This study was funded in part by start-up funds from Texas A&amp;M University

Preparation before dissection
Surgical tools: sterilize all dissection scissors and forceps by autoclave
Coverslip cleaning:
<ol>
 <li>Soak all coverslips (15mm in diameter for 24-well plate) in 1L grass beaker in 33% HCl for at least 24 h</li>
 <li>Wash with running water for 10 min with occasional shake to remove all residual HCl</li>
 <li>Rinse with Millipore water</li>
 <li>Drain water off</li>
 <li>Soak coverslips in 95-98% ethanol</li>
 <li>Transfer coverslips to a clean tissue on a flat plate and air dry the coverslips</li>
 <li>Transfer coverslips to a glass beaker, cover with aluminum foil and autoclave 
 Note: Coverslips can be stored at this stage</li>
 <li>Place individual coverslip into culture plates. Ready for coating</li>
 </ol>
Coverslip coating:
 <ol>
 <li>Dilute 100 x stock of poly-D-lysine (PDL, 10 mg/ml in 0.5% bovine serum albumin in PBS, stored as aliquots at -20&deg;C) with PBS and filter-sterilize (0.22 &mu;m) </li>
 <li>Coat coverslips with 1 x PDL solution (~ 0.15 ml/cm2) for 2 h at 37&deg;C in an incubator </li>
 <li>Remove PDL solution and wash 3 times with sterile ddH2O and dry coverslips completely </li>
 </ol>
Note: Coat enough coverslips/plates for each dissection. PDL-coated coverslips can be stored for a few weeks at 4&deg;C.
Media and solutions
Dissection medium (DM): Use sterile ice cold Hank's Balanced Salt Solution (w/o Ca2+, Mg2+) (HBSS, Invitrogen 14175) supplemented with 10 mM HEPES (Invitrogen 15630).
Aggregate culture media: The DMEM/NBB27 medium contains DMEM/Neurobasal (1:1 vol:vol), 2% B27, 1 x Sato, 0.5 mM sodium pyruvate, 0.75mM GlutaMAX, 60&mu;g/ml N-acetylcysteine, 5 &mu;g/ml insulin, 10nM d-Biotin and 1% penicillin/streptomycin. To make 100 ml of the aggregate culture medium, mix 50 ml DMEM (w/o pyruvate/glutamine, Invitrogen 11960), 50 ml Neurobasal medium (Invitrogen 211034), 375 &mu;l GlutaMax (100 x, Invitrogen 35050), 5.5 mg sodium pyruvate (Sigma P2256), 2 ml B27 (Invitrogen 17504), 6.3 mg N-acetylcysteine (Sigma A8199), 1 ml of Sato stock (100 x stock, see below), 25 &mu;l of d-Biotin stock (4000 x stock, 40 &mu;M in PBS stored as aliquots at -20&deg;C. Sigma B4639), 100 &mu;l of insulin (1000 x stock, 5 mg/ml in 0.01N HCl stored as aliquots at -20&deg;C, Sigma 16634), and 1 ml penicillin/streptomycin (100 x stock, Invitrogen 15140), filter-sterilize and store at 4&deg;C.
Sato 100 x stock solution: To make 40 ml of the stock: mix 40 ml Neurobasal with 400 mg apo-transferrin (Sigma T2252), 400 mg BSA (Sigma A9647), 10 &mu;l of progesterone (25 mg/ml ethanol, stored as aliquots at -20&deg;C. Sigma P8783), 64 mg putrescine (Sigma P7505), and 40 &mu;l of sodium selenite (30 &mu;M in PBS, stored as aliquots at -20&deg;C, Sigma S5261). Filter sterilize the Sato stock solution, and store as aliquots at -20&deg;C. 
Neuron plating medium (PM): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20&deg;C, Invitrogen 25030), 25&mu;M glutamic acid (100 stock, aliquots stored at -20&deg;C) and 1% penicillin/streptomycin. 
Neuron culture medium (CM): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20&deg;C) and 1% penicillin/streptomycin. 
5-FdU stock: 100x stock, 1mM 5-fluoro-2'-deoxyuridine (Sigma F0503) and1mM uridine (Sigma U3003) in Neurobasal medium. Filter sterilized and stored as aliquots at -20&deg;C.
Papain digestion solution (make fresh prior to dissection): 
<ol>
 <li>Dissolve 3.2 mg L-Cysteine (Sigma C-7352) in 4ml DM</li>
 <li>Adjust pH to around 7.4 with 1N NaOH (test on pH test strips) and place in water bath at 37&deg;C 
Note: Perform the next step right before tissue digestion (see below).</li>
 <li>Add papain to a final concentration of 20 unit/ml</li>
 <li>Filter sterilize (0.22mm), and place in water bath at 37&deg;C </li>
</ol>
Trypsin inhibitor solution (make fresh prior to dissection):
<ol>
 <li>Dissolve 0.2 g trypsin inhibitor (Sigma T7295) in 20ml DM </li>
 <li>Check pH and adjust pH to ~7.4 with 1 N NaOH</li>
 <li>Filter sterilize, and place in water bath at 37&deg;C </li>
</ol>
