The overall goal of this procedure is to form, pattern, and encapsulate cells within synthetic polymer based extra cellular matrices. This method can be used to address critical biological questions related to the identification of extra cellular cues that regulate cell responses within a heterogeneous micro-environments. The main advantage of this technique is that a robust synthetic matrix is generated for cell encapsulation and culture using protocols and accessible materials.
This method can be used for controlled cell culture and to study cell response in vitro. It can be applied to other systems including drug delivery and tissue engineering. Prepare materials for hydrogel formation as described in the text protocol.
Attach a collimating lens to the end of the liquid filled light guide to ensure relatively even light intensity across all samples. Adjust the distance of the light guide from the samples to achieve a spot size that will cover the samples of interest. To make non-patterned hydrogels, prepare thick hydrogels molded in syringe tips by pipetting 10 to 20 microliters of the gel precursor solution into the tip of a sterile cut syringe.
Make a single gel per syringe to that it's approximately 0.5 to 1.5 millimeters thick, based on volume and syringe diameter. Prepare thin hydrogels in glass slide molds by placing the rubber gasket around the edges of the non-coated glass slide. Next, pipette five to 10 microliters of the gel precursor solution onto the non-coated glass slide.
And place the glass slide coated with anti adhesive on top of the gel solution. Secure the glass slides with small binder clips to stabilize. Set the lamp intensity to achieve 10 milliwatts per square centimeter at the gel surface for syringe tip molds or glass slide molds.
Using a radiometer to detect intensity. And place the molds under the lamp. Apply light for one to five minutes to allow complete polymerization of gels.
Use a shorter polymerization time for gels with higher polyethylene glycol terephthalate content. And a longer time for gels with a lower content to produce fully polymerized hydro gels with moduli within the range of soft tissues. Place the gels from the syringe tip molds into a sterile non-treated 48 well plate, and place glass slides into a sterile dish.
To prepare patterned hydrogels, prepare the precursor solution as described in the text protocol and pipette the solution vigorously to ensure even mixing of the solution. Next, cover preformed gels with a penton peptide into lithium phosphate photoinitiator solution, and incubate for one hour at 37 degrees Celsius. Following incubation, remove an excess solution.
If gels are molded in a syringe tip, use a spatula to carefully transfer from the 48 well plate to a sterile glass slide for patterning. Place a photo mask with the desired pattern directly on top of syringe and glass slide molded gels. Ensure that the printed part of the mask touches the gel for optimal pattern fidelity.
Place the samples under the lamp and radiate for one minute. After patterning, place syringe molded gels into sterile 48 well non-tissue culture treated plate. Place the glass slide molded gels that are adhered to the glass slides into a sterile dish.
Rinse three times, 15 minutes with cultured medium or the appropriate buffer based on the planned experiments. To preform for the detection and quantification of free thiols in photopatterned hydrogels, first prepare the solutions and perform the calculations as described in the text protocol. Form thin hydrogels for patterning as before.
Specifically, make gels with excess free thiols and pattern half of these samples with the penton peptide using a clear cover slip to fully expose the entire gel with light. Place patterned and non-patterned gels in wells of a 48 well plate. Rinse for 15 minutes, three times with Ellman's reaction buffer, to allow diffusion of the end reacted species out of the gel and equilibrium swelling to occur.
Add extra Ellman's reaction buffer to the rinse gels, so that the swollen gel volume with the reaction buffer is a multiple of 20 microliters. For example, if the predicted swollen gel volume is 15 microliters, add five microliters of reaction buffer. And if the predicted swollen gel volume is 30 microliters, add 10 microliters of reaction buffer.
Pipette the sistine standards into individual wells of a 48 well plate in multiples of 20 microliters. Depending on the volume used in the previous step. For example, if the previous step had a swollen gel volume, plus extra reaction buffer equal to 40 microliters, add 40 microliters of each standard to individual empty wells.
Next, dilute Ellman's reagent and reaction buffer. Add the diluted Ellman's reagent to wells containing standards and samples. For 20 microliter samples, add 183.6 microliters of solution to each well.
Incubate or place on a shaker for one hour and thirty minutes, or until the color of the solution matches the color of the gels by visual inspection, to ensure sufficient diffusion of the yellow 2-Nitro-5-thiobenzoic dianion, which is generated upon reaction of Ellman's reagent with free thiols from the gel. Take 100 microliters of solution from the samples and standards and place into wells of a 96 well plate. Read the absorbency at 405 nanometers on a plate reader.
To visualize the photo patterns, remove excess reaction buffer by gently wiping around the edges of the hydrogel with a tissue. Pipette Ellman's reagent directly on the surface of the gel. Immediately image on a light microscope at 10x or on a stereo microscope with a color camera.
Collect the cells and prepare them for encapsulation as described in the text protocol. Immediately after aspirating PBS, suspend the pelleted cells in the precursor solution to a final concentration of five thousand cells per microliter. Leave an appropriate concentration of free thiols for later reaction with the penton peptide.
If photo patterning is desired. Mold, polymerize, and pattern hydrogels as before. Rinse the gels for 10 minutes, three times, in growth medium after patterning to remove unreacted species and excess lithium phosphate photoinitiator.
Incubate the gels in growth medium at 37 degrees Celius and five percent CO2 until the desired time point for further analysis. Replenish the medium every 48 hours, or as determined by the applications. To determine encapsulated cell viability, first remove the growth medium from the gels and rinse for 15 minutes, three times, with PBS to allow diffusion of the medium from the gels.
Add two microliters of the thawed two millimolar ethidium homodimer-1, and 0.5 microliters of the thawed four millimolar calcium AN solutions, to one milliliter of sterile PBS. Vortex to ensure complete mixing. Add 300 microliters of standing solution to each syringe mold gel in the 48 well plates, or enough to cover the surface of the gel on a glass slide in a sterile dish.
And incubate for 45 minutes. Remove excess standing solution and rinse to remove excess dye from the gels. Image the cells on a confocal microscope.
Or on an epifluorescence microscope with capability at 10x. And 488 nanometer excitation and 525 nanometer emission. The free thiol concentration of non-patterned and patterned hydrogels quantified with Ellman's demonstrated that hydrogels initially polymerous for one minute and for five minutes have statistically similar free thiol concentrations, indicating rapid reaction and complete gelation within one minute.
Subsequent bulk patterning of the gels incubated with penton peptides for 30 minutes, and for one hour and 30 minutes, have 60 to 80 percent reduction in free thiol concentration, indicating efficient modification of the excess thiol. Photo patterns formed by shining light through samples covered with masks, printed here with lines, may be observed in two dimensions with the application of Ellman's reagent. Or, in three dimensions with the incorporation of fluorescent peptides in subsequent imaging.
Adult human stem cells encapsulated within hyrogels were treated with viability and hydrotoxicity stains. And imaged with confocal microscopy. Viable cells with no membrane damage are labeled green and indicates survival of the cells one day after encapsulation.
Cells begin to spread within the matrix six days after encapsulation. As show in the inset image. While attempting this procedure, it's important to remember to maintain sterile and careful handling procedures to prevent damage to cells after encapsulation through the introduction of contaminants or mechanical sheer.
Following this procedure, other methods like immunostaining flow cytometry and gene expressions can be performed to study cell function and fate in response to the extra cellular environment. This technique paves the way for researchers in the fields of biology, bioengineering and medicine, to studying tissue region erosion or disease progression in synthetic extracellular matrices with user defined property control, toward the development of new treatment strategies. After watching this video, you should have a good understanding of how to encapsulate cells, and pattern soft matrices for controlled cell culture studies.
