Name: magnetic isolation of microglial cells from neonate mouse for primary cell cultures
Uploaded: 2022-07-25T12:30:00
Description: Watch this Scientific Journal Video about Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures at JoVE.com

Figures were created using BioRender. Research is funded by Inserm, Universit&#233; de Paris, Horizon 2020 (PREMSTEM-874721), Fondation de France, Fondation ARSEP, Fondation pour la Recherche sur le Cerveau, Fondation Grace de Monaco, and an additional grant from Investissement d&#39;Avenir -ANR-11-INBS-0011-NeurATRIS and Investissement d&#39;Avenir -ANR-17-EURE-001-EUR G.E.N.E.

The protocol was approved, and all the animals were handled according to the institutional guidelines of Institut National de la Sante&#769; et de la Recherche Scientifique (Inserm, France). Magnetic isolation of microglia from the brains of 24 OF1 mouse pups (both male and female) at P8, divided into 6-well, 12-well, or 96-well plates, are presented. The experimental work was performed under a hood to maintain sterile conditions.
1. Preparation of sterile solutions for isolation and cel...

The current work presents a primary microglial cell culture using magnetically sorted CD11b+ cells. In addition to the microglial functional evaluation (RT-qPCR and phagocytic assays), microglial culture purity was also determined.
Classical microglia cell cultures are commonly generated from P1 or P2 rodent neonate brain and co-culture with astrocytes for at least 10 days. Microglia are then separated mechanically using an orbital shaker. The method to isolate and culture microglia in vit...

Microglia are the central nervous system resident macrophage-like cells derived from erythropoietic precursors of the yolk sac that migrate to the neuroepithelium during early embryonic development1. Apart from their immunity functions, they also play a significant role during neurodevelopment, particularly for synaptogenesis, neuronal homeostasis, and myelination2. In adulthood, microglia develop long cellular processes to scan the environment continuously. In case of homeostasis ruptures such as brain injury or brain disease, microglia can change their morphological appearance to adopt an amoeboid shape, migrate to the injured area, increase and release many cytoprotective or cytotoxic factors. Microglia have heterogeneous activation states depending on their developmental stage and the type of injury sustained3,4,5. In this study, these activation states are broadly classified into three different phenotypes: pro-inflammatory/phagocytic, anti-inflammatory, and immuno-regulatory, keeping in mind that in reality, the situation is likely to be more complex6.
Studying in vivo microglial activation and screening for neuroprotective strategies at early stages of brain development can be challenging due to (1) the fragility of animals before weaning and (2) the low number of microglial cells. Therefore in vitro studies on microglia are widely used for toxicity7,8,9, neuroprotective strategies5,10,11,12,13,14, and co-cultures15,16,17,18,19,20,21. In vitro studies can use either microglial cell lines, stem-cell-derived microglia, or primary microglia culture. All these approaches have advantages and disadvantages, and the choice depends on the initial biological question. The benefits of using primary microglia cultures are the homogenous genetic background, pathogen-free history, and control of the time when the microglia are stimulated after animal death22.
Over the years, different methods (flow cytometry, shaking, or magnetic labeling) were developed for culturing primary microglia from rodents, both neonate and adult23,24,25,26,27,28,29. In the present work, microglia isolation from mouse neonate pups is performed using previously described magnetic-activated cell sorting technology using microbead-coated anti-mouse CD11b25,27,29. CD11b is an integrin-receptor expressed at the surface of myeloid cells, including microglia. When there is no inflammatory challenge within the brain, almost all CD11b+ cells are microglia30. Compared to other previously published methods23,24,25,26,27,28,29, the present protocol balances immediate ex vivo microglial activation analyses and common in vitro primary microglial culture. Thus, microglia are (1) isolated at postnatal day (P)8 without myelin removal, (2) cultured without serum, and (3) exposed either to siRNA, miRNA, pharmacological compound, and/or inflammatory stimuli only 48 h after brain isolation. Each of these three aspects makes the current protocol relevant and rapid. First of all, the use of pediatric microglia allows obtaining dynamic and reactive viable cells in culture without requiring an additional demyelination step that could potentially modify microglial reactivity in vitro. The present protocol aims to get as close as possible to the physiological environment of microglia. Indeed, microglia never encounter serum, and this protocol does not require the use of serum either. Moreover, exposing microglia as early as 48 h after culture prevents them from losing their physiological faculties.