Brain Dissection
Setup:
<ul>
 <li>Sterilized forceps and scissors</li>
 <li>70% ethanol</li>
 <li>Clean tissue paper and diaper pad on bench</li>
 <li>10cm Petri dishes with ice-cold DM for uterus and fetus on bench</li>
 <li>Ice platform at dissection microscope</li>
 <li>2-3 6cm dishes with DM on ice</li>
 <li>Warm PM in a 37&deg;C water bath</li>
</ul>
<ol>
 <li>Euthanize a pregnant Sprague-Dawley rat (E16) according to a procedure approved by your Institutional Animal Care and Us Committee (IACUC) </li>
 <li>Lay the rat on the diaper pad and spay alcohol on the abdominal region</li>
 <li>Use tweezers to hold the abdominal skin and a pair of scissors to make a V shape incision cutting only the skin</li>
 <li>Spread apart the skin and use another sterilized tweezers and scissors to cut through the muscle layer </li>
 <li>Extract the chain of the embryo sacs and transfer them into a 10cm dish containing ice-cold DM </li>
 <li>Using a microdissection scissors, carefully remove each embryo from the sac and the brain from each embryo, and place freed brains into a clean 10cm dish with DM on an ice platform</li>
 <li>In a Laminar hood and under a dissection microscope, remove midbrain/hindbrain sections, and use fine forceps to remove meninges, and transfer the cleaned cortices/hippocampus to a new 6cm dish with DM on an ice pack 
 Note: at this stage, the tissue is ready for enzyme digestion.</li>
 <li>Make papain and trypsin inhibitor solution and filter sterilize</li>
 <li>Remove excess DM from cortices/hippocampi </li>
 <li>Add 4ml of the prepared papain digestion solution</li>
 <li>Transfer all content to a 50ml tube and place the Falcon tube in a water bath at 37&deg;C for exactly 5 min</li>
 <li>Remove papain solution with pipette</li>
 <li>Add 5ml trypsin inhibitor solution, swirl the tube, and place in a water bath at 37&deg;C for 2-3 min</li>
 <li>Remove trypsin inhibitor solution </li>
 <li>Repeat step 13 and 14 three times </li>
 <li>Add 20ml warm PM </li>
 <li>Triturate until all clumps have disappeared (~20-30 times)</li>
 <li>Centrifuge at 100xg for 7min</li>
 <li>Resuspend the pellet in 10ml PM and wash twice by centrifugation</li>
 <li>Resuspend the pellet in 10ml PM for regular neuron cultures or in NBB27/DMEM for aggregate cultures</li>
 <li>Pass the cells through a 70&mu;m cell sieves </li>
 <li>Count live cells</li>
 <li>Proceed to aggregate cultures as described below or plate the cells for regular neuron cultures 
Note: At this step, highly enriched neuron cultures can be achieved by plating the dissociated cells onto PDL-coating plates at density of 200-640 cell/mm2 depending on the purpose of experiments. After cells have attached (~ 2-4h), replace medium with warm PM. If culturing for extended periods of time, on day 4, replace half of the medium with warm CM. For highly purified neuronal cultures, mitotic inhibitor 5-FdU (final concentration 10 &mu;M) can be added at DIV2 to inhibit non-neuronal cell proliferation (i.e., during DIV2-3). After recovery in CM for 2 days, cells are pulse treated again with 5-FdU for another 2 days (DIV 6-7) followed by a complete medium change. Thereafter, replace half of the medium with fresh, warm CM every 3-4 days. Neurons are viable for up to 4 weeks.</li></ol>
Aggregates preparation and plating
<ol>
 <li>Suspend dissociated cells in NBB27/DMEM medium containing 1x Sato, 10ng/ml CNTF and 10&mu;M forskolin at a density of 2x106 cells/ml</li>
 <li>Transfer 2ml of cell suspension into each well of a uncoated 6-well plate </li>
 <li>Culture for 3 nights. Each day, gently resuspend the cells once using a P1000 Pipetman Note: Cells start to form aggregates(Figure 1).</li>
 <li>On the day before aggregate plating, coat plate/coverslips with Matrigel:
 <ul>
 <li>Dilute stock Matrigel (growth factor reduced, BD Biosciences 354230) 1:20 with cold DMEM on ice 
 Note: Matrigel aliquots should be prepared per manufacturer protocol. All tips, Eppendorf tubes and solutions for making aliquots of the stock should be cold to avoid Matrigel polymerization. Aliquots of Matrigel are kept at -80&deg;C and thawed at 4&deg;C.</li>
