Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The overall goal of this protocol is to prepare an injectable chitosan hydrogel for three-dimensional cell culture to ultimately use in therapeutic treatments. This method can answer question in the field of self-healing hydrogel, such as how to make a self-healing hydrogel by immersing whole way and how to use it for 3D cell culture. The my advantage of this technique is, by compatible self-healing hydrogel can be prepared by a very simple way, and the use of a 3D cell culture.

This hydrogel is also injectable, so it can be used as a carrier for cell therapy. Now Mr.Yongsan Li will show you how to make the hydrogel and how to do the 3D cell culture using this hydrogel. Yongsan Li is our graduate student of our lab.

To make the hydrogels, first synthesize DF PEG. In a fume hood weigh out four grams of molecular-weight 4, 000 PEG into a round-bottom flask, and add 100 milliliters of toluene. Then, to dissolve the polymers, warm up the solution.

After all the polymers dissolve, use an evaporator to remove all the solvents. Then, repeat the process of mixing in toluene and evaporating it twice more to prepare the dry PEG polymers. Next, using a magnetic stirrer and 100 milliliters of DHF, dissolve the polymers.

Then, dissolve 0.9 grams of 4-Carboxybenzaldehyde into the solution. Next, dissolve 0.07 grams of dimethylaminopyrimide into the solution. Now add 1.25 grams of DCC in a drying tube filled with anhydrous calcium chloride.

Now allow the reaction to stir at room temperature for around 12 hours. The next day, use 500 milliliters of cold diethyl ether to precipitate the white solids, and use vacuum filtration to collect the solids. Discard the solute, and dry the solids under good ventilation.

Next, dissolve the white solids in 100 milliliters of THF. Any insoluble white solids must be filtered out before proceeding. Now precipitate the dissolved solids using fresh cold diethyl ether.

Then use vacuum filtration to collect the solids, and let them dry out again. Repeat this process two to three times, and then place the white solids in a vacuum-drying oven to dry them completely. The resulting white powder is the final product:benzaldehyde-terminated DF PEG.

To encapsulate the cells in the hydrogel for culturing, first dissolve 0.44 grams of DF PEG into two milliliters of cell-growth media in a four-milliliter tube. Vortex the mixture into a 20%solution by weight. Next, sterilize the solution.

Load it into a 10-milliliter syringe, and press it through a 0.22-micron bacteria-retentive filter. Now mix 0.165 grams of the glycol-chitosan with four milliliters of cell-culture media, using a vortex to make a 4%by-weight chitosan solution. Then, sterilize the solution using another 0.22-micron bacteria-retentive filter.

Next, harvest the cells, such as L929 cells using trypsin. Wash the collected cells, and resuspend them in media at 3.75 million cells per milliliter. In the following step the cells are planted into a 3D matrix.

Following these steps precisely is critical to produce success. Now mix 0.4 milliliters of the cell suspension with 0.4 milliliters of chitosan solution using a vortex. Pipet this mixture into the center of a two-centimeter-diameter petri dish, and pipet 0.2 milliliters of the DF PEG solution into the dish.

Then, use gentle pipeting to induce the formation of the hydrogel. Assess the hydrogel's formation by tilting the dish and observing the solution's viscosity. To test the injectability of the encapsulated cells, make the same preparation in a 10-milliliter syringe.

Then, after the hydrogel forms, slowly expel the hydrogel through a 48-gauge needle into the petri dish. Next, add one milliliter of culture media to the hydrogel-cell mixture in the plate, and transfer the plate to an incubator for culturing. Every other day, change the media, overlaying the 3D hydrogel cell culture.

To prepare the cells encapsulated in hydrogels for imaging, first rinse the hydrogels with one milliliter of PBS two times. Then, stain the hydrogels with fluorescein-diacetate solution and propidium iodide solution. Overlay 0.5 milliliters of both solutions onto the hydrogel, and incubate the hydrogel for 15 minutes at room temperature.

After the brief incubation with the stain solutions, aspirate away all the solvents. Now observe the hydrogels using confocal microscopy, with excitation at 488 nanometers to view living cells, and excitation at 543 nanometers to view dead cells. Collect image stacks with two-micron slices to validate the distribution of cells in the hydrogel.

L929 cells. A typical mouse-fiberglass cell line with an in-vivo environment tissue stiffness of about 5, 600 Pascals were encapsulated in a hydrogel. Their viability was extremely high throughout the culture process, and they achieved a remarkable cell density.

After seven days of culture in the hydrogel, the cell number increased by 200%Hydrogels that were expelled onto their cell-culture dish through a 48-gauge needle could self-heal to reform an integrated hydrogel after about one hour. After seven days in culture, these cells also proliferated dramatically. Evidence could be found of cell-fission events in which some cells appeared divided into two.

The live-dead assay showed very high viability, demonstrating the successful cell proliferation in the 3D hydrogel after injection. This cell-proliferation rate was about 3/4 that of uninjected cells after seven days of culturing. So, although the sheering force during injection has a measurable negative effect, the injected cells were still quite proliferative.

After watching this video you should know how to prepare a self-healing hydrogel and how to use this hydrogel for 3D cell culture and how delivery the cell with this hydrogel. Once mastered, this technique can be done within one hour if performed properly. Following best procedure, other method, like in-vivo injection for cell therapy or drug delivery, can be performed.