The overall goal of this human preclinical model derived from surgically resected tumor tissue is to obtain the establishment of a primary culture of patient-derived soft tissue sarcoma. This method can help answer key questions about the pathophysiology of soft tissue sarcoma and it represents an important preclinical tool for predicting response to therapy. The main advantage of this technique is that it is a fully human preclinical model.
The implications of this technique extend towards therapy of soft tissue sarcoma because this preclinical model can be tested with conventional innovative drugs to investigate their activity and mechanism of action. Although this method can provide insight into soft tissue sarcoma, it can also be applied to the study of cancer cell soft tissue cell interaction. Individuals new to this method will struggle because of the heterogeneity of the tumor tissues and in isolating tumor cells.
Visual demonstration of this method is critical as the initial soft tissue sarcoma cell selection and CD steps requires an amount of experience before proficiency can be achieved. Tumor specimens should be received in a 100 milliliter sterile urine container with 50 milliliters of DMEM low glucose medium sealed with a paraffin film. The samples may be kept at four degrees Celsius for a maximum of three hours after tumor resection.
Sterilize the tissue culture hood with 70%ethanol and sterilize steel INOX tweezers with 70%ethanol. Remove the paraffin film from the urine container and discard. Clean the exterior of the urine container with 70%ethanol.
Open a square Petri dish, then open the urine container and use the sterilized steel INOX tweezers to transfer the tumor specimens into the dish. Add PBS and wash the tumor specimen twice. After washing, finely shred the tumor specimen into pieces of one to two millimeters in size.
Transfer one-quarter of the total sample to a two milliliter tube. Then add one milliliter of TRIzol reagent to the tube and immediately freeze at minus 80 degrees Celsius. Collect the rest of the finely shredded tumor material in a 15 milliliter tube and add four milliliters of collagenase type one solution.
Then dilute the collagenase type one solution with four milliliters of culture medium, close the tube, and seal with paraffin film. Incubate at 37 degrees Celsius on a rolling shaker for 15 minutes to activate the digestion. After 15 minutes, place the tube with the rolling shaker at room temperature and leave it overnight.
The following day, gently filter the cell suspension through a 100 micron sterile filter into a 50 milliliter tube. Then wash the filter with culture medium and centrifuge the filtrate at 225 times g for five minutes at room temperature. Discard the supernatant by inverting the tube and resuspend the cells with one to two milliliters of culture medium.
After performing a cell count and calculating the cell number, plate the cells in a standard monolayer culture at a density of 80, 000 cells per square centimeter. To prepare a cytological sample, begin by collecting 100, 000 cells in 200 microliters of culture medium. Pipette the solution into a disposable funnel equipped with a filter and mounted with a glass slide.
Open the cytocentrifuge and insert the samples. Following cytocentrifugation, discard the funnel equipped with the filter. Then fix the slide in acetone for 10 minutes, followed by chloroform for five minutes.
Passage the STS cells when confluence is around 90%After washing the cell sheet with PBS, add two to three milliliters of 1X Trypsin in PBS and incubate for three to five minutes at 37 degrees Celsius. After the incubation, use a 200 to 1, 000 microliter pipette to collect the detached cells and transfer to a 15 milliliter tube. Add four milliliters of medium to stop the enzymatic reaction.
Then centrifuge at 225 times g for five minutes at room temperature. After discarding the supernatant, resuspend the cell pellet by adding one milliliter of medium and seed the cells into a new culture plate or a flask. Tumor cells were isolated from a liposarcoma surgically removed from a 54-year-old patient.
Fluorescent in situ hybridization was performed to detect MDM2 amplification in the patient tissue specimen. Cytological analysis of MDM2 amplification in isolated tumor cells confirm the establishment of a patient-derived primary culture. Morphological changes from base were induced when cultures were treated with Ifosfamide, Epirubicin, Ifosfamide and Epirubicin, Trabectedin and Eribulin.
Three independent replicates were performed for each viability experiment. The results confirmed the sensitivity of the cultures to chemotherapy. The most active treatment was combined Ifosfamide and Epirubicin, a first-line therapy used in advanced or metastatic ALT-DDLPS.
Once mastered, this technique can be done in 15-16 hours if it is performed properly. While attempting this procedure, it's important to remember to finely shred the tumor specimen. Following this procedure, a variety of downstream analysis including gene expression profiling, immunohistochemical analysis, or cytotoxicity assay can be performed in order to answer additional questions about soft tissue sarcoma biology.
After its development, this technique paved the way for researchers in the field of soft tissue sarcoma to study the natural history of these poorly explored malignancies. After watching this video, you should have a good understanding of how to establish a primary culture of patient-derived soft tissue sarcoma. Don't forget that working with biological samples and drugs can be extremely hazardous and precautions such as wearing gloves and working with a fume hood should always be taken while performing this procedure.
