Microfluidic Vasculature for Cell Culture - Lilin Zhao, Melissa Deschamps, Marissa Burgess, Matthew Tiller, Jacob Kellett, Tina Leong, Katelyn Mullen, Daniel Bell, Anna Comperchio, Evelyn Moore

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CHEM-ENG 535: Microfluidics and Microscale Analysis in Materials and Biology

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Introduction

It is extremely important to create in vitro models of vasculature in order to study cancer (including metastasis and tumor cell circulation), drug delivery, diseases, and to supply oxygen and remove waste to systems too large to depend on diffusion. Vascular diseases are the leading cause of death worldwide causing around 17 million deaths per year. There are limited treatments, and therefore a dire need to better understand the vascular system [1]. Blood vessels are responsible for delivering nutrients and oxygen and for disposing wastes to and from all parts of the body. Vessels therefore end up controlling the microbiome. [2]. Currently, the best experiments are conducted on animal models and/or cell culture, but even these methods are poor indicators for what occurs in vivo [1].

Cell culture experiments fail to consider the complex, diverse, vascular system. Rather than being a single type of cell, capillaries and arteries are composed with different layers of cells. The inner layer is made up of endothelial cells (ECs) which act as a semipermeable membrane between the vessel wall and the fluid from the outside. The middle layer is comprised of smooth muscle cells (SMCs), which give the vessels their mechanical properties and provide structure by producing collagen. Fibroblasts and connective tissue line the outer layer [1]. Depending on what researchers are investigating, all of the layers of the blood vessels may need to be included. If the goal is to supply oxygen or deliver waste or maybe includes topics not associated with the actual biology of the blood vessel, then simpler fabrication methods can be used.


Figure 1. Schematic depiction of the general idea of 3D printing with multiple extruders necessary to create many vascularized microfluidic devices [3] (Figure Adapted from Kolesky et al.).

The vascularization of microfluidic devices is the creation of channels within a device capable of transporting blood in one end and completely out the other. As blood vessels range in size from the smallest capillaries of 5 micrometers to the aorta of 25 mm, microfluidic devices are excellent tools in order to provide the proper channels for blood flow [4]. The first generation of these devices relied on 2D manufactured devices stacked on top of one another in order to create a 3D vascularized device [5]. Now technology allows for the creation of detailed 3D vascularized microfluidic devices with the proper channel size for the type of blood vessel being recreated and type of tissue being replaced.

The vascularization of microfluidic devices has a vast number of applications as the ability to successfully move blood in an artificial tissue construct is important in the effectiveness of the device. Vascularized microfluidic devices can be used in both the medical field as a tissue implant in a number of areas of the body or for more research applications as a way of studying blood flow in vitro [3]. A number of considerations must be taken into account when creating these devices because of their use within the body. Biocompatibility of the materials used in these devices is very important as cells must adhere to the surface of the device and each device should accurately model the body [4]. Over the past few decades, several different techniques for the construction of vascularized microfluidic devices have been developed including traditional methods of construction using photolithography[1] and soft lithography[2] as well as emerging technologies of 3D printing through both dissolvable sacrificial moldings and hybrid method bioprinting. As these technologies continue to develop, vascularized microfluidic devices will continue to grow in importance for organ and tissue repair.

There are several challenges that must be considered when using microfluidics to create a vascular system. For example, many microfluidic devices are inexpensive and easy to make because of the heat curing polymer polydimethylsiloxane (PDMS). It is typically considered a great material for biological applications because it is gas permeable, bio-compatible, and clear. However, PDMS adsorbs hydrophobic molecules, making it difficult to study drugs. Some decide to work with thermoplastic materials in place of PDMS for drug studies, but these are expensive, auto-fluorescent (bad for imaging), and not gas permeable (so cells will die quicker than in PDMS) [1].

Additionally, the geometry of the channels may not always be perfect with lithographic techniques. Even though chips can function on the micro and nano scale, the vessel walls in vivo are extremely thin, and consequently difficult to study. Soft lithography also typically will create rectangular channels by adhering a design to a flat surface [1]. This may cause discrepancies between fluid interaction with cells in vivo and in vitro. Molding the channels a new way or taking advantage of 3D printing technologies can help create cylindrical channels.

Mimicking Geometry of Blood Vessels

Soft lithography techniques typically involve binding one side of the channels to a flat surface, creating rectangular or straight-edged channels. To properly model vascularization, standard fabrication methods need to change to make cylindrical channels that more accurately represent blood vessels. Sharp corners at intersections and bifurcations as well at the corners of a rectangular cross-section make it extremely difficult to properly seed them with endothelial cells. Non-cylindrical channels will have various shear stress depending on location, while a circular cross-sectional area will promote even shearing on all areas along the circumference. Correctly mimicking shear stress is critical when creating these microfluidic systems because shear stress determines endothelial cells functional phenotype and gene expression. Therefore, a microfluidic chip with a rectangular cross-section and uneven shearing will create a very different environment than what occurs in vivo [6].

Figure 2. Method for how to turn a leaf into a microfluidic device that can mimic blood vessels (Adapted from He et al.) [7].

