Name: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies (Video) | JoVE Journal
Uploaded: 2009-09-30T23:59:00.000+00:00
Duration: 12 min 3 s
Description: Learn to prepare rat brain aggregate cultures for studying neuron and glia development. This protocol isolates E16 fetal brain tissue, generates single-cell suspensions, and cultures multipotent progenitors that differentiate into neurons, astrocytes, and oligodendrocytes.

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5 minutes

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°C) with PBS and filter-sterilize (0.22 μm) </li>
 <li>Coat coverslips with 1 x PDL solution (~ 0.15 ml/cm2) for 2 h at 37°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°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, 60μg/ml N-acetylcysteine, 5 μg/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 μl 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 μl of d-Biotin stock (4000 x stock, 40 μM in PBS stored as aliquots at -20°C. Sigma B4639), 100 μl of insulin (1000 x stock, 5 mg/ml in 0.01N HCl stored as aliquots at -20°C, Sigma 16634), and 1 ml penicillin/streptomycin (100 x stock, Invitrogen 15140), filter-sterilize and store at 4°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 μl of progesterone (25 mg/ml ethanol, stored as aliquots at -20°C. Sigma P8783), 64 mg putrescine (Sigma P7505), and 40 μl of sodium selenite (30 μM in PBS, stored as aliquots at -20°C, Sigma S5261). Filter sterilize the Sato stock solution, and store as aliquots at -20°C. 
Neuron plating medium (PM): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20°C, Invitrogen 25030), 25μM glutamic acid (100 stock, aliquots stored at -20°C) and 1% penicillin/streptomycin. 
Neuron culture medium (CM): Neurobasal Medium, 2% B27, 2mM Glutamine (100x stock, aliquots stored at -20°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°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°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°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°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°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°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°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 70μm 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 μM) 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 10μM 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°C and thawed at 4°C.</li>
 <li>Coat previously PDL-coated coverslips with 300μl/well of the diluted Matrigel solution overnight in a 37°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 200μm 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 (500-1000μl) 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 50μl 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 μl) 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="Cell culture growth over three days; microscopy images showing aggregation and post-washing results." data-altupdatedat="1787630408000" data-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="Neuron growth comparison, phase contrast microscopy, DIV 2 vs DIV 12, cellular network analysis." data-altupdatedat="1787630408000" data-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/pubmed?term=((Myelination+in+rat+brain+aggregating+cell+cultures%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li>
<li>Chen, Y. ing <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">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/pubmed?term=((Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">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. ei-S. ong, Colman, D. avidR., Pedraza, L. iliana <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Research%22%5BJournal%5D)">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

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. 

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) ...

