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.
