The Effect of a Y-Splitter on the Scale Up of Microsphere Formation
ABSTRACT
Hydrogel microparticles (HMPs) are made up of polymers that range in size from 1 to 1000 µm. Small HMPs are called microspheres. Microspheres encapsulate cells so that they can live in a controlled environment. The encapsulated cells secrete extracellular vesicles (EVs), which are nano-sized particles that act as carriers throughout the body. EVs have therapeutic potential in many conditions due to their various cellular components. Encapsulated cells secrete more EVs than 2D cultured cells. Therefore, we aim to scale up the production of HMPs to improve EV production. A Y-splitting microfluidic device split the flow from the syringe pumps to scale up the fabrication of HMPs. Using a Y-splitter microfluidic device to scale up the production of HMPs will produce a greater volume of HMPs. We found that HMPs made from the Y-splitting microfluidic device were significantly larger (P<0.005) compared to the control microfluidic device. In conclusion, we scaled up the production of HMPs by splitting the flow of production into two separate tubes using a Y-splitting microfluidic device efficiently and at minimal cost.
INTRODUCTION.
Cancer is one of the deadliest diseases in the world and there is no cure for it. While many scientists are looking at multiple pathways to cure this deadly disease, some are specifically studying extracellular vesicles. Extracellular vesicles (EVs) are nano-sized lipid bilayer particles that are secreted by cells, including cancer cells, into the space around cells. EVs are important because they contain proteins, lipids, RNA, and DNA that can be transferred to recipient cells to change their function. Furthermore, EVs tend to accumulate in tumors. [1] Therefore, research into EVs will provide more insight into the function of cancer cells. HMPs (Hydrogel microparticles) are micron scale particles that vary between 1 to 1000 µm. HMPs are usually composed of natural or synthetic polymers. They can form many different shapes and sizes with these HMPs [2,3]. This paper will specifically look at the formation of microspheres. Microspheres are important because they can encapsulate cells inside of them. [2,3]. Once cells are encapsulated, they have many different uses: chemotherapy, gene delivery, drug delivery, and vaccine delivery [3]. Cell encapsulation is important because it creates a stable 3D environment for the cells. The environment of these encapsulated cells is easier to manipulate than other methods [4]. Furthermore, cell encapsulation is one of the best ways to enhance EV secretion. When cells are encapsulated, the secretion of EVs is greater than in 2D culturing. More EVs would allow for more efficient research of cancer cells. Microspheres are formed by using a microspheric device and pumping an emulsion made up of mineral oil, 2% span 80, 7% GelMA (Gelatin methacryloyl, Sigma-Aldrich), and 2% LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate, Sigma-Aldrich) into it. This emulsion then forms microspheres. My lab previously used a Flow-focusing microfluidic device (S1), which was slow and inefficient. Many methods to scale up microsphere formation, like utilizing a 2 multi-channel syringe pump, are expensive. To address this, we studied a new microspheric device called a Y-splitting microfluidic device (S2) [5] and tested the effects of it compared to the Flow-focusing microfluidic device. These devices differ because the Y-splitting microfluidic device is meant to split the flow of the emulsion into two Flow-focusing microfludic devices. On the other hand, the Flow-focusing device is meant to create microspheres using just one pathway. The purpose of using the Y-splitting device is to scale up the production of microspheres.
MATERIALS AND METHODS.
Instruments.
The instruments used in the study were a Y-splitting microfludic device (S2), a Flow-focusing microfludic device (S1), an “InfusionONE Single Channel Syringe Pump,” a “Harvard Apparatus 11 Elite Programmable Syringe Pump,” a 1.5 mm biopsy punch, a vacuum chamber, and a versatile fluorescent microscope the EVOS m5000.
Mold process.
To make the microfluidic devices PDMS (polydimethylsiloxane, SYLGARD 184) and a curing agent (dimethyl, methylhydrogen siloxane copolymer) were combined at a 9:1 ratio. This ratio was then mixed thoroughly, cast into the microfluidic device molds, that came from a CAD design, and put into a vacuum chamber for 60-120 minutes. After this, the microfluidic devices were cured in an oven at 55C ° overnight. After this, the microfluidic device molds were taken out of the oven and the cured PDMS was removed from the microfluidic device molds. Then, inlet and outlet holes were punched with a 1.5 mm biopsy punch. To finish this process the devices were bonded to prepared glass microscope slides by O2-plasma surface activation
Flow-focusing microsphere formation microfluidic device microsphere formation.
The flow-focusing microsphere formation microfluidic device was set up with two syringe pumps. One syringe was filled with a solution of mineral oil and 2% span 80 and ran at a flow rate of 0.66 μL/s. Span 80 is a (W/O) mixture. The other pump was filled with a 7% GelMA (Gelatin methacryloyl, Sigma-Aldrich) and 2% LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate, Sigma-Aldrich) solution with 60 μL of green fluorescence into a volume of 200 μL of LAP solution and ran at a flow rate of 6.67 μL/s. The “%” is percent by volume. These solutions flow out of the pumps and into the channel of the flow-focusing microsphere formation microfluidic device to create microspheres. This was placed into a 24 well plate to collect data. The pumps ran into the flow-focusing microsphere formation microfluidic device for three minutes. Once they were in the 24 well plates, the microspheres were UV crosslinked. Finally, the microspheres were analyzed under a versatile fluorescent microscope called the EVOS m5000 at multiple magnifications.
