The overall goal of this procedure is to produce modular micro tissues that can be used for tissue engineering experiments. This is accomplished by first pulling the collagen solution into a polyethylene tubing, using a syringe, and then gelling the collagen. The second step is to cut the polyethylene tubing into two millimeter lengths to form the micro tissue modules.
The third step of the procedure is to separate the modules from the tubing, and lastly, the modules are coated with endothelial cells. Ultimately, the resulting micro tissues can be used for in vitro cellular assays to study the interaction of cells via a microfluidic chamber. In addition, the micro tissues can be implanted to generate tissues for studying the interaction and remodeling of the micro tissues in vivo.
Hi, my name is Brendan Long from the Sepan Lab here at the University of Toronto. A major obstacles in creating large 3D tissue substitute is a lack of rapid vascularization after implantation. In this video, we will show you how to create modular tissue coated with endothelial cells to help overcome this limitation.
Demonstrating the procedure will be deemed Chamberlain, a post-op fellow from the Septin lab. The amount of collagen to neutralize will depend on the length of the polyethylene tubing. In this demonstration, we'll use a two meter polyethylene tubing with an inner diameter of 0.76 millimeter and outer diameter of 1.22 millimeter.
To prepare the tubing, thread a 20 G one needle into one end of the tubing, and then place the tubing in a converter self-heal pouch for gas sterilization. Since one milliliter of collagen fills about two meters of polyethylene tubing, we will neutralize one milliliter of collagen. Place the collagen into a 15 milliliter conical tube and keep the tube on ice.
Add 128 microliters of 10 x alpha, MEM per milliliter of collagen to the tube and mixed by repeatedly pipetting the solution. Taking care to minimize making bubbles, the solution should turn yellow as the alpha MEM is mixed into the acidified collagen. Next, neutralize the collagen by adding 0.8 molar sodium bicarbonate For a general estimate of the pH, add sodium bicarbonate until the yellow collagen solution turns a light pink color.
It typically takes 30 to 60 microliters per milliliter of collagen to achieve a pH of 7.4. To prevent the neutralized collagen from gelling, keep it on ice until it is ready to be used. To begin the procedure for jelling the collagen, take the bag containing the sterilized polyethylene tubing to a biological safety cabinet and cut off the sealed end of the bag while keeping the tubing in the bag.
Use sterilized tweezers to attach a three milliliter syringe to the 20 gauge needle that was previously inserted into one end of the tubing. Insert the other end of the tubing into the neutralized collagen until the tip is at the bottom of the conical tube, which is kept on ice. This next step you have to do slowly, otherwise, you introduce air bubbles into the collagen.
Retract the plunger of the syringe slowly to pull the collagen into the polyethylene tubing. When the collagen has been pulled through the tubing, pull the 20 gauge needle out and put both ends of the tubing back into the bag. After taping the bag shut, incubated at 37 degrees Celsius for 60 minutes.
A preassembled cutting device will be used to cut the tubing. To begin this procedure, place tubing in a sterile tray in front of the cutting device and load the tubing into the cutting device at the outlet of the cutting device, set up a 50 milliliter conical tube containing 25 milliliters of endothelial cell media. To collect the cut modules, set the cutting device to cut two millimeter lengths of tubing and then cut the tubing.
Incubate the cut modules in the 50 milliliter conical tube at 37 degrees Celsius for 60 minutes. To remove collagen modules from the tubing, vortex, the 50 liter conical tube containing the modules at medium speed. For 10 seconds, let the module settle to the bottom of the tube, which takes about five minutes.
A loose pellet of modules will form at the bottom of the tube while the tubing will form a layer on top of the media. After the modules have settled, use a wide mouth, 10 milliliter serological pipette to transfer them to a 15 milliliter conical tube. Vortex, the 15 milliliter conical tube, and then allow the modules to settle for two more times.
The settled modules from the second and third vortex are also transferred to the 15 milliliter tube. After all the modules have been removed from the tubing and pooled, bring the volume of media in the tube to five milliliters. The modules are now ready for coating with endothelial cells, which will be shown next to prepare endothelial cells for coating the modules.
First, remove the media from the cells and wash with PBS. After removing the PBS, add three milliliters of trypsin EDTA and incubate for five minutes at 37 degrees Celsius. Next, resus, suspend the cells in seven milliliters of media and transfer to a 15 milliliter tube.
Count the cells using a hemo cytometer, you will need five times 10 to the sixth endothelial cells per milliliter of packed modules at the bottom of the conical tube. Pellet the cells at 300 G for five minutes. Resuspend the cell pellet in five milliliters of endothelial cell media.
Now add five milliliters of media containing endothelial cells to the modules. The modules are incubated with the endothelial cells, both statically and dynamically for static incubation, mixed modules with endothelial cells by inversion, and then set the tube upright at 37 degrees Celsius for 15 minutes. After 15 minutes, invert the tube again and incubated 37 degrees Celsius for another 15 minutes.
To incubate the modules with the endothelial cells dynamically, gently rock the 15 milliliter tube at 37 degrees Celsius for 60 minutes. Once incubation with the endothelial cells is complete, transfer the modules to a non tissue culture treated plate and incubate overnight at 37 degrees Celsius on the following day, place the modules in a new non tissue culture treated plate with fresh media to remove unattached endothelial cells. Incubate modules that are coated with endothelial cells at 37 degrees Celsius until the cells cover 100%of the surface of the modules.
This usually takes about seven days. The modules will look cylindrical when they're first removed from the tube. If they're coated with endothelial cells, they can contract up to 50%in volume and develop an oval shape, as well as become denser and more opaque when viewed by light microscopy.
In these images, the diameter and length of the modules are indicated by d and l respectively. This next image shows embedded Hep G two cells uniformly distributed within modules at the time of fabrication. The cells retained high viability as indicated by the green color of live cells.
Dead cells would be red in color when the endothelial cells are confluent. On the surface of the modules, there's a formation of tight junctions, which can be seen by immunofluorescence. Staining of VE cadherin mass transfer analysis has demonstrated that modules are capable of supporting high cell densities without developing a necrotic core due to inadequate oxygen transport, which is often problematic in larger tissues.
In this example, massen Tri chro staining of typical small modules show that the modules retained a uniform and high distribution of live cells seven days following fabrication. In contrast, a large number of dead cells had formed within the core of the large modules leaving only a thin rim of viable cells. Modules have also been produced with a mixture of collagen and a ine polymer or containing drug eluting microspheres.
Here when h hm EC one cells are seated on palin collagen modules, the modules retain their cylindrical shape and there is limited cell attachment properties compared to collagen only modules. A light microscope image of a collagen module containing PLGA based biodegradable microspheres is shown here. Several in vitro assays have been shown to be compatible with modules.
For example, in an angiogenesis assay, capillary like formations on a module seated with endothelial cells can be easily detected and quantified after five days of incubation with adipose derived stem cells. In another example, confocal microscopy images of a live dead assay on modules shows live cells stained green and dead cells stained red. Finally, modules have also been used in microfluidic chambers and implanted into mice and rats to study the interaction between different cell types and how the micro tissues are remodeled by the host response.
Following the procedure, the modules can be cultured in a Petri dish or other vessels such as a bioreactor or a microfluidic device. This method can also be adapted to create different tissue by using various combinations of cell type or using tubings with different diameter. Thanks for watching.
