Photocrosslinking of OkaGel Microspheres
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GelMA microspheres are a useful method for delivering specialized cells to various locations, both in vitro and in vivo while maintaining high cell viability. Here, a flow-focused microfluidic device is used to create cell-laden GelMA microspheres. The cell-laden GelMA acts as the aqueous phase and an oil solution is prepared to emulsify the GelMA into droplets.
- Prepare Okagel solution to include cells and photoinitiator of choice.
- See “Embedding cells in Okagel” protocol
- Recommended concentration contains a 7.5 wt% GelMA (between 4.0 wt% and under 8.0 wt%) and 1.0 wt% photoinitiator
- Form an oil solution of perfluorinated oil (3M HFE 7500). This becomes the oil phase.
- a. Add a biocompatible triblock perfluorinated copolymer surfactant to stabilize the droplets (for example 1.0 WT% of, Krytox-PEG, RAN Biotech).
- flow GelMA into a capillary microfluidic flow-focusing device. This will create the microspheres.
- Oil phase flow rate should be kept constant at 10mL/h.
- GelMA flow rate should vary from 100mL/h – 2000mL/h as to control the size of microsphere., This will result in droplet diameters varying between 90 and 230 um.
- Subject the resultant droplets to UV light (~365nm) for 20 seconds.
- Wash the microspheres in 20% perfluorooctane in HFE oil to remove surfactant.
- Wash the microspheres in 1:1 volume distilled water.
- Repeat Steps 7 and 8 once more to ensure complete removal of residual oil and immerse thecrosslinked microspheres in water for 24 hours.
– 20 seconds of UV exposure time was selected to be long enough to allow full conversion but short enough to ensure high cell vitality.
– The size of microspheres was selected so that it is larger than 60 um to ensure a sufficient number of encapsulated cells to promote cell contact and proliferation and smaller than 200 um to allow ready oxygen exchange across hydrogels for cell viability.
– The resultant GelMA microspheres can be easily injected through a syringe head. GelMA microsphere stiffness can be measured with the nanoindentation technique AFM.
Zhao, X., Liu, S., Yildirimer, L., Zhao, H., Ding, R., Wang, H., Cui, W., & Weitz, D. (2016). Injectable stem cell-laden photocrosslinkable microspheres fabricated using microfluidics for rapid generation of osteogenic tissue constructs. Advanced Functional Materials, 26(17), 2809–2819.https://doi.org/10.1002/adfm.201504943