The overall goal of this experiment is to prepare either unmodified or oligopeptide or extra-cellular matrix-grafted polyvinyl alcohol-co-itaconic acid hydrogels with the optimal elasticity for culturing and passaging stem cells. This method can help answer key questions in the stem cell research field about long term content of stem cells under xeno-free conditions. The main advantage of this technique is that it provides a platform to investigate a different hydrogel elasticity on stem cell differentiation into specific lineages.
Demonstrating the procedure will be me, Jia-Sin Yang and Yeh-Chia Tseng, graduate students from Professor Higuchi's laboratory. To begin the procedure, place 20 grams of P-IA and 200 milliliters of ethanol in a 500 milliliter conical flask. Stir the mixture for 24 to 30 hours, replacing the ethanol every eight to ten hours.
Collect the purified P-IA on Buchner funnel. Dry the P-IA under vacuum at room temperature for 24 hours. Next, over the course of 15 minutes, slowly dissolve 15 milligrams of purified P-IA in 100 milliliters of ultra-pure water.
Heat the solution to above 60 degrees Celsius and stir for one hour. Confirm that the solution has become clear before proceeding. Then, turn off the heat.
Allow the P-IA solution to cool to room temperature on the cooling hot plate while stirring. Continue stirring the solution at room temperature for 48 hours. Then, leave the P-IA solution undisturbed at room temperature for 20 to 24 hours to ensure that visible air bubbles are not present in the solution.
Next, dispense one milliliter of the P-IA solution into each of eight 35 milliliter tissue culture-treated dishes. Dry the dishes in an oven at 45 degrees Celsius for two days to produce P-IA films in the dishes. Next, add to 10 milliliters of ultra-pure water 100 microliters of a high-purity aqueous 25%glutaraldehyde solution, two grams of sodium sulfate and 100 microliters of sulfuric acid.
Stir the mixture well to obtain the crosslinking solution. Gently add one milliliter of the crosslinking solution to each dish, being careful not to damage the fragile P-IA films. Leave each film immersed in the crosslinking solution for half, one, two, four, six, twelve, twenty four or forty eight hours to obtain the hydrogels.
Rinse each hydrogel with one milliliter of ultra-pure water after its crosslinking period. Sterilize each hydrogel by immersion in an aqueous 75%ethanol solution for one minute. Rinse each sterilized hydrogel with ultrapure water six times.
Cover the hydrogels in fresh ultra-pure water, place the lids on the dishes and store the hydrogel dishes in a clean area until ready for activation and grafting. Next, activate each sterilized hydrogel by immersion in one milliliter of an aqueous solution containing 10 milligrams per milliliter each of EDC and NHS, either for one hour at 37 degrees Celsius or four hours at four degrees Celsius. Then, rinse each activated hydrogel with one milliliter of Ph 7.2 phosphate-buffered saline three times.
Immerse each hydrogel in one milliliter of a solution of either oligopeptides or ECMs in PBS for 24 hours at four degrees Celsius. Finally, wash each hydrogel with one milliliter of ultra-pure water three times after grafting is complete. Use standard method to culture human ES or IPS cells on a membrane matrix in six centimeter culture dishes until near confluent.
Then, incubate the cells in DMMF-12 supplemented with two milligrams per milliliter of dispase-2 at 37 degrees Celsius for eight to ten minutes. Rinse the cells twice with DMMF-12. Add two milliliters of DMMF-12 to each culture dish and attach weekly adherent colonies with the pipette or cell scraper.
Collect the cells in 15 milliliter centrifuge tubes and centrifuge the cells at 160 times G for five minutes at 37 degrees Celsius. Discard the supernatant and re-suspend the cells in one milliliter of xeno-free eight component medium. Count the cell density in the suspension.
Then, obtain culture dishes or multi-well plates containing sterilized P-IA hydrogels grafted with either oligopeptides or ECMs. Load the appropriate volumes of the cell suspension onto the hydrogels. Passage the cells on grafted P-IA hydrogels until 80%confluent the desired number of times.
To begin the immuno-staining process for a 24 well plate of passaged human ES or IPS cells on grafted hydrogels, add 0.5 milliliters of four per cent paraformaldahyde by volume to each well. Incubate the cells at four degrees Celsius for 15 minutes to fix the cells. Aspirate the paraformeldehyde solution from the wells.
Add one milliliter of PBS to each well and aspirate the PBS. Perform this PBS rinse three times in total. Then, add 0.5 milliliters of 0.3%octoxynol 9 by volume in PBS to each well.
Incubate for 30 minutes at room temperature. Aspirate the octoxynol 9 solution. Add 300 microliters of two per cent BSA and PBS to each well and incubate for another 30 minutes at room temperature.
Aspirate the two per cent BSA solution from the cells and add the primary antibodies. Incubate the cells for one day at four degrees Celsius. Then, aspirate the primary antibody solutions and wash the cells with 300 microliters of 05 per cent polysorbate 20 in PBS three times.
Add 300 microliters of secondary antibodies specific to the primary IDG subtype in two per cent BSA to each well. Incubate the cells for one hour at room temperature in the absence of light. Finally, wash the cells with 300 microliters of 05 per cent polysorbate 20 and PBS three times in the dark.
Image the stained cells with fluorescence microscopy. P-IA hydrogels crosslinked for 24 hours and grafted with vitronectin-derived oligopeptides, supported cultures of human ES and IPS cells for at least 10 to 20 passages. Human ESLs cultured on commercially available coated dishes were found to be more easily differentiated at the edges of the colonies.
Differentiated cells were not observed on the hydrogels, indicating that human ESLs maintained their pluripotency. Pluripotency proteins were satisfactorily expressed on human ES cells cultured on hydrogels grafted with three different Vitronectin-derived oliogopeptide designs for ten passages. Satisfactory expression was also observed on recomibinent Vitronectin-coated dishes.
Similar results were observed for human IPS cells. Both human ES and IPS cells differentiated into cells derived from all three germ layers after cultivation on P-IA hydrogels and on recombinant Vitronectin-coated dishes for at least ten passages. Human ES cells cultivated on hydrogels grafted with Vitronectin-derived oligopeptides were injected into NOD mice.
The harvested teratomas exhibited cells originating from all three germ layers indicating that pluripotency is maintained in vivo. Following this procedure, methods like media screening can be performed to answer additional questions like the effect of calcium arterial and stem cell differentiation and proliferation. After this development, this technique paved the way for researchers in the field of stem cell co-division to explore tissue or organ regeneration in neurodegenerative medicine.
Once mastered, this technique can be done in four days if it's performed properly. After watching this video, you should have a good understanding of how to prepare P-IA hydrogel dishes, both with and without ECM. While attempting this procedure, remember to set up a clean environment for preparing P-IA hydrogel dishes.
Don't forget that working with ethanol, hot water and the sulfuric acid can be extremely hazardous. The appropriate lab coat, gloves and the protective glasses should always be worn while performing this procedure.
