The overall goal of this procedure is to build a 3D tumor test system with primary cells on a decellularized biological scaffold. This is accomplished by first preparing the scaffold using a decellularization process in which a porcine al segment is pumped with decellularization solution. The second step is to isolate primary human cells from a patient's biopsy using a standard isolation protocol.
Next, set up the tumor test system by cutting the tubular small intestinal submucosa open on one side, fixing it between two metal rings and seeding with tumor cells and associated stromal cells in defined cell densities. The final step is to culture the cell loaded construct in a suitable setup under static or dynamic conditions. Ultimately, immunohistochemical microscopy is used to assess the characteristics of tumor growth.
The main advantage of this technique over existing methods like TT tumor test systems, is that with this method, it's possible to create 3D tissue models that simulate the in vivo conditions of a tumor more accurately than common 2D models. The advantage of the dynamic culture is that cell specific characteristics Are maintained. This method can help answer key questions in the tumor biology field, such as how cancer cells form cell cell and cell matrix interactions in a 3D environment, which will help elucidate cancer related processes such as tumor progression and metastasis.
The biological vascularized scaffold or biova used in this tumor test system is derived from a porcine jaal segment. Rinse the vascular system of the porcine al segment and the intestinal lumen with PBS via cannulated arterial access. Repeat until it is completely clean.
Prepare a 200 millimeter diameter glass tank with four adapters and connect them via silicon tubes to the peristaltic pump. The pressure controlling unit can be monitored via a pressure sensor that is connected to a sterile disposable dome. Fill the reservoir bottles with decellularization or DZ solution.
Check the tubing system for possible air bubbles. Connect the intestinal lumen with cable ties to the glass connectors for luminal flow. Pump 500 milliliters of DZ solution into the red arterial access of the vascular system.
Interrupt the pumping process every 15 minutes shortly to manually press out the entire intestinal lumen. It is important to monitor the pressure of the buffer solution during the decellularization process. The pressure should be between 80 to 100 millimeters of mercury modeled after the natural blood pressure.
Wash the biova with PBS until it is free of cell remnants and the vascular structure is completely white in color. Begin this procedure by cutting the skin biopsy into strips of two to three millimeters in width with a scalpel and rinsing them three times with PBS solution. After incubating the tissue with disbe solution, use two tweezers to separate the epidermis from the dermis.
Transfer both separately into Petri dishes filled with PBS to isolate primary dermal microvascular endothelial cells or MV eecs rinse dermis strips Once with ene, add 10 milliliters trips in EDTA solution to the dermis strips and incubate at 37 degrees Celsius for 40 minutes. Stop the enzyme reaction with 1%FCS and transfer skin strips to a Petri dish filled with vascu life. Scratch out each strip with the scalpel eight times on each side, adding a little pressure to produce a cell suspension.
Subsequently centrifuge the cell suspension and resus, suspend the cell pellet with vascu life. To isolate fibroblasts. First, use a scalpel to chop the dermis strips into little pieces.
Transfer the dermis pieces to a Falcon tube and add 10 milliliters of collagenase solution incubated 37 degrees Celsius for 45 minutes. Next, centrifuge the solution and carefully remove the supernatant. Wash the pellet with DMEM plus 10%FCS plus 1%pen strep.
After centrifugation, carefully remove the supernatant resuspend the pellet in culture medium, and transfer to a T 75 culture flask to allow cells to grow out of the tissue incubate under static conditions. To set up the tumor test system first, cut the tubular small intestinal submucosa or SIS muc, open on one side and fix it between two metal rings. These self-constructed cell crowns have a diameter of 10 millimeters.
Cover the SIS mu in cell culture medium overnight on the following day, seed primary MVE CS onto the basal later surface of the SIS, the former CI.Incubate at 37 degrees Celsius, 5%carbon dioxide for three hours. Three hours later, fill the well with medium to ensure submersed culture and return it to the incubator. Allow endothelial cells to adhere for three days after three days.
Flip the static culture system by 180 degrees and transfer it to a 12 well plate. Next, see a mixture of primary dermal fibroblasts and tumor cells on the apical surface of the SIS. The side of the former lumen.
Allow cells to adhere for three hours. Fill the well with medium to submerse the culture culture, the tumor test system under static conditions at 37 degrees Celsius, 5%carbon dioxide for an additional 14 days, changing the culture medium every two to three days for the dynamic culture. Fix the SIS mu between two metal rings and seed primary MV eecs as demonstrated earlier.
After three days, remove the SIS mu from the metal rings and insert the membrane into the flow reactor with a syringe and cannula. Apply the primary dermal fibroblasts and tumor cells onto the matrix in the bioreactor. Allow the cells to adhere for three hours before filling the bioreactor system with culture medium on the following day, place the bioreactor in a self-constructed incubator system and connect it to a peristaltic pump.
This setup allows the dynamic culture with either pressure regulated pulsatile flow or constant flow. In this experiment, a constant medium flow of 3.8 milliliters per minute is used. Maintain the dynamic culture for 14 days, changing the culture medium after seven days.
At the completion of the experiments, tissues are fixed, paraffin, embedded, and stained, and then imaged using an inverse microscope. Representative results are shown here. Panel A gives an overview of the statically cultured S 4 62 tumor cell line in 2D monoculture stained with hemat tolin.
The 3D co-culture is shown in panels B, C, and D.This H and e stained image shows the triple culture of tumor cells S 4 62 and primary fibroblasts on the apical side of the SIS MU and MVEC. On the basal lateral side, the arrows mark the endothelial cells. Different cell types can be identified by staining cell type specific markers such as von Willebrand factor to label MVEC shown in panel C and P 53 shown in panel D.The P 53 positive S 4 62 cells can be distinguished from the P 53 negative primary fibroblasts, and the 3D distribution of cells can be analyzed in the same way.
Different cell types can be identified in the dynamically cultured triple culture panel A is an H and E stained image where the arrows mark the endothelial cells. Panel B shows immuno histological staining for von Willebrand factor, and panel C shows staining for P 53 Following this procedure. Other methods such as drug tests can be performed in order to answer additional questions like how to find new cancer therapies or the analysis of important signaling pathways in tumorgenesis.
Also, primary cells can be used to define the best personalized treatment for individual patients.
