chance of integration of the viral genome. 
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• Micro-injection enables injection directly into the cytosol or nucleus. However, traditional approaches of micro-injection utilizing glass capillaries have a major disadvantage by causing stress to the cell membrane, yielding low cell survival rates. Next to that, most micro-injection setups need to be operated manually, which makes it more challenging to inject in the desires subcellular compartment and highly time intensive.

FluidFM is a new type of injection technology that can specifically inject cargo into desired subcellular compartments such as the nucleus or the cytosol of a specific neuron. In contrary to traditional micro-injection with glass pipettes, FluidFM utilizes force sensitive probes that minimize the pressure needed to penetrate the cytosolic or nuclear membrane. As such, significant less stress is exerted on the cells compared to traditional use of glass capillaries, resulting in higher cell survival rates. Another advantage of our FluidFM OMNIUM system is that injection is done semi-automatic making it highly reproducible, straightforward and less user-dependent.