Name: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays (Video) | JoVE Journal
Uploaded: 2011-01-13T23:59:00.000+00:00
Duration: 10 min 17 s
Description: Learn in vitro assays to study Schwann cell-astrocyte interactions for spinal cord injury repair. These methods reveal how boundary formation and migration affect axonal regeneration and therapeutic integration.

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5 minutes

We would like to thank Philippa Warren for her kind help.

1. Boundary Assay:
<ol>
<li>Basic preparation: Chamber slides are coated with Poly-D-Lysine overnight and sterile glass slides are prepared before starting the experiment. Medium is prepared using Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum (FCS), and 1% Penicillin-Streptomycin-Fungizon(PSF). Also a bottle of Calcium/Magnesium free Hanks balanced salt solution (HBSS) is prepared.</li>
<li>Primary rat Schwann cells cultured in flask are trypsinized for 3 minutes using 0.1% Trypsin. Primary rat astrocyte culture is trypsinized for 5 minutes using 0.1% trypsin. 
Trypsin is inactived by adding DMEM supplemented with 10% FCS and 1% PSF.</li>
<li>Trypsinized Schwann cells and astrocytes are transferred to separate 15 mL falcon tubes and centrifuged at 300G for 5 minutes.</li>
<li>Schwann cells and astrocytes are re-suspended at the density of 2 x 106 (see representative results section for variations in cell density).</li>
<li>50 μL drop of Schwann cell suspension is placed at one end of PDL coated chamber slide wells. A glass strip was used to smear the drop towards the centre of the chamber to generate a straight edge. (Using a glass cutter, a rectangular glass coverslips are cut longitudinally so that it would fit the width of chamber slides used for boundary assays)</li>
<li>A second drop of 50 μL of astrocyte suspension was placed at the opposite end of the same well and smeared towards the centre to create a straight edge parallel to the first drop smear with a small 0.2mm gap between them.</li>
<li>The chamber slides containing the drops are placed in the incubator at 37°C for 2 hours.</li>
<li>After 2 hours, cultures were washed with DMEM to remove non-attached cellular debris and fresh medium containing DMEM/ 10%FCS/ 1% PSF added. 
Note: DMEM/ 10%FCS/ 1% PSF can be supplemented with forskolin (2μM) and Bovine pituitary extract (BPE) (10μg/ mL) stimulate Schwann cell proliferation. BPE will supply the growth factors necessary to allow long term Schwann cell culture and forskolin raises the cyclic adenosine monophosphate levels within the Schwann cells to induce proliferation</li>
<li>Cells are cultured for approximately 8-10 days at 37°C , 7% CO2 incubator. The medium requires to be changed every 3 days. The two cell fronts will eventually reach each other and a sharp boundary will form between the two cell types. At this stage experiments can be carried out analyzing factors involved in boundary formation.</li>
<li>Co-culture can be fixed using 4% formaldehyde and Schwann cells can be immunostained with anti-P75 antibody and astrocytes with anti-GFAP antibodies to identify the cells in co-culture. P75 antibody recognizes low affinity NGF receptor on Schwann cells which astrocytes lack. Anti-GFAP antibody recognizes glial firbrillary acidic protein which localizes to astrocyte cytoplasm. (Please see Figure 1A for representative boundary)</li>
</ol>
2. Migration Assay:
<ol>
<li>4 well plates (15mm wells) are PDL coated overnight and prepared for creating astrocyte monolayers.</li>
<li>Primary astrocyte culture is trypsinized for 5 minutes using 0.1% trypsin.
Trypsin is inactivated by adding DMEM supplemented with 10% FCS and 1% PSF and cell centrifuged at 300G for 5 minutes. (See Figure 2)</li>
<li>Astrocytes are re-suspended at 1x105 cells/ mL. 1 mL of this solution is added to each PDL coated well in 4 well plates. Astrocytes are cultured for 24-48 hours until the monolayer is completely confluent.</li>
<li>To assess Schwann cell migration on astrocyte monolayers, parallel to creating astrocyte monolayers, circular PDL-coated glass coverslips are placed in a 50 mL tube and crushed using a plastic pipette to create small fragments of glass.</li>
<li>Glass fragments are transferred into a well in 6 well plate dish and using forceps suitable sized fragments are chosen and placed in other wells. 5 glass fragments are placed in each well in 6 well plates.</li>
<li>Primary Schwann cells cultured in flask are trypsinized for 3 minutes using 0.1% trypsin. Trypsinized Schwann cells are transferred to separate 15 mL falcon tube and centrifuged at 300 G for 5 minutes. Schwann cells are re-suspended at 2x 106 cells/ mL in DMEM/10%FCS/1%PSF.</li>
