The overall goal of this filter insert-based 3D culture system for patient derived primary cells is to establish a more biologically relevant in vitro model system for prostate cancer research. By using primary human cells, this method can help answer key questions in prostate developmental biology as well as in prostate cancer. The main advantage of this technique is that it is a relatively rapid in vitro methodology for the establishment of differentiated primary prostate cells which also allows for the rapid isolation of biomolecules such as RNA, DNA and protein.
Visual demonstration of these methods are critical as the processing of cells on the filter insert for histology or for RNA and protein collection are delicate and time sensitive. To help limit diffusion between the chambers in a biological safety cabinet, apply a thin layer of 0.1%gelatin to the bottom side of the inserts and allow them to dry. Repeat this procedure two more times.
Then apply the cells to the inner chamber of the gelatin coated inserts and culture them for up to two weeks. After two weeks apply the cultured filter inserts directly to one of the appropriate downstream applications detailed in the followings. To begin this procedure, aspirate PBS from the outer chamber and carefully remove PBS from the inner chamber.
The cultured inserts can be kept briefly in PBS until the application is ready to begin but do not allow the membrane to dry out. Next add 100 microliters of 0.25%Trypsin EDTA to each inner chamber. Then incubate them for five minutes at 37 degrees Celsius.
Afterward briefly and carefully scrape the cells on the membrane surface with a 200 microliter pipette while pipetting up and down. Then incubate them for one minute at 37 degrees Celsius. After one minute add 250 microliters of conditioned media to the first inner chamber and pipette up and down gently to rinse the filter.
Following this transfer the resuspended cells to the adjacent filter chamber that contains the same media conditions and repeat pipetting up and down. Repeat this procedure for each of the inserts of a single condition. Then collect the cells and transfer them to a 1.5 milliliter centrifuge tube.
Rinse each inner chamber with an additional 100 to 200 microliters of conditioned media to collect any remaining cells and add them to the 1.5 milliliter centrifuge tube. Next spin the cells in a microcentrifuge at four degrees Celsius for five minutes. Afterward aspirate the supernatant and wash the cell pellet once with 1, 000 microliters of PBS.
Spin the cells again in a microcentrifuge at four degrees Celsius for five minutes. After five minutes aspirate the PBS and freeze the sample at minus 80 degrees Celsius for later use. In this procedure add 200 microliters of guanidinium thiocyanate phenol chloroform or similar extraction reagent to the inner chamber and briefly agitate the cells on the membrane surface by pipetting up and down.
Then incubate the cells in the extraction reagent for five minutes at room temperature and leave the outer chamber dry. Afterward wash the filter membrane by pipetting the extraction solution up and down. Collect as much of the extraction reagent as possible by tilting the six well plate and aspirating any residual liquid.
Please note that the filter may dissociate from the insert. Wash the dissociated filter membrane if attached. Then collect as much of the extraction reagent as possible and follow the manufacturer's protocol for purification and recovery of DNA, RNA or protein.
Care should be exercised when washing the cells and filter with guanidinium thiocyanate as excessive agitation can yield poor quality RNA. In order to isolate protein from the filter insert, place the filter insert in the six well plate on ice. Then add 10 microliters of protein lysis buffer to the inner chamber.
Agitate and scrape the cells on the membrane surface with a 20 microliter pipette while pipetting up and down and avoid generating bubbles in the lysis buffer. Afterward incubate the six well plate with filter inserts on ice for ten minutes. Repeat scraping and then rinse the membrane surface with the lysis buffer.
Collect the lysates from the six inserts and transfer them to a 1.5 milliliter centrifuge tube on ice. Next rinse the first membrane with an additional 10 microliters of lysis buffer and transfer it to the next filter. Repeat this procedure for all the inserts of a given experimental condition, collecting any residual lysis buffer solution and add to the 1.5 milliliter tube.
Incubate the sample on ice for an additional five minutes. Then spin it at maximum speed for 15 minutes at four degrees Celsius. When it is done pipette the lysate into a fresh 1.5 millimeter tube.
Then proceed to the analysis of protein concentration or store the lysates at minus 80 degrees Celsius. In this procedure add 500 microliters and one milliliter of 10%NBF to the inner and outer chamber respectively. Incubate them overnight at four degrees Celsius.
The next day aspirate NBF from the outer chamber and add one milliliter of HEA processing gel to a 1.5 milliliter centrifuge tube. Slowly melt the agarose in a microwave using low power and repeated 10 seconds pulses until the agarose is melted. Keep the HEA in a 37 degrees Celsius warm bath to prevent solidification until ready to use.
Next remove NBF from the inner chamber. Apply 25 microliters of molten HEA to the inner chamber and allow the agarose to solidify for two to five minutes. Then wet two foam histology pads in 10%NBF and place one pad in an embedding cassette.
Safeguarding the integrity of the cell layers on the filter insert using HistoGel is a critical step for preservation of the intact cell layer for histological sectioning. After that use a number 11 blade scalpel to score the filter from the bottom side of the plastic insert chamber, partially releasing it. Place 100-200 microliters of NBF in a petri dish and immerse the partially dislodged filter in the NBF.
With a number 10 blade scalpel, gently press against the middle of the filter from the inside of the insert chamber to fully dislodge the filter from the insert barrel. If there is any part of the filter that is still attached to the barrel, sever it with the scalpel. Subsequently cut the HEA coated filter in half.
Place each half of the filter onto the foam pad in the prepared histology cassette. Then add the second 10%NBF soaked sponge to the cassette, sandwiching the filter. Next snap seal the cassette.
Place it in NBF and incubate it overnight. This bright field image shows multilayered cell strata on top of the porous membrane. The individual fluorescent images for nuclei, P63 and the androgen receptor are shown, as is the merge of all three fluorescence markers.
Finally a composite bright field and immunofluorescence overlay is shown, establishing the localization of the fluorescent signal relative to the cells and the filter. Here a cross sectional image of our filter insert is shown, compared to the ductal epithelial layers of an intact prostate. Once mastered this technique can be done in two weeks with a final isolation of the cells and filter insert or the desired biomolecule taking less than 30 minutes if performed properly.
While performing this procedure, it's important to always remember to exercise caution when working with the filter inserts, so that no damage to the cell layer or underlying filter occurs. Following this procedure, other methods such as Western blots, RNA microarray and proteome analyses can be performed in order to answer additional questions pertaining to pathway analysis and cancer cells. This technique will help pave the way for researchers in the field of prostate cancer to better explore prostate biology as well as underpinnings of prostate cancer using primary prostate cells.
After watching this video you should have an excellent understanding of how to properly set up and recover cultured primary prostate cells from this 3D culture system.