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			<td>Latex beads, amine-modified polystyrene, fluorescent red</td>
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			<td>Macrophage-SFM serum-free medium</td>
			<td>Thermo Scientific</td>
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			<td>MACS BSA Stock Solution</td>
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			<td>130-091-376</td>
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			<td>MACS SmartStrainers (70 &#956;m), 4 x 25 pcs</td>
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			<td>Mouse IgG1 PE</td>
			<td>Millipore</td>
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			<td>Sony Biotechnology</td>
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			<td>Mouse IL1 beta</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-684</td>
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			<td>Miltenyi Biotec</td>
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			<td>REA control (S) Vio Bright B515</td>
			<td>Miltenyi Biotec</td>
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			<td>R&#38;D System</td>
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			<td>RIPA Buffer</td>
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magnetic isolation of microglial cells from neonate mouse for primary cell cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Microglia is the CNS resident macrophage that gets activated when exposed to environmental challenges (trauma, toxic molecules, inflammation )4,5,6,34&#160;(Figure 3A). In vitro studies on microglia are commonly used to evaluate cell-autonomous mechanisms related to those environmental challenges and characterize activation state after pharmacological or g...

Watch this Scientific Journal Video about Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures at JoVE.com

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

This protocol allow us to culture microglia in conditions that closely mimics the in vivo characteristics. Using magnetic cell sorting technology, our protocol allow us to stimulate microglia only two day in vitro, without using any serum in the culture medium. To begin, use small scissors to cut the skin from the neck to the nose, following the sagittal suture of 15 to 20 millimeters.Insert the tips with the foramen magnum parallel to the skull. Cut from each side to the eyes. Cut between the eyes with small scissors to detach the skull and brain from the head.With two forceps, grab the skull close to the olfactory bulbs, and carefully tear the skull. With a razor blade, remove the cerebellum and olfactive bulb, and cut the brain into two pieces. Place the brain pieces in a Petri dish containing 40 milliliters of HBSS without calcium and magnesium.Prepare dissociation mixture according to this table. Transfer 12 brain pieces into a C tube for a total weight of 1.2 grams per dissociation tube. Then place C tubes on the dissociater with heating.Start the optimized NTDK program in the dissociater. Centrifuge for 20 seconds. Complete the mechanical dissociation by pipetting three times.Transfer the cells to four 15-milliliter tubes with attached strainers. Rinse the strainers with 10 milliliters of HBSS with calcium and magnesium. Centrifuge for 10 minutes, and remove the supernatant with a 10-milliliter pipette.Carefully add 10 milliliters of HBSS with calcium and magnesium, and re-suspend the pellet. Again, centrifuge and remove the supernatant. Re-suspend the pellet with six milliliters of sorting buffer.Repeat the centrifugation and discard the supernatant. Then add 200 microliters of CD11b microbead solution and incubate the tubes for 15 to 20 minutes at four degrees Celsius. After incubation, re-suspend the pellet with six milliliters of sorting buffer.Repeat the centrifugation and re-suspend the pellet with eight milliliters of sorting buffer. Next, follow the Possel program on the separator to prepare eight columns. Pass the cells through the column by adding one milliliter of cell suspension at a time.With one milliliter of sorting buffer, elute CD11b-positive cells on a sterile elution plate. Pool the cells in a 50-milliliter tube. Centrifuge and re-suspend the pellet with 10 milliliters of cold microglia medium.Count the CD11b-positive cells. Re-suspend the cells in cold microglia medium to get a final concentration of 650, 000 to 700, 000 cells per milliliter. Dispense the suspension in cell culture plates.Incubate the plates overnight at 37 degrees Celsius with 5%carbon dioxide. On the next day, replace the medium with preheated microglia medium, and repeat the overnight incubation. Stimulate the cells before this step, and perform phagocytic assay during the last three hours of the stimulation.Calculate the number of beads according to this table at a ratio of 50 beads per cell. Prepare the beads mixture. Incubate the tube for one hour in a water bath at 37 degrees Celsius.Vortex every 10 minutes. To each well, add the calculated volume of the bead solution and incubate for three hours. Using this approach, the phagocytic activity of microglia was evaluated after stimulation by pro-inflammatory factors, such as interleukin-1 beta, plus interferon gamma, or lipopolysaccharides.After stimulation for six to 24 hours, fluorescent Cy3 microglia beads were analyzed by confocal microscope. After six hours, microglia start to phagocyte Cy3 beads only under interleukin-1 beta plus interferon gamma condition. After 24 hours, there was an increase of Cy3 fluorescence for both kinds of stimulation, highlighting increased phagocytic activity.The purity of microglial culture was elevated by flow cytometry, which showed an increase in cell viability after sorting. Using flow cytometry and RT-qPCR cell population markers were analyzed before and after sorting CD11b-positive cells from mice brains at postnatal day eight. These analyses distinguished various brain cell populations, such as CX3CR135 for microglia, O4, or OLIG2 for oligodendrocytes, NeuN, or synaptophysin, for neurons, and ACSA-2, or GFAP for astrocytes.After cell sorting using CD11b antibody, only microglia were obtained. It is important to pipette very gently while adding the suspension to the column to prevent clogging and avoid mechanical cell death. After this method, transcriptomic proteomics, such as western blood, or Luminex, and even immunochemistry can be performed in order to see the effect of microglial stimulation.

magnetic-isolation-microglial-cells-from-neonate-mouse-for-primary

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...