High-throughput Fabrication of Cell-laden Gelatin Methacrylate Microgels for Tissue Engineering

Mohamed G. A. Mohamed (School of Engineering, University of British Columbia, Kelowna, BC V1V1V7, Canada)
Keekyoung Kim (School of Engineering, University of British Columbia, Kelowna, BC V1V1V7, Canada)

Introduction

   Tissue engineering applies a combination of material, mechanical, chemical and biological sciences with the aim of creating systems that are able to repair of even replace a biological function. Microfluidics principles have been utilized as powerful tools to fabricate controlled monodisperse cell-laden hydrogel droplets for various biomedical applications, most importantly for tissue engineering applications.

Materials and Methods

   A flow focusing microfluidic device as shown in Figure 1 was fabricated by applying photolithography combined with softlithography methods. Gelatin methacrylate (GelMA) prepolymer was synthesized from porcine skin gelatin and used to encapsulate cells. Two cell types, NIH 3T3 mouse fibroblast cells and MCF-7 breast cancer cells, were mixed with 8 % (w/v) GelMA prepolymer solution and 2 % (w/v) VA-086 photoinitiator.

   Cells were treated with bovine serum albumin (BSA) before mixing with the GelMA prepolymer solution to prevent cell aggregation. A magnetic mixer was applied to a syringe to evenly distribute cells in the GelMA prepolymer solution (Figure 2). The prepolymer-cell mixture was then applied as the disperse phase of the flow focusing microfluidic device. A 405 nm laser was used as a light source illuminating over the serpentine channels of the device to crosslink GelMA prepolymer.

Results

   Applying the magnetic mixer and BSA was able to reduce cellular aggregation during the droplet generation process. And hence, reasonable number of cells were encapsulated in GelMA droplets. The BSA occupied cellular receptors responsible for prohibiting cell aggregation1. While the magnetic micromixer evaded cells from settling down by the effect of gravity during the microdroplets formation process. We applied the recently developed ultrafast hydrogel photocrosslinking method2 (10 - 15 seconds) using a 405 nm laser for fast on chip photocrosslinking of cell-encapsulated GelMA droplets. Incorporating the on chip crosslinking process enabled a high throughput generation of cell-laden droplets with the continuous process flow. In addition, the laser allowed us fast crosslinking of the droplets and in turn incorporating the gelation step on the chip was possible.

   Encapsulating cells to hydrogel prepolymer droplets have been problematic for the stable droplet generation process with uniform microgel size. The problem is mainly due to the non-uniform distribution of cells throughout the hydrogel solution because of the cellular sedimentation and aggregation. By controlling this cellular distribution using the BSA and magnetic mixer, we could stably generate uniform size of droplets. (Figure 3).

Discussion and Conclusion

   Several microfluidic devices and techniques were previously used to produce cell-laden hydrogel droplets. However, a practical distribution of cells among the generated droplets was not achieved while it is essential in order to have uniformity and reproducibility in any subsequent application. In addition, gelation step always requires a relatively long time (in minutes) and was undergone off-chip in a separate step, which makes the throughput very low for any subsequent practical application. A cell-laden hydrogel droplet generation in a continuous high-throughput way was established with a practical cellular distribution among uniform sized microgel droplets.


Figure 1: The device consists of two main parts: the flow focusing cross junction for droplets generation and the serpentine channel . The formed pre-gel microdroplets are illuminated with 405 nm wavelength laser for crosslinking in the serpentine channels.

Figure 2: Experimental setup with a device to stir cell-GelMA prepolymer for preventing cell aggregation in a syringe.

Figure 3: A snapshot image shows stable cell-encapsulating in microdroplets.

References

1. E. Cheng, H. Yu, A. Ahmadi, and K. C. Cheung, “Investigation of the hydrodynamic response of cells in drop on demand piezoelectric inkjet nozzles.,” Biofabrication, vol. 8, no. 1, p. 015008, Jan. 2016.

2. Z. Wang, X. Jin, R. Dai, J. F. Holzman, and K. Kim, “An ultrafast hydrogel photocrosslinking method for direct laser bioprinting,” RSC Adv., vol. 6, no. 25, pp. 21099–21104, Feb. 2016. 

3. R. Samanipour, Z. Wang, A. Ahmadi, and K. Kim, "Computational and experimental study of microfluidic flow-focusing generation of hydrogel droplets," Journal of Applied Polymer Science, published online (DOI: 10.1002/app.43701), 2016.

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