Name: using primary neurosphere cultures to study primary cilia
Uploaded: 2017-04-14T12:30:00
Description: Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Work in S.M.'s laboratory is funded by recruitment grants from CPRIT (R1220) and NIH (1R01GM113023-01).

1. Isolation of Neurospheres from the Adult Mouse Brain
<ol>
	<li>Euthanize an adult mouse (around 2 months old) by an overdose of isoflurane. Double-check that the mouse has stopped breathing and dissect immediately after death.</li>
	<li>Using scissors, make a midline incision to open the skull. Remove the brain.</li>
	<li>Place the brain in cold PBS in a 10 cm dish on ice. Follow the whole-mount dissection method to obtain the SVZ from the lateral ventricle48.</li>
	<li>Place the lateral vent...

Here, we describe a method to generate and maintain neurosphere cultures from adult mouse SVZ. A few pertinent points regarding the cultures are as follows. First, the sizes of the spheres are typically between 50 - 200 &#181;m. In our experience, when a neurosphere gets larger than 300 &#181;m in diameter, the optimal time for passaging has been missed. These larger spheres contain dead cells in the core. Second, as neurospheres are commonly used to study neural stem/progenitor cells, it is important to use EGF and FGF-...

The primary cilium is a microtubule-based dynamic subcellular compartment that functions as a sensory antenna in coordinating cellular signaling pathways, including the Sonic Hedgehog (Shh) pathway during embryonic neuronal development1,2, and compartmentalized subcellular signaling in adult neuronal function3,4. Signaling components of these pathways, such as the Shh receptor Patched5; the pathway activator Smoothened (Smo)6; and Gpr1617, an orphan G Protein-coupled Receptor (GPCR) that negatively regulates the Shh pathway, localize to cilia in a dynamic fashion. Multiple GPCRs have been reported to localize to the cilia in neurons in the brain7,8,9,10,11,12,13,14,15,16. Defects in cilia and cilia-generated signaling pathways affect multiple tissues and are collectively known as ciliopathies17,18,19. The ciliopathy disease spectrum frequently includes neurodevelopmental defects, such as craniofacial abnormalities20,21,22. In addition, primary cilia in hypothalamic neurons regulate central satiety pathways, and defects result in central obesity23, mirroring obesity in syndromic ciliopathies such as Bardet Biedel syndrome24. In addition, neuropeptide receptor signaling in cilia regulates central satiety pathways11,14. Ciliary localization of Adenylyl Cyclase III (ACIII) and GPCRs such as somatostatin receptor 3 in hippocampal neurons result in novel object recognition defects and memory deficits25,26 and parallels a lack of ciliary integrity27. The developmental aspects of cilia-generated signaling are closely tied to tissue homeostasis; in particular, cilia are important to the progression of Shh-subtype medulloblastomas arising from granule progenitors in the cerebellum28,29. Thus, primary cilia play important roles during embryonic, postnatal, and adult neuronal development and function.
Neural Stem Cells (NSCs) reside in the subventricular zone (SVZ) of the lateral ventricle, the subgranular zone of the dentate gyrus of the hippocampus, and the ventricular zone of the third ventricle in the hypothalamus in mammals30,31,32. NSCs are multipotent, possess the capacity for self-renewal, and are important for brain development and regenerative medicine30. Most NSCs in the SVZ are quiescent and possess a solitary primary cilium that, in many cases, extends out to the lateral ventricle33. The primary cilium signals via the localization of various receptors, inducing downstream cellular responses, particularly in relation to Shh, TGF&#946;, and receptor tyrosine kinase pathways2,34,35,36. Since primary cilia extend into the lateral ventricle, it is hypothesized that primary cilia detect cytokines in the cerebrospinal fluid (CSF) to activate NSCs37. Recent studies suggest that the Shh signaling pathway and primary cilia are critical for the activation of stem cells in the repair and regeneration of multiple tissues, including the olfactory epithelium, lung, and kidney38,39,40,41. However, the mechanisms by which CSF communicates with NSCs and whether primary cilia are involved are not known. Adherent NSCs in culture are ciliated; localize Shh pathway components, such as Smo and Gpr161 in cilia; and are Shh responsive42. Thus, NSCs can serve as an important model system to study the Shh pathway, ciliary trafficking, and neuronal differentiation pathways. In addition, neurons differentiated from NSCs can also be used for ciliary trafficking assays.
Neurospheres are constituted of clusters of free-floating cells arising from the proliferation of neural stem/progenitor cells that grow in the presence of specific growth factors and nonadhesive surfaces43,44. Neurospheres serve as important in vitro culture models to study neural stem/progenitor cells in normal development and disease31,45,46,47. Here, we describe a neurosphere-based assay for culturing neural stem/progenitor cells and for differentiation into neurons/glia. We particularly emphasize the trafficking of signaling components to cilia of neural stem/progenitor cells and differentiated neurons (Figure 1). As opposed to culturing primary neurons, primary neurospheres are relatively easy to culture, are amenable to multiple passages and freeze-thaw cycles, and can undergo differentiation into neurons/glia. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Neurosphere-based culturing methods can serve as an ideal model system for studying ciliogenesis and ciliary trafficking in NSCs and differentiated neurons.