We introduce a system for culturing CNS neuro progenitors as they develop and differentiate into mature neurons and glia. The procedure begins with isolating four brains from E 16 rat fetuses and digesting them with propane. The tissues are ated into a single cell suspension, filtered, counted, and dispersed into a six.Well plate cells are gently resuspended once per day for two to three days to allow aggregate formation. Aggregates are then collected and sied through a 200 micrometer mesh and small clumps and cell debris are removed by washing. Finally, aggregates are counted and plated onto matrigel coated cover slips.Hi, I'm Coto from the laboratory of Dr.John L in the Department of Veterinary Integrated Biosciences at the Texas a and m University. Today we will show you a procedure for brain aggregate cultures. We use this procedure in our level three to study endocyte lineage, cell development and accelerate interactions.So let's get started. Before you start, have ready on the bench sterilize tools, 70%ethanol a diaper pad to work on and 10 centimeter petri dishes of dissection. Medium on ice.In the hood, prepare a dissecting microscope with an ice platform and several 10 centimeter dishes of dissecting medium also on ice. Lastly, make up the solutions you will use and warm the plating medium to 37 degrees in a water bath. You begin this protocol by harvesting embryonic day 16 E 16 embryos from a Sprague dolly rat.First euthanize a pregnant female according to a procedure approved by your institutional animal care and use committee. Lay the euthanized rat on a diaper pad and spray its abdomen with alcohol to clean it. Next, use tweezers to pinch the abdominal skin and with the other hand, use scissors to cut only the skin, spread apart the skin, and use another set of sterilized tweezers and scissors to cut through the muscle layer.Next, remove the uterus and transfer it into a 10 centimeter dish containing ice cold dissection medium. The next step takes place in the hood using micros dissecting scissors. Carefully remove each embryo from the uterus.Now it is time to harvest and begin. Work with the embryonic brains. Harvest the brain from each embryo and place the freed brains into a clean 10 centimeter dish of dissection medium on an ice platform Under the dissecting microscope, finish dissecting the brains.Remove the midbrain and hind brain sections. Use fine forceps to remove the menes transfer what remains the forebrain to a 50 milliliter falcon tube containing dissection medium. Now the tissue is ready for the next step.Dissociation of cells by enzymatic digestion. Just before you are ready to begin the digest, make up fresh solutions of propane and trypsin inhibitor filter. Sterilize them and warm them to 37 degrees Celsius in a water bath.Remove excess medium from the dissected brains. Then add four milliliters of the prepared propane digestion solution to your 50 milliliter tube. Incubate the tube in a 37 degree Celsius water bath for exactly five minutes.When time is up, use a pipette to remove propane's solution. Next, add five milliliters of freshly prepared trypsin inhibitor solution and put the tube back in the 37 degree Celsius water bath for two to three more minutes. When time is up, remove the trypsin inhibitor solution.Repeat the incubation with trypsin inhibitor solution three more times. After you remove the trypsin inhibitor solution for the last time, add 20 milliliters. Warm plating medium pm to your tube.Now it is time to iterate and wash your cells. Iterate the cells with a 10 milliliter pipette until all clumps have disappeared approximately 20 to 30 times. Then centrifuge at a hundred GS for seven minutes.At this point, your cells should be well separated from each other. After seven minutes, remove the super Dayton and resuspend the pellet. In 10 milliliter plating medium to wash the cells.Then spin them down again. Remove the supernatant and repeat the washing and centrifugation. Step after the second wash.Resuspend the pellet in 10 milliliters of aggregate culture. Medium, pass the cells through a 70 micrometer cell sieve. Finally count live cells using a hemo cytometer.And now you are ready to allow the dissociated brain cells to form aggregates. After counting, suspend the dissociated cells in aggregate culture medium at a density of two times 10 to the sixth cells per milliliter. Transfer two milliliter of cell suspension into each well of an uncoated six well plate culture.The cells for three nights each day gently resuspend the cells. Once using a P 1000 pipette men, the cells will begin to form aggregates on the day of aggregate plating. After the cells have grown for three nights, gently resuspend cells again and CVE the suspension through a 200 micrometer mesh into a 50 milliliter falcon tube.Allow aggregates to settle to the bottom of the tube for approximately three to five minutes. Then carefully remove the supernatant, 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 the microscope to check whether the supernatants still contains un aggregated cells and debris. Once you have thoroughly washed the aggregates, gently resuspend them in the same medium and count aggregates to determine their concentration. Then adjust the density of aggregates to around 25 to 30 aggregates per 50 microliters.Now it is time to seed cover slips with aggregates. Transfer 1000 microliter aliquots of the aggregate suspension to two milliliter eend Dwarf tubes since aggregates tend to settle down to the bottom of tubes, it is crucial to make multiple tubes of the aggregate suspension for plating. The cover slips used in this step are coated in a cell culture substrate matrigel by an overnight incubation at 37 degrees Celsius.After you have made Alec watts of the aggregate suspension, take the culture plate containing the matrigel coated cover slips out of the incubator. Remove the solution from each. Well wash the cover slips with warm PBS and then DD H2O.They are now ready for aggregate seeding to seed the cover slip with aggregates. Invert the tube of aggregate suspension to mix and evenly distribute the aggregates. Then load 50 microliters of aggregate suspension into the center of each cover slip.Gently put the plate back to incubator without any further disturbance to allow aggregates to attach evenly to the cover slip. The density of aggregates is crucial if aggregates are seated too close to each other or if the seating density is high, they tend to merge together as they grow four to six hours after initial seating. Add 500 microliters of culture medium to each well.To maintain the aggregate culture, half change the medium every three to four days. When you change the medium, add it along the sidewall of the well to avoid disrupting the aggregates. A few hours after aggregates attached to the cover slip, neite outgrowth from the aggregates can be observed.Axons grow rapidly in the first two weeks and form abundant connections among aggregates. Ggl progenitors migrate radially outta the aggregates and differentiate over time into astrocytes and mature oligodendrocytes. We've just shown you how to culture CS aggregate.When doing this procedure. It's important to remember to quote capacity properly and see the aggregate evenly. So that's it.Thanks for watching and good luck with your experiment.

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Learn to prepare rat brain aggregate cultures for studying neuron and glia development. This protocol isolates E16 fetal brain tissue, generates single-cell suspensions, and cultures multipotent progenitors that differentiate into neurons, astrocytes, and oligodendrocytes.

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

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