<li>20 μL droplets of Schwann cells suspension is added over each 5 coverslip fragments placed in 6 well plates and cells are incubated at 37°C , 7% CO2 for 2 hours.</li>
<li>After 2 hours, the Schwann cells are attached to the coverslip fragments and DMEM/10% FCS/1% PSF (Supplemented with 2μM of forskolin and 10μg/ mL of BPE is added to each well so that the fragments are completely covered with medium).</li>
<li>The 6 well plates containing the fragments are then placed in the incubator for 24-48 hours until the glass fragments are fully confluent with Schwann cells.</li>
<li>Once fragments are confluent with Schwann cells, each fragment is picked up carefully using sharp forceps and dipped in HBSS to remove unattached cells and then inverted face down on to astrocyte monolayer.</li>
<li>Each well is then covered with 1 mL of DMEM/10%FCS/1%PSF and Schwann cells are allowed to migrate for 24 to 48 period. During this period experimental reagents such as growth factors can be added to assess their effect on the number and distance of migration of Schwann cells.</li>
<li>Following the migration period, the glass fragments are fixed to the bottom of each well by adding 4% paraformaldehyde (PFA) for 30 minutes. Following fixation, immunostain the Schwann cells for p75 (recognizing low affinity NGF receptor) then stain them using ABC strepavidin kit-diaminobenzidine(DAB), to allow visualization of the Schwann cells under light microscope.</li>
</ol>
3. Representative Results:
Following 8-10 days Schwann-astrocyte co-cultures show a sharp boundary between the two cell types. This boundary is more pronounced if forskolin and BPE is used in the culture as Schwann cell proliferation is enhanced.
If no boundary is observed during the 10 day period, the time of culture can be increased further until the boundaries are established. Changing the ratio of cell density is an alternative way to increase the boundary formation. A ratio of 3:1 Schwann: astrocyte cell density can be used when preparing cell suspensions 8,18.
Following immunostaining to identify the Schwann cells and astrocytes, to assess cell intermingling a line can be drawn where the boundary is established and the number of Schwann cells migrating into the astrocyte territory can be counted under fluorescence microscope.
Below are two examples of images obtained boundaries, one with well-formed segregation of Schwann cells and astrocytes (Figure 1A) and one where the cells have not segregated well into different territories due to poor technique (Figure 1B).
<img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Neuronal network formation in fluorescence microscopy; green and red staining, cell interaction study." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Boundary assay: Schwann-astrocyte interaction. A) A 10 day co-culture assay in the presence of forskolin and BPE to stimulate Schwann cell proliferation demonstrates a sharp boundary between the two cell types(red=P75, green=GFAP). B) A 10 day co-culture assay where the boundary is not formed and the two cell fronts mixed. This was due to mixing of the two droplets while placing astrocyte and Schwann cell droplets next to each other during the preparation of the boundary (red=P75, green=GFAP).
<img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Cell culture microscopy image, examining cellular morphology and structure, biological research tool." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Representative image of confluent astrocyte monolayer
Migration assays assess movement of one cell type over the surface of another cell type and therefore assess a different phenomenon from that of boundary formation.
Practice is needed to ensure inverting the coverslips onto monolayers does not lead to damage to the Schwann cell-covered glass fragments and astrocyte monolayers by the sharp forceps. To assess the migration of Schwann cells, the maximum distance of migration and the number of migrating cells from the edge of coverslip can be measured under light microscope.
Below is the image of Schwann cells stained with P75 antibody using DAB staining on the surface of astrocyte monolayer (Figure 3). 
<img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Microscopic image of fungal hyphae growth; microbiology analysis, structural study, cell morphology." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Migration assay using inverted coverslip assay.
A) Schwann cells (brown, immunostained with p75 antibody) migrating away from the edge of coverslip. Arrow points to the direction of the cell migration away from the coverslip.

