Elastin benzeri Protein Hydrogels üretimi için saklama ve Immunostaining hücre 3D

The overall goal of this protocol is to express and purify recombinantly derived elastin-like proteins for use as a tunable hydrogel for 3D cell encapsulation. Additionally, this protocol presents the methodology for downstream fluorescent labeling in confocal microscopy of encapsulated cells. This method can help answer key questions within the fields of biomaterials, regenerative medicine, and cell biology, such as understanding the role of cell-extracellular matrix interactions in 3D.

The main advantage of this technique is that it provides a reproducible, tunable, and modular material platform that is amenable to the study of multiple cell types. Overall, the implementation of this technique can improve our understanding of the critical cell ECM signaling parameters that may dictate the success of future cell-based regenerative therapies. Streak an ampicillin and chloramphenicol agar plate with a premade bacterial stock containing a vector that encodes for the ELP of interest.

Then incubate the streaked plate upside down at 37 degrees Celsius overnight to allow for bacterial growth. The following day, pick a single colony from the plate and inoculate the prewarmed starter culture. Incubate the culture in a shaker at 37 degrees Celsius for 16 hours.

After the incubation period, use a serological pipette to transfer 20 milliliters of the starter culture to each expression flask. Leave the expression flask shaking for one hour. After one hour, measure the optical density at 600 nanometers of an aliquot from the expression flask.

As soon as the reading reaches 0.6, reduce the temperature of the shaker to 32 degrees Celsius. When the reading reaches 0.8, add one milliliter of one molar sterile filtered IPTG to induce ELP expression in the expression flask. Allow the culture to express for seven hours.

After seven hours, centrifuge the expression media at 12, 000 x g for 15 minutes at four degrees Celsius in a floor centrifuge. After the centrifugation, use a spatula to collect the cell pellet from the centrifuge container and place into a preweighed ziploc bag. Next, dissolve the cell pellet in sterile filtered TEN buffer.

Massage the cell pellet to form a homogenous solution. Freeze the ziploc bag with resuspended cell pellet in a secondary container at 80 degrees Celsius. To the thawed cell pellet, add approximately 30 to 40 milligrams of deoxyribonuclease and one milliliter of 100 millimolar PMSF for every 100 milliliters of cell lysate.

Incubate the lysate at four degrees Celsius on a shaker overnight. Following the third freeze-thaw cycle, centrifuge the samples at at least 15, 000 x g for one hour at four degrees Celsius. Post centrifugation, pour off the supernatant from the centrifuge container and transfer the solution to a new centrifuge container.

To every 100 milliliters of supernatant, add 5.84 grams of sodium chloride in three parts to a final one molar concentration. Incubate the suspension in a shaking incubator at 37 degrees Celsius for three hours. Following the incubation, centrifuge the samples at at least 15, 000 x g for one hour at 37 degrees Celsius.

Post centrifugation, discard the supernatant from the centrifuge container and mass the pellet. Next, add 10 milliliters of autoclaved ultrapure water per gram of pellet obtained after centrifugation. To ensure that the protein dissolves completely, use a metal spatula to mash and resuspend the pellet.

Incubate the resuspended pellet at four degrees Celsius overnight on an orbital shaker. Repeat the cold and hot centrifugation steps for a total of three cycles to further purify the ELP. Next, dialize the residual supernatant in a 3.5 kilodalton dialysis membrane against four liters of prechilled ultrapure water at four degrees Celsius for three days to desalt the protein solution.

Centrifuge the final dialyzed solution in a precooled centrifuge. Freeze the supernatant at 80 degrees Celsius in a preweighed conical tube. Finally, lyophilize the frozen solution for three days and then weigh the final lyophilized product to determine protein yield.

With the help of a biopsy punch, create holes in a 0.5 millimeter thick silicone sheet. Then cut a square around each hole. Next, use tweezers to remove the plastic wrap on each side of the individual molds.