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			<td>12 mm round cover glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80&#160;</td>
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			<td>24 well plate</td>
			<td>Falcon</td>
			<td>353047</td>
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			<td>4 % paraformaldehyde</td>
			<td>Affymetrix</td>
			<td>19943</td>
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			<td>50 ml tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
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		<tr>
			<td>95 mm X 15 mm petri dish, slippable lid</td>
			<td>Fisherbrand</td>
			<td>FB0875714G</td>
			<td>10 cm dish</td>
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			<td>70 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
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			<td>Alexa Fluor 488 Affinipure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>711-545-152</td>
			<td>Donkey anti Rabbit, Alexa 488 secondary antibody</td>
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			<td>Arl13B, Clone N295B/66</td>
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			<td>B-27 Supplement (50X), serum free</td>
			<td>ThermoFisher Scientific</td>
			<td>17504001</td>
			<td>B27</td>
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			<td>Centrifuge</td>
			<td>Thermo scientific</td>
			<td>ST 40R</td>
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			<td>Cryogenic vial</td>
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			<td>430488</td>
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			<td>DAPI</td>
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			<td>D9542-10MG</td>
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			<td>Deoxyribonuclease I from bovine pancrease</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td>Dnase I</td>
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			<td>Dimethyl sulfoxide</td>
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			<td>D8418-100ML</td>
			<td>DMSO</td>
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			<td>Disposable Vinyl Specimen Molds</td>
			<td>Sakura Tissue-Tek Cryomold</td>
			<td>4565</td>
			<td>10 mm X 10 mm X 5 mm</td>
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			<td>Dulbecco&#39;s Phosphate Buffered Saline 10&#215;, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td>PBS</td>
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			<td>Dumont #5 Forceps</td>
			<td>Fine science tools</td>
			<td>11254-20</td>
			<td></td>
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			<td>FBS&#160;</td>
			<td>Sigma</td>
			<td>F9026-500ML</td>
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			<td>Fluoromount-G solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>mounting solution</td>
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			<td>GFAP</td>
			<td>DAKO</td>
			<td>Z0334</td>
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			<td>Goat anti Mouse IgG1 Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21127</td>
			<td>Goat anti Mouse IgG1, Alexa 555 secondary antibody</td>
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		<tr>
			<td>Goat anti Mouse IgG2a Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21137</td>
			<td>Goat anti Mouse IgG2a, Alexa 555 secondary antibody</td>
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		<tr>
			<td>Gpr161</td>
			<td>home made</td>
			<td>N/A</td>
			<td></td>
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			<td>human bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td>FGF</td>
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			<td>hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>0.100 mm deep</td>
			<td>improved neubauer</td>
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			<td>Isothesia</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0500-2</td>
			<td>Isofluorane</td>
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			<td>Laminin from Engelbreth-Holm-Swarm Sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Laminin</td>
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			<td>L-Glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
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			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000</td>
			<td></td>
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		<tr>
			<td>Mr. Frosty</td>
			<td>Nalgene&#160;</td>
			<td>5100-0036</td>
			<td></td>
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			<td>N-2 supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502001</td>
			<td>N2</td>
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		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
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			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
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		<tr>
			<td>OCT compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td>OCT</td>
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			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td></td>
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			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
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			<td>Recombinant human EGF protein, CF</td>
			<td>R and D systems</td>
			<td>236-EG-200</td>
			<td>EGF</td>
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		<tr>
			<td>Scissor</td>
			<td>Fine science tools</td>
			<td>14060-10</td>
			<td></td>
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		<tr>
			<td>Superfrost plus microscope slide</td>
			<td>Fisher scientific</td>
			<td>12-550-15</td>
			<td>slides</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Bio-Rad</td>
			<td>161-0407</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution (10X)</td>
			<td>Sigma</td>
			<td>T4174-100</td>
			<td>Trypsin</td>
		</tr>
		<tr>
			<td>COSTAR 6 Well Plate, With Lid Flat Bottom Ultra-Low Attachment Surface Polystyrene, Sterile</td>
			<td>Corning</td>
			<td>3471</td>
			<td>ultra-low binding 6 well plate</td>
		</tr>
		<tr>
			<td>&#946;-tubulin III</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>TUJ1</td>
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using primary neurosphere cultures to study primary cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

After plating the cells from the SVZ in NSC medium for a week, floating neurospheres were observed (Figure 2A). The sizes of the spheres varied between 50 and 200 &#181;m. To examine if the spheres were derived from neural stem/progenitor cells, the neurospheres were plated onto PLL- and Laminin-coated coverslips in NSC medium for 2 days. They were then immunostained against the neural stem/progenitor cell marker, Nestin. Two days were needed to allow the spheres to attac...