<ol><li>Assouline, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+astrocytes+and+Schwann+cells+in+culture+synthesize+nerve+growth+factor-like+neurite-promoting+factors%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors.</a> Brain Res. 428, 103-118 (1987).</li>
<li>Taylor, J. S., Bampton, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factors+secreted+by+Schwann+cells+stimulate+the+regeneration+of+neonatal+retinal+ganglion+cells%5BTitle%5D)+AND+%22J+Anat%22%5BJournal%5D)">Factors secreted by Schwann cells stimulate the regeneration of neonatal retinal ganglion cells.</a> J Anat. 204, 25-31 (2004).</li>
<li>Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrins+and+cell+adhesion+molecules%3A+neuronal+receptors+that+regulate+axon+growth+on+extracellular+matrices+and+cell+surfaces%5BTitle%5D)+AND+%22Dev+Neurosci%22%5BJournal%5D)">Integrins and cell adhesion molecules: neuronal receptors that regulate axon growth on extracellular matrices and cell surfaces.</a> Dev Neurosci. 11, 332-347 (1989).</li>
<li>Chiu, A. Y., Monteros,, Espinosa de los, A., Cole, R. A., Loera, S., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laminin+and+s-laminin+are+produced+and+released+by+astrocytes%2C+Schwann+cells%2C+and+schwannomas+in+culture%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Laminin and s-laminin are produced and released by astrocytes, Schwann cells, and schwannomas in culture.</a> Glia. 4, 11-24 (1991).</li>
<li>Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remyelination+of+CNS+axons+by+Schwann+cells+transplanted+from+the+sciatic+nerve%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Remyelination of CNS axons by Schwann cells transplanted from the sciatic nerve.</a> Nature. 266, 68-69 (1977).</li>
<li>Andrews, M. R., Stelzner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+olfactory+ensheathing+and+schwann+cells+after+implantation+into+a+dorsal+injury+of+adult+rat+spinal+cord%5BTitle%5D)+AND+%22J+Neurotrauma%22%5BJournal%5D)">Evaluation of olfactory ensheathing and schwann cells after implantation into a dorsal injury of adult rat spinal cord.</a> J Neurotrauma. 24, 1773-1792 (2007).</li>
<li>Iwashita, Y., Fawcett, J. W., Crang, A. J., Franklin, R. J., Blakemore, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Schwann+cells+transplanted+into+normal+and+X-irradiated+adult+white+matter+do+not+migrate+extensively+and+show+poor+long-term+survival%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Schwann cells transplanted into normal and X-irradiated adult white matter do not migrate extensively and show poor long-term survival.</a> Exp Neurol. 164, 292-302 (2000).</li>
<li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li>
<li>Lakatos, A., Barnett, S. C., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+ensheathing+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li>
<li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte-Schwann+cell+interactions+in+culture%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li>
<li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chondroitin+sulfate+proteoglycan+staining+in+astrocyte-Schwann+cell+co-cultures%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Chondroitin sulfate proteoglycan staining in astrocyte-Schwann cell co-cultures.</a> Glia. 14, 145-152 (1995).</li>
<li>Plant, G. W., Bates, M. L., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitory+proteoglycan+immunoreactivity+is+higher+at+the+caudal+than+the+rostral+Schwann+cell+graft-transected+spinal+cord+interface%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">Inhibitory proteoglycan immunoreactivity is higher at the caudal than the rostral Schwann cell graft-transected spinal cord interface.</a> Mol Cell Neurosci. 17, 471-487 (2001).</li>
<li>Fishman, P. S., Nilaver, G., Kelly, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrogliosis+limits+the+integration+of+peripheral+nerve+grafts+into+the+spinal+cord%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord.</a> Brain Res. 277, 175-180 (1983).</li>
<li>Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migration+of+oligodendrocyte+precursors+on+astrocytes+and+meningeal+cells%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Migration of oligodendrocyte precursors on astrocytes and meningeal cells.</a> Dev Biol. 171, 1-15 (1995).</li>
<li>Wilby, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Cadherin+inhibits+Schwann+cell+migration+on+astrocytes%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">N-Cadherin inhibits Schwann cell migration on astrocytes.</a> Mol Cell Neurosci. 14, 66-84 (1999).</li>
<li>Grimpe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+proteoglycans+in+Schwann+cell%2Fastrocyte+interactions+and+in+regeneration+failure+at+PNS%2FCNS+interfaces%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">The role of proteoglycans in Schwann cell/astrocyte interactions and in regeneration failure at PNS/CNS interfaces.</a> Mol Cell Neurosci. 28, 18-29 (2005).</li>
<li>Afshari, F. T., Kwok, J. C., White, L., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li>
<li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FGF%2Fheparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes%3A+a+role+in+astrocytosis%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li>
<li>Afshari, F. T., Kwok, J. C., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte-produced+ephrins+inhibit+schwann+cell+migration+via+VAV2+signaling%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling.</a> J Neurosci. 30, 4246-4255 (2007).</li>
</ol>