After removing the plastic wrap, use the tweezers to position the identical number of bare silicone molds and glass cover slips in alternating rows on the lid of a 48 well plate for subsequent plasma bonding. After all cell culture materials have been fabricated, the preparation of the cell type of interest for downstream hydrogel encapsulation may begin. After dissociating the cells into a single cell suspension, aliquot the desired number of cells into a 1.5 milliliter centrifuge tube.

Then, centrifuge the cells at approximately 200 x g for three minutes. Post centrifugation, aspirate the supernatant and store the pellet on ice. Resuspend the cell pellet in ELP stock solution to 80%of the final volume.

Then use a pipette to mix the solution into a homogenous suspension of cells in ELP. For the remaining 20%final volume, add THPC stock solution to the cell ELP suspension. Pipette several times to ensure the formation of a homogenous mixture, while avoiding the formation of air bubbles.

Next, in a circular motion, pipette the cell ELP THPC mixture into each mold in a 24 well plate. Then incubate the samples at room temperature for 15 minutes. After 15 minutes, incubate the samples at 37 degrees Celsius for an additional 15 minutes.

Once the incubation is over, carefully add 750 microliters of warm cell culture medium to each well in a 24 well plate. Leave the hydrogel at 37 degrees Celsius for the desired culture duration. First, aspirate the medium from each well.

Then wash the wells with one milliliter of warm DPBS. After washing is completed, add 750 microliters of fixation solution to each well. Then, incubate the 24 well plate at 37 degrees Celsius for 30 minutes.

After the incubation, aspirate the fixation solution from each of the wells and discard the fixative in a hazardous waste container. Next, add one milliliter of DPBS to the sample and immediately discard the DPBS into a hazardous waste container. Next, add 750 microliters of permeabilization solution to permeabilize the sample for one hour at room temperature on the rocker at 15 revolutions per minute.

After one hour, aspirate the permeabilization solution and add 750 microliters of blocking solution to the sample. Leave the sample on the rocker at room temperature for three hours. Then dilute the desired primary antibodies with the antibody dilution solution.

Next, add 500 microliters of the antibody solution to each sample. Incubate the samples overnight at four degrees Celsius. The following day, aspirate the primary antibody solution, then wash the samples with PBST for one hour thrice at room temperature at 15 revolutions per minute.

Next, dilute the appropriate secondary antibodies in five milligrams per milliliter of DAPI stock solution to achieve a final concentration of 2.5 micrograms per milliliter. Then, add 500 microliters of the solution to each sample. Next, use aluminum foil to cover the 24 well plate and incubate the plate at four degrees Celsius overnight.

The next day, aspirate the secondary antibody solution and wash the samples with PBST thrice each for 30 minutes at room temperature on a rocker. Next, position a drop of hardset mounting medium on the surface of a glass slide and place the sample on the mounting medium. Finally, use nail polish to seal the samples to the glass cover slide to prevent contamination or sample movement.

Here, expression of the target protein with the full length ELP under controlled conditions is validated by the presence of bands at 37 kilodaltons in both SDS-PAGE and western blot analysis. However, under uncontrolled conditions, several low molecular weight bands are also visible using western blot. The lower molecular weight bands correspond to proteins with less than four elastin-like domain repeats, indicating that the protein is not fully translated during expression.

This result is common when conducting the expression above 32 degrees Celsius. To vary the cell adhesive ligand concentration while keeping the hydrogel mechanical properties constant, the ratio of ELP variant containing the fibronectin-derived cell adhesive and the non-cell adhesive sequences are tuned. Further, murine neural progenitor cells are also viable over a period of seven days when encapsulated in a 3%ELP hydrogel.

The live cells are identified in green due to the calcein AM stain, whereas the red ethidium homodimer is preferentially taken up by the dead cells. To validate the stem cell phenotype of neural progenitor cells, expression of canonical markers such as Sox2 and nestin are also visualized by immunostaining when the cells are encapsulated in the 3D ELP hydrogels. After watching this video, you should have a good understanding of how to express and purify elastin-like protein for downstream encapsulation of various cell types, and in addition, investigate the role of the 3D microenvironment in modulating cellular phenotype.