The stepwise process for encapsulating cells starts with preparation and sterilization of hydrogel molds and other materials, followed by isolation of cells into individual populations, into tubes for each gel set. Ation is then performed in the presence of cells, followed by the in vitro or in vivo incubation period. Downstream analysis for cell behavior may include tests for viability, morphology, proliferation, and differentiation.
Hi Amidio Kahan from the laboratory of Dr.Jason Burdick in the Department of Bioengineering at the University of Pennsylvania. Today we will show you a procedure for cellular encapsulation in 3D hydrogels for tissue engineering. We use this procedure to study the effects of hydrogel composition and structure on the behavior of encapsulated cells.
So let's get started. To begin this procedure, prepare a 0.5%weight solution of the photo initiator air cure 2 9 5 9 or I 2 9 5 9 in PBS. Mix and incubate for two to three days in the dark at 37 degrees Celsius.
When ready, use a syringe filter to sterilize the solution and store it at room temperature. Next, use a spreadsheet program such as Microsoft Excel to calculate the amount of polymer and crosslinker needed demonstrated. Here is hyaluronic acid functionalized with acrylate reactive groups.
Also calculate the amount of the penant peptide containing the functional adhesive domain with amino acid cystine, arginine, glycine, and aspartate. The cystine is for tethering to the polymer. Now that the required amounts of polymer crosslinker and pent were determined, weigh them out and place each in an einor tube.
Adjust the calculation spreadsheet accordingly. For the actual mass of polymer obtained, proceed To prepare gel molds. Use a razor blade to cut off the tips of individually wrapped sterile.
One milliliter disposable syringes. Ensure a flat cut is made for molds to be used for photo pattern to gels. Finally, sterilize the syringe molds EOR tubes with their caps open and any other necessary materials by placing them beneath a germicidal light source built into the biological safety cabinet for 30 minutes.
Once sterilization is complete, proceed to prepare the cells following sterilization of the experimental equipment. Work in the hood and add sterile TEA to the polymer used in the mical type cross linking and sterile PBS to the polymer used for the free radical cross linking. ADD matching buffer to the pendant moiety tubes and vortex.
To dissolve the pendant moiety binds to the alkylated hyaluronic acid via a thiol from a INE group on the peptide to attach the pendant moiety to the polymer, add it to the polymer solution and seal with param. Briefly, vortex and incubate at 37 degrees Celsius with mixing. While preparing the cells, while the pendant moiety is attaching, prepare the cells to be encapsulated tryis, human mesenchymal stem cells or HMCs by washing them with PBS.
Then incubate with trypsin for one minute at room temperature, remove the trypsin incubate five more minutes at 37 degrees. Then add HMSC growth media to neutralize the trypsin and release the cells from the surface of the plate. Count the cells with a hemo cytometer.
Separate the counted cells into portions containing the appropriate number for each set of gels. For a set of three 50 microliter gels at a density of 10 million cells per milliliter, separate 1.5 million cells into an individual conical tube. Do not prepare more than four gels in an individual set due to the timing of ation.
Next centrifuge the cells at 500 RCF for six minutes. At room temperature while the cells are spinning down, prepare the polymerization mix for the mical edition crosslinking. Add buffer to the crosslinker to achieve the appropriate concentration.
For the light initiated free radical crosslinking, add the previously prepared I 2 9 5 9 solution to the polymer solution. At 10%of the total gel set volume for a final initiator concentration of 0.05%weight. Now the cells and the polymerization mix are ready for encapsulation in the hydrogel.
Start by aspirating the snat from the centrifuge cells. Wait for the liquid to settle after the initial aspiration and re aspirate to remove as much media as possible without disrupting the cell pellet Resus. Suspend the cell pellet with the polymer solution to prevent air bubbles.
Slowly pipette the solution up and down about 10 times, which should be sufficient to distribute the cells evenly. For the mical type addition reaction, use a wide orifice pipette tip to add the appropriate volume of crosslinker solution to the polymer cell.Mix. Set the pipette to the desired individual gel volume.
Pipette the solution up and down to homogenize and Eloqua into the syringe tip molds. Perform the step rapidly to ensure an even distribution of cells and allow 10 to 30 minutes incubation in the culture hood for cross-linking. For the light initiated free radical cross-linking.
Aliquot the appropriate volume into syringe tip molds. Expose the syringes to UV light for radical cross-linking a 10 minute exposure to 365 nanometer. Four milliwatts per centimeter squared UV light is common for ACRL functionalized polymers.
Perform the step rapidly to minimize cell settling before UV light exposure. Following cross-linking plunge the gels into the wells of a tissue culture plate containing media for incubation and analysis. Cross-linking of the hydrogel can also be done sequentially.
First, add the functionalized dye metholated rumine to a final concentration of 20 nanomolar in the gel. The dye helps visualize the patterning since it incorporates preferentially in radically cross-linked regions of the gel. Then start with the mic type polymerization reaction yielding a fraction of the theoretical cross-links for the following.
Free radical cross-linking one will need to use a mask designed in a program such as Adobe Photoshop or Illustrator and printed directly onto transparency masks. The mask dimensions must match the round syringe top. Use sterilized tweezers to apply a sterile mask directly to the gel surface and exposed to light to cross link the gel.
Further shown here are HMCs within HA based hydrogels one hour following encapsulation. Because synthesized hydrogels are typically transparent cells can be visualized for morphology using light microscopy. The same cells were visualized for viability 24 hours post encapsulation using a fluorescent live dead staining kit.
We used calcium M to detect the live cells and atherium homodimer for the dead cells and observed greater than 95%viability. We also used the alamar blue assay to determine the level of cell proliferation measured by how well the cells metabolize the assay reagent resulting in a color change. Cell staining was performed on H HMCs seven days post encapsulation within a degradable and adhesive HA based hydrogel and using standard fixation and permeable procedures.
Cellular acton is visible using fluorescein labeled phin and nuclei restrained with dpi. Immunostaining against cullin is used for visualization of focal adhesions within the hydrogel matrix. Histological staining is performed following dehydration, paraffin embedding, and sectioning of the hydrogels using standard protocols radically polymerized HMSC encapsulated HA hydrogels.
After one week in culture, were stained with hemat toin marking the nuclei and ASIN marking the connective tissue A and blue stains for glycosaminoglycans.Realtime. PCR is used to quantify differentiation of encapsulated stem cells After subtracting the background, the expression level of a gene of interest is determined by the cycle number where threshold fluorescence was reached compared with a passive reference signal, which is a constitutively active housekeeping gene. So we've just shown you how to encapsulate cells in 3D hydrogels and study the behavior of cells in gels.
When using these procedures, it's important to ensure complete sterility materials, either by keeping sterile buffer solutions and reagents long term or sterilizing with the germicidal UV lamp. So that's it. Thanks for washing and good luck with your experiments.