No conflicts of interest declared.

The assays described above have been used in various studies demonstrating the role of multiple factors involved in boundary formation between the Schwann-astrocytes and limited Schwann cell migration in astrocytic environment.
Understanding the mechanisms underlying these events is essential as it would allow development of strategies to optimise and enhance the integration of Schwann cell grafts following transplantation and in doing so facilitate the exit of the regenerating axons from the...

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>41966-029</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14170-088</td><td></td></tr><tr><td>FCS</td><td>Invitrogen</td><td>10091-148</td><td></td></tr><tr><td>PSF</td><td>Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>BPE</td><td>Invitrogen</td><td>13028-014</td><td></td></tr><tr><td>Forskolin</td><td>Calbiochem</td><td>344273</td><td></td></tr><tr><td>Coverlips</td><td>VWR international</td><td>631-0149</td><td></td></tr><tr><td>Coverlips(rectangle)</td><td>Menzel-Glaser</td><td>BB022050A1</td><td></td></tr><tr><td>Chamber slides</td><td>Nalge Nunc international</td><td>177380</td><td></td></tr><tr><td>4 well plates, 6 well plates</td><td>Nalge Nunc international</td><td></td><td>4 well plates6 well plates</td></tr><tr><td>rat p75 antibody</td><td>EMD Millipore</td><td>MAB365</td><td></td></tr><tr><td>GFAP antibody</td><td>Dako</td><td>Z0334</td><td></td></tr><tr><td>Secondary antibodies (Alexa-conjugated)</td><td>Invitrogen</td><td>A-11004 A-11034</td><td>Goat anti mouse 568Goat anti Rabbit 488</td></tr><tr><td>Secondary biotinylated </td><td>Vector Laboratories</td><td></td><td>Goat anti mouse</td></tr><tr><td>DAB tablets </td><td>Sigma-Aldrich</td><td>D4418</td><td></td></tr><tr><td>ABC elite kit</td><td>Vector Laboratories</td><td>PK-6100</td><td></td></tr><tr><td>Fine Forceps </td><td>Fine Science Tools</td><td>11295-10</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6168</td><td></td></tr><tr><td>Fluorosave</td><td>Calbiochem</td><td>345789</td><td></td></tr></tbody></table>