 <li>Coat previously PDL-coated coverslips with 300&mu;l/well of the diluted Matrigel solution overnight in a 37&deg;C incubator</li>
 <li>Wash with warm PBS, then wash plate with warm sterile ddH2O and leave the plates in the incubator. </li>
 </ul></li>
 <li>3 days after aggregate formation, gently resuspend cells again and sieve the suspension through a 200&mu;m mesh into a 50ml Falcon tube. Allow aggregates to settle to the bottom of the tube by gravity (~3-5min)</li>
 <li>Carefully remove supernatant</li>
 <li>Add medium, gently resuspend the aggregates and let them settle again. Repeat this procedure several times to remove dead cells, individual cells and debris. Use microscope to check whether the supernatant still contain non-aggregated cells and debris</li>
 <li>Gently resuspend the aggregates in the same NBB27/DMEM medium (Figure 1) and count aggregates </li>
 <li>Adjust the density of aggregates to around 25-30 aggreggats/50ul </li>
 <li>Transfer aliquots (500-1000&mu;l) of the aggregate suspension to 2ml Eppendorf tubes for plating 
Note: Since aggregates tend to settle down to the bottom of tubes, it is crucial to make multiple tubes of the aggregate suspension for plating. Gently resuspend the aggregates often before seeding them onto coated coverslips or culture plates to ensure similar number of aggregates plated on each coverslip.</li>
 <li>Take out the culture plate containing Matrigel-coated coverslips from incubator. Remove solution from each well, wash the coverslips with warm PBS and ddH2O, and then dry briefly. They are now ready for aggregate seeding</li>
 <li>Invert a tube containing aggregate suspension to allow them to be distributed evenly before seeding. Load 50&mu;l of aggregate suspension into the center of each coverslip, and gently put the plate back to incubator without any further disturbance to allow aggregates to attach evenly to the coverslip 
Note: The density of aggregates is crucial. If aggregates are seeded too close to each other or if the seeding density is high, they tend to merge together as they grow.</li>
 <li>Add additional DMEM/NBB27medium (500 &mu;l) to each well 4-6 h later or early the next morning</li>
 <li>To maintain the aggregate culture, half change the medium every 3-4 days. When change medium, be careful not to disrupt the aggregates and this is done by gently adding medium along the side wall of the well. 
Note: Few hours after aggregates attached to the plate, neurite outgrowth from the aggregates can be observed. Axons grow rapidly in the first two weeks and form abundant connections between aggregates (Figure 2). Glial progenitors migrate radially out of the aggregates and differentiate over time into astrocytes and mature oligodendrocytes.</li>
</ol>
<img src="/files/ftp_upload/1304/1304fig1.jpg" alt="figure 1" /> Figure 1. Phase contrast images of cells forming aggregates at different days and aggregates after washing.
<img src="/files/ftp_upload/1304/1304fig2.jpg" alt="figure 2" /> Figure 2. Phase contrast images of an aggregate culture 2 and 12 days after seeded on Matrigel-coated coverslips.

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

Early studies reported formation of synapses and mature myelin in rotation-mediated free floating aggregate cultures1. The CNS aggregate culture system described here combines serum-free growth of multipotent progenitor cells in three dimensional aggregates with the convenience of traditional 2D cultures to facilitate analyses of cell development, migration and differentiation in vitro. The system can be modified and used for neural precursor cell research and for investigation into neuron-glia intera...

preparation of rat brain aggregate cultures for neuron and glia development studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

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JoVE Journal

Biology

12.4K ビュー.  Texas A&M University (TAMU). 胎児ラット脳の集合体の培養系のためのプロトコルが記述されています。凝集体の多能性前駆細胞は、開発し、神経細胞、アストロサイト、オリゴデンドロサイトに分化することができる。

ビデオ: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...