The overall goal of this procedure is to create a multi-layered cell culture scaffold. This video will demonstrate how to produce a 2D cell culture matrix, incorporating the adhesion peptide RGDS in alternate layers to separate regions of C two C 12 cells. This is accomplished by first tethering RGDS peptide to an acro loyal macro.
The macro peptide combination is then tagged with the LOR dye to allow for visualization of the peptide containing layers of the multilayered gels. The second step is to form the molds by cutting them from a silicon sheet and sandwiching them between two glass slides next after prem, mixing individual components of each layer. Solutions of peg di acrl and peg di acrl with PEG RGDS Alexa three 50 are alternatively layered within the molds.
Gels are polymerized by UV irradiation. The final step is to seed C two C 12 cells on 2D multilayered gels in order to examine how matrix composition affects growth and attachment of the cells. Ultimately, brightfield and fluorescence microscopy are used to visualize the selective growth of C two C 12 cells on the scaffolds.
The main advantage of this technique over existing methods is that it's a simple and a cost effective method for creating multi-layered 2D and 3D cell culture matrices. It does not rely on need for expensive instrumentation. While the interfaces between layers are contiguous and structurally integral.
There is no mixing between components of individual layers. This method can help answer key questions such as how cells migrate and polarize in response to patterned biological chemical and mechanical cues. This method can provide gentle insight into cell migration and differentiation and can be applied to other systems such as directing new right outgrowth and synaptogenesis of neuronal stem cells.
To begin this procedure, combine RGDS peptide and acro oil peg succinyl carbo methyl ester in a one to two to one molar ratio in dry DMSO under Argonne. Then add NN diop, propyl methylamine or D-I-P-E-A at a two to one molar ratio relative to the aquilo PEG SCM to help facilitate the reaction, confirm conjugation of the aquilo PEG RGDS molecule using moldy TOF mass spectrometry. For this separately, add one microliter of the AQUILO PEG RGDS reaction solution, and one microliter of unreactive aquilo PEG SCM to different spots on the moldy target.
Allow them both to dry. Then prepare a saturated solution of universal moldy matrix in THF vortex for one minute and add one microliter to the same two spots. Allow them both to dry.
Next, load the samples. Perform moldy tough analysis with a 60 hertz laser at approximately 19%power using SAVIS gole smoothing top hat baseline subtraction and OID peak detection algorithms. The molecular weight of acro PEG RGDS should be shifted to the right, corresponding to the change in mass due to covalently bound peptide.
The next step is to conjugate the Fluor by adding an equimolar amount of LOR three 50 carboxylic acid sucks in a midal ester relative to the aquilo PEG SCM dissolved in a minimal volume of DMSO to the aquilo PEG RGDS reaction solution Under argon incubate overnight at room temperature. Then purify the acro oil peg RGD S3 50 by dialyzing against dye water at four degrees Celsius using a 3, 500 DAU molecular weight cutoff dialysis cassette. Continue dialyzing for 48 hours using a 1001 volumetric ratio, exchanging cold dialysate at least twice per day.
The product is further purified by filter sterilization through 0.22 micron syringe filter. Finally, in a sterile environment, aliquot the sterile purified acro oil peg RGD S3 50 into sterile pre weighted micro centrifuge tubes. Seal the open micro centrifuge tubes within sterile breather tubing.
Freeze dried the samples and store each sealed with paraffin film at minus 20 degrees Celsius. Pre-wash slides in 100%methanol and dry in an oven at 80 degrees Celsius until the liquid evaporates. Then place a dish containing the glass slides into a fume hood and add 250 microliters of sigma coat to each slide.
Gently rock the slides for 30 seconds in order to coat the entire surface. Next, thoroughly rinse the coated slides with 100%methanol, followed by washing and distilled water, by soaking them twice for five minutes in at least 10 milliliters of water. Once rinsed, let the slides air dry.
Prepare 0.8 millimeter thick silicone spaces by trimming a sheet of silicone to the same dimensions as the glass slides. Then using biopsy punches, make 10 millimeter or eight millimeter holes in the silicone to hold the scaffolds using a razor blade. Make around two millimeter channels at the top of the mold to allow for the addition of the scaffolding material.