Watch this Scientific Journal Video about Using Primary Neurosphere Cultures to Study Primary Cilia at JoVE.com

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

The overall goal of this procedure is to utilize neurosphere based culturing methods for primary neural stem and progenitor cells to study primary cilium biogenesis and trafficking. This method can help answer key questions in the primary cilia field such as cilia biogenesis and trafficking of cilia molecules to the primary cilia. In primary neural stem cells, neural progenitors and neurons.The main advantage of this technique is that we can use the primary cell criteria of the neural stem cells, neural progenitors and neurons to analyze biogenesis and ciliary pathways in the context of the primary cilium. Generally, individuals new to this method will struggle because culturing neural stem cells is technically challenging for beginners. We'll show a demonstration of this method is critical, as dissection of the lateral ventricle and steps such as culturing neurospheres and quantification of primary cilia are easily learned by observing the critical steps.Begin this procedure with a subventricular zone of the lateral ventrical dissected from an adult mouse using the whole mount dissection method. Place the tissue containing the SVZ into a 1.5 milliliter tube. Add 500 microliters of 0.05%Trypsin-EDTA in PBS and incubate 15 minutes at 37 degrees Celsius in a water bath.Following the incubation, add 500 microliters of stopping medium and gently pipette 20 to 30 times using a 1 milliliter tip. Avoid forming air bubbles during pipetting. After this, spin down the cells at 500 times G for eight minutes.Discard the supernatant then add 1 milliliter of PBS and resuspend the cells by gently pipetting five times using a 1 milliliter tip. Centrifuge at 500 times G for eight minutes. Then aspirate the medium using a 1 milliliter tip and add 1 milliliter of basal medium.After counting the cells, plate the cells from one SVZ into a 10 centimeter dish with 10 milliliters of NSC medium and culture at 37 degrees Celsius with 5%carbon dioxide. After five to seven days, neurospheres can be observed. To analyze cells in attached neurospheres by immunostaining, transfer 1 milliliter of medium containing multiple neurospheres to a 1.5 milliliter tube and spin down at 500 times G for eight minutes.After removing the medium, fix the spheres with 4%paraformaldehyde for 15 minutes. After removing paraformaldehyde incubate the spheres with 30%sucrose, overnight. And then centrifuge again.Wash with PBS. Then, after centrifuging as before, remove and discard the supernatant. Then, fit a pipette with a cut 1 milliliter tip and pipette 500 microliters of OCT solution onto the neurospheres.Transfer the OCT solution containing the neurospheres into a disposable plastic freezing mold. Then, freeze the mold on dry ice for at least 15 minutes. Cut sections with a cryostat.To visualize the stem cells in the neurosphere perform immunostaining against the stem cell marker Nestin. To visualize the primary cilia in a neurosphere, perform immunostaining against ARL13B. Begin this experiment one to two days after seeding adherent dissociated neural stem cells onto Poly-L-Lysine and Laminin coated cover slips.Change the medium in the experimental wells to starvation medium, and to fresh NSC medium in the control wells. Incubate for a further 24 hours. The next day, fix the cells with 4%paraformaldehyde in PBS for 15 minutes at room temperature.After fixation, wash with PBS twice for five minutes each, at room temperature. And then perform immunoflourescence using standard protocols. Mount the cover slips with mounting solution.Then, dry the slide glass overnight at room temperature in the dark. The next day, acquire images on a compound microscope at the necessary magnification. The microscope camera and objectives should be controlled using the accompanying software.Take sufficient z-sections at 0.5 to 0.8 micron intervals. For the quantitative analysis of ciliary localization in adherent cells, select three to eight consecutive fields with confluent cells by viewing the DAPI channel. Acquire stacks of images from each field.Quantify the number of primary cilia using ImageJ. Open the Plugins Analyze dialog box and select Cell Counter tool to count the cells with GPCR positive cilia. Use similar image intensity and contrast parameters for all images from the same experiment for counting and exporting purposes.24 hours after culturing dissociated cells in NSC medium or starvation medium cells were fixed and immunostained against GPR161 shown in green, ARL13B which appears red, and DNA shown as blue. GPR161 positive cilia are observed in control cells on the left, and starved cells on the right. As seen in these enlarged views.The white arrows and arrowheads indicate GPR161 positive and negative cilia, respectively. This image shows quantification of ARL13B positive ciliated cells. The quantification is performed from two fields of view each from two technical replicates of a single experiment.The percentage of ARL13B positive ciliated cells increases upon starvation. This image shows quantification of GPR161 localizing in ARL13B positive cilia in control and starved cells. The percentage of GPR161 localizing in ARL13B positive cilia, also increases upon starvation.Once mastered this technique can be done in less than two hours if it's performed properly. Following this procedure, another method of culturing primary tumor derived cells can be performed in order to answer additional questions like the role of cilia in tumor genesis. After its development, this technique paved the way for researchers in the field of primary cilia to explore ciliary biogenesis and trafficking in primary neurosphere based systems.After watching this video, you should have a good understanding of how to analyze primary cilia in neural stem cells, neural progenitors and neurons that are derived from neurosphere based cultures.

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Research

JoVE Journal

Developmental Biology

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...