The overall goal of this procedure is to create a cellular co-culture system of two cell types using inserts with a permeable membrane, thus allowing the diffusion of secreted soluble factors. This is accomplished with a series of steps that finely placed inserts containing one cell type in a multi-welled tissue culture plate containing a second cell type. We start my seeding cell type number one in inserts, and seeding cell type number two in a multi-well tissue culture plate.
The second step is to transfer the inserts into the wells of the plate containing cell type number two. Ultimately, this gives the ability to harvest the two cell types as well as the respective supernatants. Therefore, it is possible to evaluate the effect of secreted soluble factors on the cell type of interest.
In multi-cellular organisms secreted soluble factors elicit responses from different cell types as a result of paracrine signalling. As such, the value of an insert co-culture system resides in its ability to offer an original way to assess the changes of cellular parameters mediated by secreted soluble factors in the absence of cell-cell contact. Insert co-culture systems offer various advantages over other co-culture techniques, such as:one by directional signaling;two conserved cell polarity;and three population specific detection of cellular changes.
A specific protocol to measure the toxic effects of cytokine secreted by lipopolysaccharide activated N9 microglia on neuronal PC12 cells will be detailed hereafter, thereby providing a concrete understanding of insert co-culture methodology. To begin, unwrap the 24-well tissue culture plate and the 0.4 micron por polytetrafluoroethylene inserts from their packaging. Then, place the inserts in the empty wells of the tissue culture plate by gripping the uppermost edge of the insert using sterile tweezers.
Next, fill the inserts with routine N9 medium until the membrane is completly covered. Incubate the inserts for at least one hour, or overnight, to improve the ability of the cells to attach. Once the inserts are ready, split the N9 microglia at 80 to 90%confluence using routine N9 medium to obtain a cell suspension that will be used to seed the inserts.
Then, remove the routine medium from the inserts taking care not to perforate the membrane with the micropipette tip. Next, seed each insert at 60, 000 cells per square centimeter with 50 microliters of the cell suspension, or according to the insert manufacturer's protocol. While distributing the cell suspension, ensure that it remains homogeneous by occasionally inverting the tube.
When all the inserts are seeded, gently rock the plate back and forth, then left to right, in order to distribute the cells evenly. Avoid making circular motions as this will cause the cells to accumulate in the center of the insert. Subsequently, incubate the plate containing the inserts for 24 hours before pursuing with N9 microglia activation.
Begin by weighing 10 milligrams of lipopolysaccharide and store it in a 1.5 milliliter Eppendorf Tube for later use. Since lipopolysaccharide is a potent pro-inflammatory endotoxin and requires particular safety precautions, glasses, gloves and a particulate respirator are strongly recommended. Afterward, create a set of serial dilutions of lipopolysaccharide using warm N9 treatment medium to obtain working solutions of four, two and one micrograms per milliliter.
Finally, remove half of the culture medium from the inserts, taking care not to disturb the cells. Then, gently add 25 microliters of the lipopolysaccharide working solutions to the inserts to yield final dilutions of two, one or 0.5 micrograms per milliliter. Incubate the plate for another 24 hours before proceeding with the co-culture experiments.
This step requires PC12 cells plated at 30, 000 cells per square centimeter differentiated interneurons, and N9 microglia activated with lipopolysaccharide for 24 hours as described in the previous section. Start by gently removing all of the medium from the inserts, and by replacing it with 50 microliters of fresh, warm N9 treatment medium. This is necessary to remove all traces of lipopolysaccharide, leaving only activated N9 microglia.
Keep in mind that the inserts are loose in the wells of the tissue culture plate and may move around if the tip is pressed too hard upon the wall. This step should be executed one insert at a time in order to prevent the cells from drying out due to prolonged contact with the air. Subsequently remove the neuronal PC12 cell medium completely and replace it with 0.6 milliliters of fresh, warm PC12 treatment medium.
Do this one well at a time to ensure that the cells do not dry out. Using tweezers, grip the uppermost edge of an insert and gently place it into the well containing neuronal PC12 cells. When all of the inserts are transferred, check for the presence of air bubbles beneath the membranes of the inserts.
Air bubbles will prevent any exchange across the membrane of the insert and can jeopardize the entire experiment. Remove any air bubbles by very gently lifting the insert from the well using tweezers and placing it back into the cell culture medium, this action should get rid of most bubbles. However, for persistent bubbles try gently dipping the insert back into the cell culture medium at an angle.
Do not knock or stir inserts, as this has a tendency to cause the dissociation of adherent cells. Afterwards, check the volumes of media in both the inserts and the wells, the liquid in both compartments should be roughly at the same level. When all air bubbles are removed, and the media volumes are appropriate, incubate the plate.
The cells, culture medium, or both, can then be harvested to measure the effects of paracrine signalling between the N9 microglia and the neuronal PC12 cells. Enzyme-linked immunosorbent assays were performed on the cell culture medium of the lower compartment to quantify proinflammatory cytokines. The results illustrate that 24 hours after the activation period, N9 microglia treated with two micrograms per milliliter of lipopolysaccharide secreted significantly more proinflammatory cytokines, such as interluken-6, tumor necrosis factor-alpha, and interferon gamma, in comparison to microglia that were not treated.
In addition, N9 microglia treated with one microgram per milliliter of lipopolysaccharide secreted pointedly more interferon gamma after 24 hours, while it took 48 hours to see a significant increase in cell medium levels of interluken-6 and tumor necrosis factor-alpha. In contrast, the 0.5 microgram per milliliter condition failed to enhance cytokine expression in N9 microglia at both 24 and 48 hours after the activation period. Moreover, the data at 24 hours in the two micrograms per milliliter group suggest that there was microgliosis, an increase in the microglial population.
However, it was only after 48 hours that the cellular number was significantly increased. Finally, in order to assess the consequences of microglial activation leading to enhanced cytokine secretion and microgliosos, cytotoxicity was assessed in the co-cultured neuronal PC12 cells. By measuring the levels of Extracellular Lactate Dehydrogenase, which is released by damaged cells, neuronal PC12 cells were found to exhibit more cell death as the concentration of lipopolysaccharide on the N9 cells increased.
As such, these results show that secreted soluble factors are able to cross the membrane of the co-culture inserts, and can cause cytotoxic damage to neuronal PC12 cells in the absence of cell-cell contact. While only one scenario of insert co-culture was presented here, it is a very flexible technique. In this protocol, one cell type was pretreated with the molecule responsible for reducing the secretion of soluble factors that, upon transferring the insert to the well, affect the behavior of second cell type.
However, it is possible to incubate both cells types together in a co-culture system to detect the increased weakness, or resistance, of one or both cell populations to a later treatment. This technique can simply make use of two population of cells incubated together without any treatment, to assess the basal paracrine reciprocal signalling, for example. In addition of being valued in the field of neuroinflammation as demonstrated here, insert co-culture systems can be used to answer a vast range of questions pertaining to tumor regenesis, neurolathyrisma, apoptosis signalling, or any other topic with a component of paracrine signalling.
After watching this video, you should now have a good understanding of how insert co-culture systems can offer a simple way to assess the changes mediated by secreted soluble factor. And we hope to have convinced you of the high potential of this insert co-culture systems in the study of multicellular paracrine signalling in the absence of cell-cell contact.