Then autoclave the silicone spaces and sigma coat treated glass slides to sterilize them. Also sterilize additional materials for casting the gels such as einor tubes and forceps. In a tissue culture hood filter sterilize a stock solution of Peg di aquil using a sterile one milliliter syringe and 0.2 micron filter.
Begin by formulating PEG pre polymer solutions at 15%weight to volume ratio for each respective layer in individual amber micro refuse tubes by combining the 50%PEG D acrl with different amounts of sterile 60%IO DAL stock solution PBS and photo initiator to yield varying final concentrations of 10%20%30%and 40%I oal thoroughly mix the solutions to prepare the fluorescently labeled 15%PEG pre polymer solution, combine 50%PEG di aquil and aquilo PEG RGD S3 50 at a final concentration of eight millimolar with IO Dal stock solution PBS and photo initiator in amber Microview tubes to yield concentrations of 15%25%and 35%IO IAL thoroughly mix the solutions. Next, assemble the mold set up by placing a precut spacer between two sigma co treated glass slides and secure with clamps. Once the mold is assembled, cast a layered gel by adding 10 microliters of the 40%solution first, followed by 10 microliters of the fluorescently labeled 35%solution.
Repeat the alternate layering to achieve several layers. Next, irradiate the mold with 365 nanometer light for three minutes. Using a portable UVR 9, 000 lamp, allow the polymerized hydrogels to cure for five minutes.
Then remove the clamps and gently lift the top glass slide and the mold. The stratified density gradient. Multi laid polymerization or DG MP gels will remain on the slides.
Using a sterile spatula, carefully remove the gels from the mold and place them into a 50 milliliter tube containing sterile DMEM. Wash the polymerized gels using a 1000 to one volumetric ratio of DMEM for 48 hours with two buffer exchanges per day. This will remove any density, modifier, photo initiator, and on react pre polymer.
Then store the DGMP gels in DMEM supplemented with 1%penicillin streptomycin. To visualize alternating layers, arrange DGMP gel along a ruler on the sample tray of a gel documentation unit. Then expose and image the gels using Alexa three 50 alternating dark and blue bands in the DGMP hydrogel demonstrate formation of discreet layers of distinct chemical composition.
To prepare the DGMP gels for cell culture, gently insert them into the wells of a 48 well cell culture plate. Using a sterile cell scraper and rinse em three times in cell culture medium. Next, harvest C two C 12 myoblasts from a 100 millimeter cell culture plate when they are 60%confluent.
To do this, rinse the plate three times with prewarm PBS. Then add one milliliter of 0.25%trips in EDTA and incubate the plate at 37 degrees Celsius for two minutes. Once the cells have detached, add one milliliter of prewarm culture medium and transfer the cells into a 50 milliliter conical tube for centrifugation.
After the cells have been pelleted Reeses, pen them in growth medium and count the live cells by adding triam blue and using a TC 10 cell counter. Then seed the DG MP scaffold with C two C 12 myoblasts and incubate for 24 hours at 37 degrees Celsius with 5%carbon dioxide. Confirm the attachment of C two C 12 myoblasts on the RGDS containing layers of DGMP gels using epi fluorescence and phase contrast microscopy.
The alternating layers of DGMP gels may contain various peptides such as RGDS shown on the right, which supports cellular attachment and growth. The layer on the left, however, did not contain any cellular attachment peptides and the cells did not survive in that region. IO diol can affect the gel stiffness.
Here, the elastic modulus was measured using atomic force microscopy. A 60%increase in stiffness was seen when using 30%IAL in this gel formulation, the effect IAL on gel stiffness can vary based on the materials being used While attempting this procedure, it's important to remember the sequence of the layers. The layer containing the highest amount of IEX null will be at the bottom, while the layer with the least amount of IEX null will be at the top.
Always remember to add the photo initiator at the end to prevent polymerization from ambient light. With our collaborators, we're adapting this technique to organize neurite outgrowth in 3D. This will allow comparisons of neuro progenitor cells derived from healthy individuals and those from patients with neurodevelopmental disorders.
Don't forget that working with ultraviolet light can be hazardous. Wear UV protective eyewear while performing the cross-linking step.
