The overall goal of this procedure is to demonstrate the fabrication and operation of a microfluidic device that recreates tumor microenvironments in vitro and can be used for testing chemotherapeutic drugs. This is the microfluidic device. This device can be useful in the following areas, easy testing for chemotherapeutic drugs in vitro.
It provides consistent and accurate results for testing three dimensional tumor tissue, and this device provides a very accurate representation of in vivo tumor. Microenvironments tumors exist in 3D microenvironments and the ability to test in one is significant. The first step of the procedure is to assemble the device, which involves fabrication of microfluidic chip using soft lithography, punching holes at the inlets and outlets, bonding to a glass slide and connecting tubing for flow inlet and outlet.
Next, the uniform multicellular tumor steroids are generated for use in the device using the hanging drop method. The spheroids are then introduced into the device, followed by apoptosis, detecting agents and chemotherapeutic drugs. Finally, time lapse microscopy is performed, followed by mathematical estimation of drug diffusivity coefficients to quantify the penetration ability of the chemotherapeutic agent.
Ultimately, results can be obtained that show tissue growth in the device formation of heterogeneous microenvironments within the tissue and drug diffusion into the tissue through the use of time lapse fluorescence microscopy. Limited penetration of cancer drugs into solid tumors is an important cause of their failure. Most current cancer drug testing models do not mimic the three dimensional nature of solid tumors.
We have developed a microfluidic device consisting of continuously perfused three dimensional tumor tissues. This model more accurately represents the delivery of drugs to solid tumors and their systemic clearance. Multicellular tumor steroids are a well-established model of three dimensional tumor tissues.
In this procedure, the hanging drop method is used to create steroids for use in the microfluidic device. This method allows formation of consistent steroids in a short period of time. Testing chemotherapeutic drugs in vitro in three dimensional tissue can help screen for drugs in a more efficient manner compared to monolayers of cells.
Mix the PDMS and curing agent from the silicon elastomer in a nine to one weight ratio. Then pour this mixture over the master to form a four millimeter thick glare Degas. To remove air bubbles cure at 60 degrees Celsius for five hours.
Peel the cured PDMS from the mold to obtain a stamp of flow features on the elastomer. Then using a one And a half millimeter biopsy punch mounted on a drill, press punch holes for inlets and outlets. Pull out any debris.
Subject the feature side of the stamp and a clean glass slide to oxygen plasma for eight minutes. In an oxygen plasma etcher, bring the treated surfaces in contact immediately to form a bond between them. Maintain the assembly at 60 degrees Celsius on a slide warmer for at least five hours.
To strengthen the bond connect 0.032 inches ID PTFE tubing to the inlets and outlets of the microfluidic device. Using an interface of male lure lock connectors attached to barbed female lure lock connectors, these specific connectors are used to allow to easily pass through them without breaking. Finally, set up the flow assembly using shutoff valves and a Y connector, mount the device onto the microscope After mounting the device, connect tubing to appropriate inlets and outlets to complete the flow setup.
As shown in this figure, this configuration allows for the ease and versatility of packing a spheroid into the device or create a flow of medium through the device to pack. Open the packing inlet valve VPN and the packing outlet valve VP out a tumor. OID can then pass through the inlet and flow into the culture chamber where the post in this chamber will hold the tumor in place to flow medium.
Close the packing valves and open the flow inlet valve VF in and the flow outlet valve VF out to initiate the formation of uniform PHE. Using the hanging drop method trypsin ice cells under a culture hood. Take the resulting trypsin cells from the centrifuge.
Dilute the stock solution to a desired concentration by referring to table one. Next, remove the cover of a 48 well plate and place it upside down. In a sterilized hood, fill each well of the plate with one milliliter of sterilized water.
To maintain humidity, put a 20 microliter droplet of dilute cell solution in each circular region on the cover. Using a micro pipette, carefully invert the cover and place it over the plate, ensuring that drops Do not touch the edges of the wells. Incubate the plate at 37 degrees Celsius.
Refer to table one for the duration of the incubation based on the particular cell used, a steroid will form in Each hanging drop. Start By sterilizing the device by flushing it with 70%ethanol through the flow inlet. Follow with a flush each of PBS, then H-E-P-E-S buffered cell culture medium.
Leave the medium syringe, attach to the tubing. Next, draw two to three Steroids into the syringe tap syringe. To remove Air bubbles, Attach the syringe to the packing inlet of the device.
