Cancer Research
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Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions
Chapters
Summary January 7th, 2019
We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.
Transcript
This method can help answer key questions in the stem cell field about the role of adult stem cells in the regulation of cancer cell behavior. The main advantage of this technique is that it allows the evaluation of cancer cell and stem cell interactions in vitro without the use of living organisms. This technique has potential for cancer metastasis as it models the promoting role of adult stem cells in cancer metastasis to hard tissues such as bone.
Demonstration of the procedure will be carried out by Huseyin Apdik, a post-doctoral in my laboratory. Begin by using sterile extraction forceps to remove the pulp tissue from the center of wisdom teeth obtained from young adults aged 17 to 20 years old. Place the tissue in cold complete DMEM in 10-centimeter tissue culture dishes and use a scalpel to mince the samples into two to three-millimeter fragments.
Transfer the pieces from each dish into individual wells of a tissue culture treated six-well plate and cover the tissues in each well with 200 microliters of complete DMEM. Allow the tissues to attach to the well bottoms with an at least two-hour incubation in a humidified, 37 degree Celsius and 5%CO2 cell culture incubator. At the end of the incubation, feed the tissues with two to 2.5 milliliters of fresh complete medium and culture the cells for an additional eight to nine days.
When the culture has reached 80%confluency, wash each well with two milliliters of PBS followed by detachment of the cells with two milliliters of trypsin at 37 degrees Celsius. After two minutes, stop the reaction with two milliliters of complete DMEM per well and collect the cells by centrifugation. Then, resuspend the pellets in 15 milliliters of fresh complete medium per two wells of cells and passage the cells into T75 flasks for their subsequent culture.
After at least eight passages, visualize the cells by light microscopy. The dental pulp stem cells should have attached to the culture dishes and should display a spindle-like cell morphology. For surface marker analysis, after trypsinization is demonstrated, fix the cells with 4%paraformaldehyde for 20 minutes at room temperature in 1.5-milliliter tubes followed by three washes in 500 microliters of PBS per wash.
After the last wash, label the cells with the appropriate antibodies of interest in 100 microliters of PBS per tube for one hour at four degrees Celsius. At the end of the incubation, wash the cells three times in PBS as demonstrated and label the cells with the appropriate secondary antibodies in 100 microliters of PBS per tube for 30 minutes at four degrees Celsius. Then wash the samples three times in PBS and analyze the cells by flow cytometry according to standard protocols.
For differentiation of the dental pulp stem cells, seed one times 10 to the four cells into individual wells of 24-well plates in 500 microliters of complete DMEM for a 24-hour incubation in the cell culture incubator. The next day, replace the medium in each well with the appropriate differentiation medium and return the cells to the incubator, refreshing the medium twice a week for two weeks. Differentiation can be confirmed by von Kossa and Alcian blue staining according to standard protocols.
After two to four passages, collect the conditioned medium supernatants from the cultured dental pulp stem cells when the cultures reach 80%confluency. Then, centrifuge the collected medium to remove any waste tissue material and cell debris and store the medium at negative 20 degrees Celsius until use. For cancer cell treatment, seed one times 10 to the five tumor cells into individual wells of a 12-well plate for overnight incubation at 37 degrees Celsius and 5%CO2.
The next morning, use a sterile 200-microliter tip to make a scratch injury in each well and immediately replace the supernatant in each well with fresh medium containing various concentrations of conditioned medium. Then, immediately observe the scratches under an inverted microscope and obtain images of the injured cultures at regular time intervals. At the end of the analysis, measure the scratch closure over time in ImageJ.
To assess dental pulp stem cell migration, first seed three times 10 to the four dental pulp stem cells onto individual 24-well plate inserts with 0.4 micron pores in 250 microliters of DMEM and incubate the inserts overnight at 37 degrees Celsius. Next, seed five times 10 the four tumor cells into individual wells of a 24-well plate in 500 microliters of DMEM for overnight incubation at 37 degrees Celsius. The next morning, scratch the tumor cells as demonstrated.
Replace the supernatants with 500 microliters of fresh DMEM and place one dental pulp stem cell seeded insert into each well fed with fresh medium. Then immediately observe cells on an inverted microscope and image the cells at regular intervals to assess dental pulp stem cell migration. To assess the direct interactions of dental pulp stem cells with cancer cells, trypsinize each labeled culture to obtain single-cell suspensions and collect the dissociated cells by centrifugation.
Resuspend the pellets in distinct cell linker dye solutions prepared in diluent C buffer, supplied according to the manufacturer's instructions, and incubate the cells are room temperature for 10 minutes. Terminate the staining reaction with 100 microliters of fetal bovine serum and collect the cells by centrifugation. Then centrifuge the cells with complete growth medium before plating five times 10 to the four dental pulp stem cell and tumor cells per well in a six-well plate at a one to one ratio in two milliliters of complete medium.
Collect the cells after 24 or 48 hours of incubation by centrifugation followed by a wash in PBS. Then, resuspend the cells in 300 microliters of fluorescence-activated cell sorting buffer in five-milliliter round-bottom flow cytometry tubes and vortex to disperse any cell aggregates before analyzing the cells according to standard flow cytometric analysis protocols. Dental pulp stem cells exhibit a fibroblast-like cell morphology after plating and express mesenchymal stem cell surface antigens but not hematopoietic cell markers.
Changes at the morphological and molecular level related to osteo, chondro, and adipogenic differentiation are also observed in the dental pulp stem cell cultures following the appropriate differentiation cocktail application. The treatment of scratch-injured tumor cell cultures with 10 and 20%concentrations of conditioned medium increases the scratch closure significantly compared to control-medium-treated tumor cell cultures. Secreted molecules from dental pulp stem cell insert co-cultures also increased scratch closure in injured tumor cells compared to control cell co-cultures.
Fluorescence microscope analysis of the interactions of dental pulp stem and tumor cell within an in vitro cell culture reveals the creation of a well-organized structure within which tumor cells rapidly proliferate after 48 hours. While attempting this procedure, it's important to remember to keep two tissue samples in cold medium and immediately transport samples to the laboratory to minimize cell death. After its development, this technique paved the way for researchers in the stem cell fields in exploring adult stem cell cancer interactions in humans.
Following this procedure, other methods like immune cell tracing for differential labeled cells of various stem cell and cancer types can be performed to answer additional question about how adult stem cells interact with cancer cells and control cancer metastasis. Don't forget that working with the human samples can be dangerous and that precautions such as analyzing patient samples for infectious diseases and obtaining written patient consent should always be implemented when performing this procedure.
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