The overall goal of this procedure is to derive epithelial cells from three dimensional mixed cell tissue cultures in order to analyze the effects of stromal fibroblasts on epithelial cell biology. This is accomplished by first establishing three dimensional mixed cell steroid co cultures. The sphe are then separated from the matrix culture in order to isolate the post co-culture epithelial cells.
The next step is to passage the post co-culture epithelial cells until they are ready for analysis. Ultimately, the effects of exposure to fibroblasts in three dimensional culture on epithelial cell behavior can be evaluated through microscopic imaging of the cultures and analysis of the post co-culture. Epithelial cells, such as the fluorescence imaging of matrix invasion and Transwell migration as shown here, Demonstrating the procedure will be Quin, a research associate from my laboratory The day before, establishing the co cultures.
Remove the matre gel from the freezer and thaw it on ice in a four degree Celsius refrigerator overnight. On the day of the experiment, place the culture medium on ice at least one hour prior to mixing it with the matra gel. To dilute the thawed matra gel.
Add half the volume of ice cold H Mac medium necessary for the co-culture to a sterile tube on ice. Then add the same half total volume of thawed matrigel to the tube to achieve a one-to-one dilution, keeping the tube on ice. Next place, 50 microliters of matra gel medium mixture mid well in designated wells of a 24 well plate and incubate the plate in a 37 degree Celsius incubator with humidified air and 5%CO2 for 10 minutes.
Then add an additional 450 microliters of matra gel media mixture to the wells and incubate for another 60 minutes. During the second incubation, prepare a one-to-one suspension of fibroblasts and epithelial cells at a concentration of 0.5 times 10 to the fifth cells in each 0.5 milliliter of H me medium. When the matrigel medium mixture has solidified, gently drop the cell suspension onto the top of the gel and return the culture to the incubator for two days.
Change the medium every two to three days by very gently aspirating the medium from the side of the well and gently replacing it with fresh HEC medium monitor s spheroid formation daily under the microscope image as appropriate using light or fluorescent microscopy. Begin by pipetting the culture up and down 10 times using a sterile two milliliter glass pipette. Then transfer the culture with the glass pipette to a sterile 15 milliliter centrifuge tube and centrifuge for 10 minutes at 350 times G at room temperature after centrifuging, the cellular steroid will sediment at the bottom of the tube.
Use a sterile pasture pipette tip to gently remove the disrupted matra gel from the top of the centrifuge.Preparation. Now add one milliliter H mac medium to the pelleted steroid pipette up and down two to three times with a sterile two milliliter glass pipette and transfer the culture to a well in a 24 well tissue culture plate. Allow the steroid to attach to the dish for at least 48 hours prior to changing the medium.
Over time, the cells will leave the spheal structure and grow as monolayers after the cell cultures have reached 50%Confluence passage them using a standardization protocol to a 35 millimeter dish for two to four days and then passage to a 100 millimeter dish for three to five days before further analysis of the cells. Use a sterile pasture pipette to aspirate the medium and then manually remove as many residual ghosts spheroids as possible. In three dimensional spheroid co cultures fibroblasts with engineered TM one silencing as demonstrated here in figures d, E, and F induce increased matrix invasion by memory epithelial cells as compared to the control fibroblasts in figures A, B, and C.Note the epithelial projections invading into the matrix in spheroids incorporating TM one silenced fibroblasts as indicated with arrows in figures D and F fibroblasts with engineered over expression of TM one shown here in figures JK and L induced decreased invasion by the breast cancer cell line sum 1 59 as compared to control fibroblasts in figures GH and I compare the lack of epithelial projections in steroids incorporating TM one engineered fibroblasts with those incorporating control fibroblasts indicated by the arrows in figures G and i once co-culture steroids have been established.
Cell populations can be isolated from steroids by replating in a two dimensional context. This composite of images shows cultures under light and fluorescence microscopy after their isolation from matrigel culture here. Day zero represents imaging immediately after isolation days one, two, and five indicate days after isolation.
Note how the corona of cells leaving this steroid increases over time. Eventually, a ghost steroid with very few cells remains as highlighted here by the arrow. Very few steroids remain after ghost removal and a passage.
Depending on the relative growth rates of the cells tested. Pure populations may emerge through serial passage or can be sorted using appropriate cell markers. Sample watts are analyzed by flow cytometry in this example, using green fluorescence and red fluorescence to identify GFP expressing fibroblasts and mCherry expressing epithelial cells respectively to determine relative populations of epithelial and fibroblasts cells.
After isolation from 3D, co-culture, epithelial cells retain the properties conferred by co-culture with fibroblasts over extended periods of time, allowing ample opportunity for analysis. In this example, exposure to TM one deficient fibroblasts induced increased migration in co cultured H max that persisted for weeks after isolation from 3D co-culture compared to exposure to control fibroblasts labeled here as C Following this procedure. Other methods like those for analyzing changes in gene expression and epigenetic marks can be performed in order to answer additional questions regarding the effects of fibroblasts on epithelial cells.