Open the inlet valve VPN and close VFN open outlet valve VP out and close VF out. Hold the packing syringe vertical with the needle pointing downward. Watch as the steroid settle to the bottom of the syringe into the lure lock connector of the needle.
Push the plunger on the packing syringe and watch PHE enter the tubing and flow into the device because only the packing outlet valve is open. A steroid will enter the chamber of the device and be retained by posts at the back. Once inside the chamber, the S spheroid conforms to the shape of the chamber and acquires a rectangular geometry.
Close inlet valve VPN and out outlet valve VP out mount the flow syringe SF on the syringe pump. This pump is run by a computer using syringe pump pro open inlet valve VFN and outlet valve VF out. Begin the flow of medium into the device at Three microliters a minute.
Incubation Of spheroid for 24 hours is followed by introduction of apoptosis detecting agents. Before introducing apoptosis, detecting, or therapeutic agents, allow equilibrium of spheroid for up to 24 hours to establish nutrient gradients and microenvironments. Then shut off valve VFN.
Stop the syringe pump. Remove syringe from pump and replace it with the syringe containing medium with 0.25 microliters per mil of CASP glow red active Caspase three marker. Wash the device with the solution by manually flushing 0.7 milliliters of it through the device.
Then mount the syringe on the pump. Restart the flow and open valve VFN. Over a period Of five to eight hours, cask glove red active cph phase three will diffuse into the tissue and fluoresce brighter in apoptotic regions apoptosis detecting agent at this concentration is maintained in all subsequent Flow solutions.
Doxorubicin Is introduced five to eight hours after introduction of apoptosis detecting agents. This treatment is cleared away from the system by flushing. To add chemotherapeutic agents, follow the procedure for apoptosis detection to introduce 10 micromolar doxorubicin hydrochloride.
Then replace the treatment syringe with the syringe with 0.7 milliliters of medium. Continue the flow of the therapeutic agent for a fixed period of time. Then shut off valve VFN and turn off the syringe pump.
Remove treatment syringe from the syringe pump and replace with the syringe with 0.7 milliliters of medium. Allow the flow of fresh medium for 24 to 36 hours while continuously monitoring the tissue under a microscope. The Entire acquisition process for time lapse microscopy was automated using a customized script in IP lab, acquire transmitted light and fluorescence images of the PAX Foid at 10 x magnification every 30 minutes For mathematical estimation of drug diffusivity coefficients, the first step is to generate average linear intensity profiles of docs fluorescence using image J.Select a rectangular region of interests, or ROI encompassing the tissue in the chamber.
Use the plot profile command to generate a profile of average intensities as a function of distance from the flow channel. Repeat for up to three different time points. Next, subtract the average background fluorescence intensity from the obtained intensity profiles and normalize each profile using the corresponding maximum intensity to obtain C as a function of X and T.Where C is the normalized concentration of doxorubicin, the value of C varies between zero to one.
The next step is to evaluate the effective diffusion coefficient of docs within tumor tissue docs. Diffusion can be represented by the following equation where ERFC is the complementary error function. X is the distance into the tissue from the channel, and T is the time after introduction of doxorubicin.
Use the following iterative scheme at each time point considered. Guess a value for D.Calculate right side of equation at each location. X.Calculate the sum of squared errors between the two sides, modify D to minimize the residual average.
The optimum values of D obtained at each time point to estimate the average effective diffusion coefficient of doxorubicin in the tissue. The microfluidic devices provided a one millimeter by 0.3 millimeter by 0.15 millimeter optically accessible culture chambers for growth of three dimensional tumor tissue. The hanging drop method allowed quick formation of tumor PHE of consistent size and shape from several cell lines.
PHE were successfully grown on the device for up to three days. Growth in the chambers was associated with the reproducible modification of microenvironments. Within phe, apoptosis occurred less in cells in close proximity to the flow channel and higher and deeper into the tissue.
The device was used to estimate the diffusion coefficient of doxorubicin and tumor tissue. The experimentally tained value agreed with the value reported previously in human breast cancer While attempting this procedure. It's important to be extra cautious of maintaining sterility when flowing PHE into the device because it has to be performed outside of cell culture.
Hood spray syringes and ends of tubing with ethanol generously to ensure sterility. After watching this video, you should have an understanding of the role of limited penetration of drugs into tumors. We have demonstrated a technique to mimic the delivery and systemic clearance of drugs on three dimensional tumor tissues and quantify their penetration.
Eliminating poorly penetrating drugs will significantly streamline the process of cancer drug development.
