The overall goal of this procedure is to demonstrate techniques for encapsulating cells in thine photo polymerized hydrogels, as well as subsequent recovery of naturally formed beta cells pH from the enzyme sensitive hydrogels from biological characterization. This is accomplished by first mixing the mins six beta cells with a forearm peg, norbertine macer, and an enzyme sensitive peptide crosslinker. The cells are then encapsulated using free radical mediated thne photo polymerizations to form a hydrogel.
The second step is to culture encapsulated cells in thine hydrogels in a carbon dioxide incubator at 37 degrees Celsius. After the cells have proliferated to form aggregates, the cell laden hydrogels are rinsed with buffer solution and then incubated in chia trypsin solution. Under gentle shaking for gel erosion, then the recovered aggregates are carefully washed to remove enzyme solution and eroded polymer.
Ultimately, these cell aggregates can be used for structural and functional analysis. So the main advantage of step growth thne photochemistry is that its reaction conditions are milder than the traditional chain growth PEG dde. Therefore, the thine hydrogels provide an excellent microenvironment for many cell types, including pancreatic beta cells.
The implication of this technique extends towards various cell-based therapies because this DIN system provides an excellent microenvironment for in vitro experiments and a great platform for cell delivery. While we synthesize the peptides used in these experiments in our lab, there are many companies from which the peptide cross linkers we described can be obtained. To begin this procedure, prepare a 20%PEG four NB solution in PBS vortex the mixture until the rimer dissolves completely.
Then pass the Kermer solution through a syringe filter to sterilize it and store Eloqua at minus 20 degrees Celsius until needed. Next, prepare a 2%solution of the photo initiator lithium arol phosphonate in PBS. Pass the photo initiator solution through a syringe filter and store the sterilized solution at room temperature protected from light.
Another preparatory step is to dissolve the chrysin sensitive peptide crosslinker in PBS and syringe filter the solution for sterilization. Extra aliquots are stored at minus 20 degrees Celsius. Determine the Sulf hydro group concentration in the prepared peptide solution using elman's reagent according to the manufacturer's protocol.
Next, prepare the ation molds by cutting off the top one centimeter portion of sterile one milliliter disposable syringes. Using a razor blade re-sterilized the syringe molds. Using an autoclave, prepare a spreadsheet containing calculations of the desired gel formulation, including volume and concentration of stock materials, including polymer peptide, crosslinker, photo initiator, and a buffer solutions.
First prewarm cell culture, medium hank's balanced salt solution, and two x trips in EDTA to 37 degrees Celsius. Next, remove flasks of min six beta cells from the incubator and place them in the laminar flow hood. After aspirating the cell culture media from the flasks, rinse the cells with prewarm tanks, balance salt solution.
Then tryin ice the cells using three milliliters of prewarm, two x trips in EDTA and incubate the flasks for four minutes. After incubation, tap the flasks on the surface of the hood gently to completely detach the cells. Confirm the detachment and dissociation of the cells under a microscope.
Next, add an equal volume of cell culture medium. To neutralize the trypsin, mix the solution well by gently pipetting up and down. Then transfer the mixture into a sterile conical tube centrifuge the cell suspension for five minutes at 200 RCF following centrifugation, aspirate the supernatant and gently resuspend the cells in four to five milliliters of prewarm culture.Media.
Finally, determine cell density by mixing some of the cell suspension with 50%trian blue and counting cells with a hemo cytometer. Once the cells are ready, mix the required volumes of PEG four and B peptide crosslinker photo initiator at a final concentration of one millimolar cells and HBSS in a micro centrifuge tube based on a one-to-one TH to EEN ratio and mix gently by pipetting. A 4%PEG four and B is used in this demonstration.
Next pipette 25 microliters of the resulting solution into each syringe mold. Expose the syringe molds to 365 nanometer UV light at a power of five milliwatts per centimeter squared for two minutes. To polymerize the gel following photo polymerization, plunge the gels into a sterile 24 well plate containing one milliliter of cell culture medium and incubate the hydrogels at 37 degrees Celsius and 5%carbon dioxide for 30 minutes to rinse away any a loosely attached cells or uncross linked macros.
After 30 minutes, remove the plate from the incubator and transfer the gels into one milliliter of fresh media. Refresh the media every two to three days. Prepare one milligram per milliliter enzyme solution by adding Hanks balanced salt solution to trypsin free alpha chimi trypsin.
Then sterilize the mixture with a syringe filter. Next, remove the culture plate from the incubator and rinse the gels with Hank's balance salt solution. Then add 500 microliters of chime trypsin solution to each well next incubate room temperature with gentle shaking on a shaker for approximately five to 10 minutes in order for complete gel erosion to be achieved.
Once the gel is completely dissociated, place the plate on ice for a few minutes to reduce the activity of the CH trypsin. The next step is to transfer the cell suspension into a one milliliter tube and centrifuge slowly at 45 RCF and four degrees Celsius for two minutes. Following centrifugation, carefully remove the supernatant using a PEI pipette and gently suspend the steroid in Hank's balance salt solution.
Then transfer the recovered steroid solution to a new 24 well plate and place the plate on ice for five to 10 minutes to allow the steroid to settle and attach to the surface for size analysis. Acquire phase contrast images of the settled spheroids using a light microscope measure recovered spheroid diameters using software such as Nikon element software or image J.This method of encapsulation ensures high initial cell viability as quantified by live dead staining. With time in cell culture, cell metabolism as measured by alamar, blue reagents increased over a 10 day period in all concentrations studied.
After 10 days in cell culture, the steroids increased in size and still maintained high cell viability. In the hydrogels, the PEG peptide hydrogel supported the survival and proliferation of encapsulated beta cells, shown here after 10 days in culture, in somatic recovery with alpha chim trypsin frees the steroids from the hydrogel matrix, producing a narrow distribution of steroid sizes that are independent of matrix concentration. These steroids can then be used for additional characterizations and applications While attempting this procedure.
It is important to maintain homogeneity in the pre polymer solution during encapsulation to ensure that the cells are well distributed. Following this procedure, recovered cells can be characterized using other techniques like whole mount immuno staining and western blot in order to study the effect of hydrogel matrix condition on cell protein expression and phenotype. After watching this video, you should have a good understanding as to how to encapsulate cells in th hydrogels, as well as how to recover the cells from the gels for additional biological characterizations.
