The overall goal of this procedure is to demonstrate the simple fabrication, manipulation, and assembly of modular hydrogel sheets for a 3D cell culture system. This method can help answer key questions in the tissue engineering field such as how to create more in vivo-like functional engineered tissues with cultured micro-environments. The main advantage of this technique is that cellular hydrogel constructs can be generated without any costly increment.
The 3D cell culture conditions will depend on each modular hydrogel sheet. The implications of this technique extend toward improved cell-based assays and biological studies because the technique can provide various geometries and composition of 3D cellular micro-and macro-environments. Though this method can provide insight into in vitro cell-based assays, it can also be applied to another system such as transplantable scaffold of 3D tissue model.
Begin by producing the desired microscale patterns using standard photolithography techniques. The example shown in this video will use a liver lobule-like mesh pattern like the one shown here. Next weigh out 2.5 grams of PDMS and 2.5 grams of the curing agent solution and mix the components together thoroughly.
De-gas the solution in a vacuum chamber and then spread the mixed solution onto the micropattern surface of the silicon wafer evenly within a foil casting dish. Next, cure the PDMS by placing the dish onto the flat surface of a 65-degree Celsius heated plate for 90 minutes. Once cured, remove the PDMS from the casting dish and carefully peel it away from the silicon wafer.
Cut away the edges of the PDMS and place the patterned portion onto a 100-millimeter diameter Petri dish so that the micropattern side faces up. Then, wash the micropatterned PDMS with 70%ethanol, followed by distilled water, for primary sterilization. Afterwards, dry the setup completely for 10 minutes in an oven at 65 degrees Celsius.
Next, place the micropatterned PDMS mold into a plasma cleaner and clean them for one minute in order to create a hydrophilic surface. This will facilitate the loading of aqueous liquids. Then coat the surface of the PDMS with 100 milliliters of the surfactant solution listed in the Table of Materials.
Incubate the samples on a laboratory rocker for at least three hours. Next, wash the surfactant solution from the PDMS molds using sterile water, and dry them completely in an oven at 65 degrees Celsius for 10 minutes. Then, sterilize each micropatterned mold by exposing it to ultraviolet radiation for 30 minutes.
Begin by culturing hepg2 cells using standard techniques. Once the cells are 70%to 80%confluent, wash the cells once using PBS. Then, harvest the cells by adding one millileter of 05%tripsin edta and incubating them for four minutes at 37 degrees Celsius.
Once detached, collect the cells in a centrifuge tube and pellet them at 250 g for three minutes. Remove the supernatant and then resuspend the cells in one millileter of PBS. Count the number of single distributed cells in PBS using an automated cell counter.
Then, pellet the cells again at 250 g for three minutes. Remove the PBS supernatant and add enough of a one percent sodium alginate solution to the cells so that the final seeding density is between five and ten million cells per millileter. Mix the cell solution gently using a pipette and then incubate the cell hydrogel suspension in a five percent CO2 humidified incubator at 37 degrees Celsius.
While the cells incubate, place the micropatterned PDMS molds into a plasma cleaner and clean them for one minute at 85 watts to create a hydrophilic surface. Mix the cell hydrogel suspension by gently pipetting up and down and then steadily load 7.2 microliters of the suspension at the edge of the micropattern in the mold. Then, gel the cell hydrogel suspension by producing a mist of the cross-linking reagent with the humidifier at a rate of 250 milliliters per hour and spraying it onto the hydrogel precursor for five minutes.
Ensure that this procedure covers the topographic surface of the PDMS mold within a range of five centimeters. Following the cross-linking process, turn off the humidifier and fill the PDMS mold with PBS. Using a 200-microliter pipette, dispense PBS gently around the edge of the hardened hydrogel sheets in order to detach each of the hydrogels.
Then, pick up each floating hydrogel sheet, using an end-cut, 1, 000-microliter pipette tip. Transfer each sheet of the hydrogel into individual wells of a 12-well plate containing one millileter of pre-warmed DMEM so that they float on the surface of the media. Culture the cells like this for one week, exchanging the culture medium every other day.
Next, produce a PDMS frame, which contains 170-micron high pillar structures at the lower surface as described in the accompanying text protocol. During fabrication, place a specialized polycarbonate mold on the silicon wafer for the PDMS frame to create an interior frame with a wide of eight millimeters, a height of nine millimeters, and a depth of two millimeters. Once sterilized, place the PDMS frame onto a quarter of a piece of nylon filter paper set in a 60-millimeter Petri dish.
Remove the hydrogel sheets from the incubator and transfer the first of the modular hydrogel sheets into the interior of the PDMS frame using an end-cut 1, 000-microliter pipette tip. Align the edge of the modular hydrogel sheet with the PDMS frame using an empty 200-microliter pipette tip. Repeat this process with additional hydrogel sheets until a stack of four to six layers is achieved.
Next, remove the culture medium by flowing it through the pillar structures at the bottom of the PDMS frame. Then, add two microliters of a two percent alginate solution at one corner of the multi-layer construct. Using a humidifier, spray a mist of the cross-linking reagent at a rate of 250 milliters per hour onto the multi-layer construct for thirty seconds to attach the edges of each layer together.
Then, rinse the multi-layered construct gently with 400 microliters of DMEM and remove the PDMS frame using a tweezer. Then, add an additional four milliliters of DMEM. Detach the multi-layered construct with the filter paper by gently wiping it with a cell lifter and then use filter paper to transfer it to a six-well plate containing three milliliters of DMEM.
Culture the cells in a floating manner with a hydrogel construct as a multi-scale cellular scaffold in the six-well plate for at least one week. Exchange the culture medium every other day. The result of a layer-by-layer assembly of liver lobule-like micropattered hydrogel sheets is shown here, where human liver carcinoma cells fluorescing green and NIH-3T3 fibroblasts fluorescing red were grown together in co-culture on a combined mesh.
Using the technique presented here, many pattern variations can be created, including hexagonal pillars, thin meshes, whole arrays, and multiple microcomb-like microfibers in the form a thin hydrogel construct on the order of 100 to 200 micrometers. These structures provide a rich 3D culture environment for many different cell types. After watching this video, you should have a good understanding of how to create cellular hydrogel sheets for engineering in vivo-like 3D culture systems from the bottom up.
