Photocrosslinking of OkaGel Microspheres

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Overview:

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.

Protocol

  1. Prepare Okagel solution to include cells and photoinitiator of choice.
    1. See “Embedding cells in Okagel” protocol
    2. Recommended concentration contains a 7.5 wt% GelMA (between 4.0 wt% and under 8.0 wt%) and 1.0 wt% photoinitiator
  2. Form an oil solution of perfluorinated oil (3M HFE 7500). This becomes the oil phase.
    1. a. Add a biocompatible triblock perfluorinated copolymer surfactant to stabilize the droplets (for example 1.0 WT% of, Krytox-PEG, RAN Biotech).
  2. flow GelMA into a capillary microfluidic flow-focusing device. This will create the microspheres.
    1. Oil phase flow rate should be kept constant at 10mL/h.
    2. 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.
  3. Subject the resultant droplets to UV light (~365nm) for 20 seconds.
  4. Wash the microspheres in 20% perfluorooctane in HFE oil to remove surfactant.
  5. Wash the microspheres in 1:1 volume distilled water.
  6. Repeat Steps 7 and 8 once more to ensure complete removal of residual oil and immerse thecrosslinked microspheres in water for 24 hours.

Notes:

– 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.

References:

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