The overall goal of this procedure is to reproducibly create bi-layered alginate hydrogel discs for investigating the relationships between different cell populations during 3D co-culture. This method facilitates the study of the interactions between multiple cell populations in spatially heterogeneous 3D cultures, which can help answer key questions in tissue engineering and the physiology of load bearing tissues. The main advantage of this technique is that it offers a straightforward, economical, and reproducible way to construct cell-seeded, multi-layered alginate hydrogels that are structurally conducive for mechanical stimulation.
Demonstrating this procedure will be Poonam Sharma, a graduate student from my laboratory. To form the alginate discs, first stack a piece of thick filter paper on top of a three by three inch end plate, followed by a cell microsieve membrane. Use a sterile spatula to flatten the membrane and filter paper.
Then, invert the stack onto a pile of paper towels and compress the materials to remove the excess calcium chloride solution. Next, place a gel formation mold with cylindrical wells on top of the microsieve membrane, and use binder clips to gently fasten the mold to the end plate on the left and right sides. Now, stack a piece of thick filter paper onto a small end plate, followed by a piece of microsieve membrane, and invert the stack to remove any excess calcium chloride, as just demonstrated.
When both of the mold halves are ready, re-suspend one times 10 to the sixth cells of interest per milliliter of sterile alginate medium. The mixture will appear homogeneously cloudy when the cells are appropriately dispersed. Then, transfer 130 microliters of cells dropwise into individual six millimeter diameter by three millimeter tall wells in the bottom half of the mold construct.
A slight convex meniscus should be visible above the edge of each well. When all of the cells have been seeded, use a sterile spatula to carefully smooth the top half of the mold construct. Then, turn the mold over so that the microsieve membrane is on top of the wells, and place the mold on top of the wells, taking care to cover the wells containing the cell and alginate mixtures completely.
When the mold is in place, lift the loaded mold construct and pressing firmly on the center, place binder clips on the remaining two sides of the construct to fasten the top and bottom halves of the mold. The cell alginate solutions should be securely nestled in the wells. Next, immerse the entire mold construct in a 102 millimolar calcium chloride bath in a cell culture hood at room temperature for 90 minutes.
At the end of the incubation, transfer the construct onto a stack of paper towels and remove the two binder clips securing the top end plate to the mold. Separate the two halves of the construct. The hydrogels formed in the wells should not have any bubbles, and should fill the wells completely.
Remove the remaining two binder clips and separate the hydrogel-containing mold from the remaining end plate. Using a spatula, carefully trace the edge of the wells containing the hydrogels to carefully loosen the gels. Then, transfer the hydrogels directly into a bath of DPBS with calcium chloride and magnesium chloride.
After one to five minutes, transfer the hydrogels into individual wells of a six well plate containing enough basil growth medium solution to fully immerse the gels for at least one hour at 37 degrees Celsius and five percent CO2. During the last 30 minutes of the incubation, use binder clips to fasten the mold with wells half of the height of the gel formation mold to the three by three inch or three by 1.5 inch aluminum end plate on the left and right sides to create a cutting mold. Then create the annealing mode by clipping the mold with three millimeter larger in diameter wells than the gel formation mold to three by three inch aluminum plate end plates on the left and right sides.
Now, transfer the hydrogels into the cutting mold wells. Each gel should fit snugly into the well with half of the gel protruding above the mold. And use a scalpel to slice along the surface of the mold to cut the hydrogel in half.
Flip the top half of the gel and place it into an open mold well. The gel piece should fit snugly into the well, but now both halves should be the height of the mold, with the cut inner surface visible. Next, place a piece of dry cell microsieve membrane over the wells, making sure that the membrane is in contact with all of the gel halves to be annealed, and cover the membrane with a piece of thick filter paper.
When the gels are completely covered, saturate the filter paper with sodium citrate supplemented with EDTA, and incubate the gels at room temperature. After one minute, discard the cell microsieve membrane and thick filter paper, and remove the binder clips. Then, open the mold and transfer a gel half into a prepared annealing mold with the cut surface facing upwards.
Using a spatula, transfer a second treated half gel onto the first gel, such that the cut surface is in contact with the cut surface of the first gel. When all of the gels have been placed, use a spatula to press down gently on the gels to remove any bubbles, and carefully submerge the annealing mold in the calcium chloride bath. After 30 minutes, transfer the annealing mold onto a stack of paper towels and remove the binding clips.
Then, separate the mold from the end plate and transfer the gels briefly into a DPBS bath, followed by their immersion in the appropriate cell culture medium. Completed bilayer gels exhibit a total initial separation of the cell populations, as observed in this image of 293 FT cells labeled with green or red cell tracker dyes. The viability of human mesenchymal stem cells embedded after annealing is high within layered hydrogels, similar to the cell viability observed in bulk hydrogels.
After isolating the peak stress from each cycle over a four hour unconfined cyclic compression, cell-free layered hydrogels exhibit a stress response to the cyclic compression that is significantly smaller in magnitude compared to bulk non-layered cell-free hydrogels, even though both types of hydrogel remain intact after seven days in culture. Once mastered, this technique, including the preparation of all the materials, can be completed in less than four and a half hours if performed properly. While attempting this procedure, it's important to work deliberately and slowly to prevent any errors, and to more easily keep track of the different samples.
Following this procedure, other methods like mechanical stimulation and immunostaining can be performed to answer additional questions regarding the interactions between the different cell populations in the extracellular matrices that may form. This technique will allow researchers in the field of mechanobiology and tissue engineering to more easily investigate the relationship between different cell populations, such as mesenchymal stem cells, chondrocytes, and nucleus pulposus cells. Don't forget that working with sharps, biological materials, and especially human cells, can be extremely hazardous, and that precautions such as wearing the appropriate personal protective wear should always be taken while performing this procedure.
