(J-364) Uniform Production of Millimeter Sized Alginate Microbeads via Microfluidics

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row J - Poster # 364

Co-Author(s)

KT

Khanh Tran

Student
California State University Long Beach, United States
Brenda Ametepe

Undergraduate Researcher
California State University, Long Beach, United States
EG

Erika L. Gomez

Undergraduate Researcher
CSU Long Beach
La Puente, California, United States
DR

Daniel Ramos

Student researcher
CSU Long Beach, United States
Clare Kim

Undergraduate researcher
CSU Long Beach, United States
PA

Perla Ayala, Ph.D.

Associate Professor of Biomedical Engineering
CSU Long Beach, United States

Presenting Author(s)

Siavash Ahrar, PhD (he/him/his)

Assistant Professor of Biomedical Engineering
CSU Long Beach
Long Beach, California, United States

Introduction:: Hydrogel microbeads are widely used in drug delivery and tissue engineering investigations as a 3D matrix to encapsulate bioactive agents. Therefore, platforms to produce uniform millimeter-sized hydrogel microbeads are essential to these studies. We investigated the uniform production of millimeter-sized alginate beads using co-flow microfluidic systems. To develop these systems, we used PDMS attached to PMMA slides. In contrast, most microfluidic systems use PDMS attached to glass slides. As measured by the contact angles, PMMA is more hydrophobic than glass (Figure 1). In our system, the hydrophobic properties of PMMA enabled an easier production of mm-sized aqueous-in-oil droplets. By adjusting the flow rates, we demonstrated the tunable generation of microbeads. Next, human umbilical vein endothelial cells (HUVECs) were encapsulated within the alginate microbeads, and their viability was verified for up to 2 days. The encapsulated HUVEC alginate microbeads' viability was further investigated for wound healing applications using a wound scratch assay. The presented platform provides a well-controlled approach to encapsulate cells within hydrogels microbeads.

Materials and Methods:: First, contact angles of 1% alginate solution were measured for PMMA and glass. The contact angle for alginate was higher against PMMA than glass (67.6° vs. 28.7°). Therefore, PMMA substrates instead of glass were used to build microfluidics for the alginate experiments. PDMS channels (2 mm wide and 15.5 mm long) were made from laser-cut molds. The inlets and outlet of the channels were hole punched using Ø: 2.5 mm and 5 mm biopsy punches. These PDMS channels were attached to the PMMA substrates via uncured PDMS as an adhesive. A 2 wt% sodium alginate (Sigma Aldrich) solution was used as the dispersed phase. Olive oil (food-grade olive oil with 1% surfactant SPAN 80) was used for the continuous phase. Droplets were generated inside the device, and the outlet tubes were placed inside a graduated cylinder containing 10 wt% calcium chloride solution to enable microbead gelation. For cell culture experiments, HUVECs were premixed with alginate (10,000 cells per mL), and the system was placed inside a biosafety cabinet to prevent contamination

Results, Conclusions, and Discussions:: First using water-in-oil droplets, behavior of both glass and PMMA systems was investigated (Figure 2). Since our droplets had an elongated shape, the size was defined as the length ratio to width (instead of a diameter). Droplet size could be tuned by adjusting the ratio of the flow rates. Droplets generated via PMMA chambers were more uniform. Next, PMMA devices were used to generate alginate-in-oil droplets. As demonstrated in Figure 3., alginate droplet size could be tuned. Utilizing this approach, HUVECs were encapsulated inside alginate droplets. Continuous flow rates of 10 mL/h and dispersed phases of 0.5 mL/h were used to generate 2 mm beads for cell-encapsulation experiments. HUVECs seeded in alginate microbeads were cultured in a 12-well plate. Cell viability was verified with a Calcein AM dye (Figure 4). A proof-of-concept wound assay (with 3T3 fibroblast) was designed to investigate the effects of HUVECs-alginate microbeads to promote recovery of 3T3 confluency (Figure 5). Our preliminary results suggested that the HUVECs-alginate microbeads accelerated the rate of gap closure after one day of culture. Alginate microbeads without HUVECs were not able to produce a similar result. Additional biological replicas are needed to verify this effect and investigate the specific biological benefits of HUVECs. Using a microfluidic system, we successfully demonstrated the formation of HUVECs-micrometer-size alginate beads. Alternative bioactive agents or cells can be used with the platform for tissue engineering or biomanufacturing applications.

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