analysis of schwann-astrocyte interactions using in vitro assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Schwan cells are glial cells of the peripheral nervous system that have been used in repair of spinal cord injuries. Basically, growth factors that support axonal regeneration and provide growth promoting adhesion and extracellular matrix molecules. In addition, they myelinate axons at the site of injury.Unfortunately, transplanted swan cells do not migrate from the site of implants and do not intermingle with the host astrocytes. This results in formation of a sharp boundary and prevents growing axons from integrating with the host tissue. In this video, two methods are used to study the interactions of swan cells and astrocytes in vitro.First, a boundary assay is demonstrated in which swan cell and astrocyte territories are established on a slide and cultured. Different reagents are then added to the slide and following additional culture. The slides are then imaged and analyzed to determine the effect of the reagents on migration of swan cells into the astrocytic territory or vice versa.Next, a migration assay is demonstrated in which cover slip fragments covered with nests of swan cells are inverted on astrocyte monolayers. Again, different reagents are added to the culture medium, and following culture imaging is performed to assess the extent and distance of migration of schwan cells on astrocyte monolayers. Ultimately, this type of assay should facilitate the development of strategies to optimize and enhance the integration of schwan cell grafts following transplantation.The advantage of these in vitro assays over existing methods where migration on pure substrates is assessed is that it is more relevant to in vivo assays where transplanted one cells come into contact with astrocytes. In addition, these in vitro assays can be used in other fields such as cell biology to answer key questions such as tumor cell invasion and migration. Prior to beginning the assay have on hand the following items, poly D lysine coated chamber slides, sterile glass slides, cell culture medium, and a bottle of calcium.Magnesium free Hanks balance source solution begin by trypsin primary rashwan cells for three minutes and primary rat astrocytes for five minutes. Once the cells have detached and activate the trypsin by adding cell culture medium. Next, transfer the schwan cells and astrocytes to separate 15 milliliter falcon tubes and centrifuge them at 300 times gravity for five minutes.Meanwhile, using a glass cutter cut rectangular glass cover slips longitudinally so that they will fit the width of chamber slides used for boundary assays and can facilitate smearing the drop of cell suspension. Following the spin Resus, suspend the schwan cells and astrocytes at a density of two times 10 to the six cells per milliliter. At one end of a polylysine coated chamber slide, well place one 50 microliter drop of schwan cell suspension.Use a glass strip to smear the drop towards the center of the chamber to generate a straight edge. Next place a second 50 microliter drop of astrocyte suspension at the opposite end of the same well and smear it towards the center. To create a straight edge parallel to the first, there should be a 0.2 millimeter gap between them.Place the chamber slide containing the drops in the incubator at 37 degrees Celsius for two hours. After two hours. Wash the cultures with DMEM to remove any non-attached cellular debris.To stimulate swan cell proliferation, add medium to the chamber slides. This medium is supplemented with four galin and bovine pituitary extract. The four galin raises the cyclic A MP levels within the schwan cells to induce proliferation and the BPE will supply growth factors necessary for long-term culture.Incubate the slide for eight to 10 days. The two cell fronts will eventually reach each other and a sharp boundary will form between the two cell types. At this stage, experiments can be carried out.Analyzing factors involved in boundary formation to examine the boundary, fix the co-culture with 4%formaldehyde. To distinguish swan cells and astrocytes perform immuno staining with an anti P 75 antibody and an anti GFAP antibody respectively. P 75 antibody recognizes the low affinity NGF receptor on schwan cells, which astrocytes lack anti GFAP antibody recognizes glial fibrillary acidic protein, which localizes to astrocytes to perform a migration assay.Begin my plating. One times 10 to the five astrocytes in one milliliter of medium in each well of a four well plate with 15 millimeter wells. Place the plate in the 37 degrees Celsius incubator for 24 to 48 hours until the monolayer is completely confluence.Meanwhile, to prepare swan cells to assess the migration on astrocyte monolayers, place, circular polylysine coated glass cover slips in a 50 milliliter tube and use a plastic perpet to crush them, creating small fragments of glass. Transfer the glass fragments into one well of a six well plate then using forceps. Choose fragments approximately five millimeters across and transfer five to each of the other wells.Trypsin i's primary swan cells for three minutes. Then transfer the cells to a 15 milliliter tube and centrifuge them at 300 times gravity for five minutes. Re suspend the schwan cells at two times 10 to the six cells per milliliter in culture medium.Then add 20 microliters of the schwan cell suspension over each fragment and incubate at 37 degrees Celsius and 7%carbon dioxide for two hours. After two hours, remove the schwan cells from the incubator. Add enough culture medium supplemented with two micromolar, four scale in and 10 micrograms per milliliter of BPE to each well.So the fragments with attached cells are completely covered with medium incubate for 24 to 48 hours until the glass fragments are fully conent with swan cells. Once the fragments are conent with swan cells, retrieve both swan cells and astrocytes from the incubator. Use forceps to carefully pick up each fragment with attached swan cells.Dip the fragment in HBSS to remove unattached cells and then invert it face down onto the astrocyte monolayer. Once all the fragments have been transferred. Add one milliliter of cell culture medium to each well.If the effects of experimental reagents such as growth factors will be assessed, they should be included in the medium. Return the plate to the incubator and allow the schwan cells to migrate for 24 to 48 hours following the migration period. The glass fragments are fixed to the bottom of each well by adding 4%para formaldehyde for 30 minutes following fixation.Immuno stain the schwan cells for P 75, then stain them using the A BC Strept ADIN kit supplied DIAM benadine to allow visualization of the schwan cells under a light microscope after 10 days. Schwan astrocyte co-culture show a sharp boundary between the two cell types.Here. Schwan cells are identified by P 75.Staining of the low affinity NGF receptor and are shown in red astrocytes are identified by GFAP staining shown in green. The boundary between these cells is more pronounced if forskolin and BPE are used in the culture because schwan cell proliferation is enhanced. In contrast, in this 10 day co-culture assay, the cells have not segregated well into different territories.This was due to the poor technique and mixing of the two cell types. While preparing the boundary assay, migration assays assess movement of one cell type over the surface of another cell type, and therefore assess a different phenomenon from that of boundary formation. This image shows swan cells migrating away from the edge of the cover slip, the arrow points the direction of cell migration.While attempting this vitro assays, it's important to remember that the effect of the reagents can be either on astrocytes or schwan cells, and therefore subsequent experiments are required in order to show exactly which cell type is responsible for the observed, increased or decreased migration. In addition, other techniques such as immunochemistry can be performed and especially in the boundary assay in order to show which molecules are upregulated or down-regulated at the boundary between Sean cells and astrocytes.

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Research

JoVE Journal

Biology

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Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

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