Y-splitting microsphere formation microfluidic device microsphere formation.
The Y-splitting microsphere formation microfluidic device [5] was set up just like the flow-focusing microsphere formation microfluidic device. One syringe filled with a solution of mineral oil and 2% span 80. The other pump was filled with a 7% GelMA and 2% LAP solution with 60 μL of green fluorescence into a volume of 200 μL of LAP solution. However, these solutions flowed into two different Y-splitters. The Y-splitter would split the flow of the fluids into two separate flow-focusing microsphere formation microfluidic devices. The emulsion in those flow-focusing microsphere formation microfluidic devices would be placed in a 24-well plate to collect data. The pumps were run into the Y-splitting microsphere formation microfluidic device for three minutes for three tests, just like the Flow-focusing microsphere formation microfluidic device. Once they were in these 24-well plates, UV crosslinked these microspheres. Finally, they were analyzed under the EVOS m5000 just like the flow-focusing microsphere formation microfluidic device.
RESULTS.
The Y-splitter microsphere formation microfluidic device was originally designed to upscale the production of microspheres. We wanted to upscale the formation of microspheres without having to purchase an expensive multi-channel syringe pump. A multichannel syringe pump is used to scale up the production of microspheres. However, these multi-channel syringe pumps can cost up to $2000. To understand the effect of the Y-splitter microsphere formation microfluidic device on microspheres, we compared it to the Flow-focusing microsphere formation microfluidic device my Lab uses.
DISCUSSION.
Extracellular vesicles (EVs) have an increased importance in the world of biomolecular engineering because of their many helpful properties. Encapsulation of cells by microspheres is becoming more and more prominent. However, many methods to scale up microspheric formation, like a multi-channel syringe pump, are extremely expensive and not worth the cost. To address this, we explored the effects of a Y-splitter on microspheric formation aiming to scale up the production of microspheres.
We saw a significant increase in the size of microspheres formed by a Y-splitting microsphere formation microfluidic device(246.9 µm) vs the Flow-focusing microsphere formation microfluidic device(126.4 µm) (Fig. 2) (p<0.05). This tells us that using a Y-splitting microsphere formation microfluidic device instead of the Flow-focusing microsphere formation microfluidic device creates larger microspheres. Larger microspheres are able to hold more cells which can then secrete more EVs. One reason why we think the Y-splitting microsphere formation microfluidic device created these bigger microspheres is because of Bernoulli’s equation:
\[P_1+\frac{1}{2}\rho v_1^2+\rho gh_1=P_2+\frac{1}{2}\rho v_2^2+\rho gh_2\tag{1}\]
Bernoulli’s equation (Eq. 1) states that if you increase the area of something that is flowing the velocity will decrease, and vice versa [6]. The channels in the Y-spliiting microsphere formation device are smaller than the channels in the Flow-focusing microsphere formation microfludic device. Therefore, the velocity of the microspheres decreased, in turn, making them larger.
We also found that the Y-splitting microsphere formation microfluidic device is able to form microspheres. Thus, telling us that a Y-splitting microsphere formation microfluidic device is an effective way to form microspheres for extracellular vesicles. However, the results compared to the Flow-focusing microfluidic device were not statistically significant (Fig. 3) (p=0.07). This shows that the Y-splitting microfluidic device is not effective at increasing production when compared to the Flow-focusing microfluidic device.
In conclusion, the data gained from this research shows an effective way to produce microspheres (Fig. 4), which introduces a different way to form HMPs that allow for more efficient EV secretion in cells. In the future, to up scale the production of microspheres we will use a 4-way Y-splitting microfluidic device to make microsphere formation as efficient as possible.
SUPPORTING INFORMATION
Supporting information includes detailed figures of the Flow-Focusing microfluidic device and the Y-splitting microfluidic device discussed in the article.
ACKNOWLEDGEMENTS.
Thank you to the School for Science and Math at Vanderbilt, and the Young Lab at Vanderbilt for allowing me to do this research. Funding support for this research was provided by the National Science Foundation(AwardCBET-2328276).
REFERENCES
[1] R. Kalluri1, and K. McAndrews, The role of extracellular vesicles in cancer. Cell. 186, 1610-1626 (2023).
[2] A. C. Daly, L. Riley, T. Segura, and J. A. Burdick, Hydrogel microparticles for biomedical applications, Nature Reviews Materials, 5, 20–43 (2019).
[3] Prasad Galande, V. Yadav, and Smita Borkar, A Review on Microspheres: Preparation, Characterization and Applications. Asian Journal of Pharmaceutical Research and Development. 10, 128–133, (2022).
[4] G. Orive et al., Cell encapsulation: technical and clinical advances. Trends in Pharmacological Sciences. 36, 537–546, (2015)
[5] A. K. Yates, H. N. Murray, and E. S. Lippmann, “Design and optimization of a fluid flow splitting device for low-flow applications,” SLAS TECHNOLOGY. 100305–100305, (2025).[6] R. Qin and C. Duan, The principle and applications of Bernoulli equation. Journal of Physics: Conference Series. 916, 012038 (2017).
Posted by buchanle on Thursday, May 14, 2026 in May 2026.
Tags: EV, microfluidic, Microsphere