Many research groups have developed interesting approaches to vascularize microfluidic systems. One lab developed microfluidic channels able to mimic blood vessels embedded in an extracellular matrix (ECM). They drew in sacrificial gelatin into a PDMS microfluidic channel, gelled it, and then removed the PDMS design. They then poured type I collagen (acting as the ECM) over the gelatin and allowed that to gel. The gelatin was then melted, leaving behind channels that can then be coated with endothelial cells to mimic blood vessels. With these types of channels, controllable spatial gradients of soluble molecules, such as growth factors, can be created [8]. This device is easy to make, and offers a platform for studying morphogenesis. The same research group later implemented another device that also features channels completely immersed within an ECM. They polymerized rat tail collagen type I around two 400-μm-diameter needles. Once the needles were removed, they coated the channels with endothelial cells leaving cylindrical channels completely surrounded by ECM [9]. This method solves the rectangular channels limitation that many microfluidic devices have. This also eliminates the issue of having part of the channel be silicon or glass, which cause devices to deviate from in vivo results further. This system was used to study gradient-driven angiogenic sprouting while allowing sprouting to occur in all directions from the preliminary channel [9].

Another interesting approach to achieving the correct geometry included electroplating wafers with copper and creating semi-circular ridges on silicon. The masters were embossed with polystyrene sheets with semi-circular channels and then aligned together to create circular channels[6].

One lab argued that microfluidic channels over simplified vascular systems and decided to use a template from nature as their template-a leaf. They believed that the complexity of the venation of the leaf was comparable to that of vascular systems and also followed the same Murray's law as blood flow [7]. They first digested the soft tissues of the leaf, and then coated the remaining venation network with chrome. The leaf was used as a photomask over a UV-curing polymer that was spin-coated on a silicon wafer. The negative pattern remained on the silicon while the UV-irradiated area was dissolved with 0.5% wt NaOH. Chrome was coated over this silicon wafer with the design, creating a master. PDMS could then be layered over the wafer, producing a negative mold of the master, and a very complicated microfluidic design comparable to blood vessels [7]. This technique exemplifies how creative researchers can be when developing methods on how to make something out of plastic mimic something that is alive.

Applications for Understanding Tumor Metastasis

Figure 3. Endothelial microvessel culture in a microfluidic device. [10] Licensed under a Creative Commons Attribution 4.0 International License.

When cancer cells spread from their origin to other areas in the body, it is referred to as tumor metastasis. This event is responsible for the greatest percentage of cancer deaths and therefore is an important area of study for improving cancer understanding and improving patient outcomes. [11] One way that researchers are looking to understand the molecular processes of metastasis is with microfluidic models of the tumor and its microenvironment.

The primary mechanism by which metastasis occurs is through the bloodstream, so in microfluidic devices aimed to model metastasis, vascularization must be properly represented. Referred to as angiogenesis, the development of new blood vessels occurs in tandem with cancer metastasis and is a primary area of study. [12] An example of such device can be seen in Figure 3. This in vitro model was designed for high-throughput screening of new drugs to protect against microvascular destabilization and provides an excellent example of three-dimension tissue modeling in a microfluidic device. Three channels are shown, a blue collagen extracellular matrix, endothelial cells in the top perfusion lane to form a microvessel, and a third channel to which perfusion of the culture spread towards when the angiogenic factors were stimulated.[10]

Conclusions

Microfluidic technology hosts immense potential for understanding in vivo phenomena such as vascularization. By understanding the methods and applications of cell culture and vascularization in microfluidic devices, scientists can enhance understanding of cancer, drug delivery, diseases, oxygen delivery, and waste removal in the human body. Using such knowledge is key to the development of organ-on-a-chip systems.


References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 Gold, K., Gaharwar, A.K., & Jain A.J. (2019). Emerging trends in multiscale modeling of vascular pathophysiology: Organ-on-a-chip and 3D printing. Elsevier, 196, pp. 2-17. http://doi.org/10.1016/j.biomaterials.2018.07.029
  2. ↑ Lee, S., Ko, J., Park, D., Lee, S., Chung, M., Lee, Y., & Jeon, N.L. (2018). Microfluidic-based vascularized microphysiological systems. Lab on a Chip, (18), pp. 2686-2709. http://doi:10.1039/C8LC00285A
  3. ↑ 3.0 3.1 Kolesky, D.B.; Truby, R.L.; Gladman, A.S.; Busbee, T.A.; Homan, K.A.; Lewis, J.A. 3D bioprinting of vascularized, heterogeneous cell‐laden tissue constructs. Advanced materials, 2014, 26(19), 3124-3130. https://doi.org/10.1002/adma.201305506
  4. ↑ 4.0 4.1 Hasan, A.; Paul, A.; Vrana, N.E.; Zhao, X.; Memic, A.; Hwang, Y.S.; Dokmeci, M.R.; Khademhosseini, A. Microfluidic techniques for development of 3D vascularized tissue. Biomaterials, 2014, 35(26), 7308-7325. https://doi.org/10.1016/j.biomaterials.2014.04.091
  5. ↑ He, Y.; Wu, Y.; Fu, J.Z.; Gao, Q.; Qiu, J.J. Developments of 3D printing microfluidics and applications in chemistry and biology: a review. Electroanalysis, 2016, 28(8), 1658-1678. https://doi.org/10.1002/elan.201600043
  6. ↑ 6.0 6.1 Borenstein, J.T., Tupper, M.M., Mack, P.J., Weinberg, E.J., Khalil, A.S., Hsiao, J.P., & García-Cardeña, G. (2010). Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate. Biomedical microdevices, 12 1, 71-9. http://doi.org/10.1007/s10544-009-9361-1
  7. ↑ 7.0 7.1 7.2 He, J., Mao, M., Liu, Y., Shao, J., Li, D. (2013). Fabrication of Nature-Inspired Microfluidic Network for Perfusable Tissue Constructs. Advanced Healthcare Materials, 2(8), pp.1108-1113. https://doi.org/10.1002/adhm.201200404
  8. ↑ Baker, B. M., Trappmann, B., Stapleton, S. C., Toro, E., & Chen, C. S. (2013). Microfluidics embedded within extracellular matrix to define vascular architectures and pattern diffusive gradients. Lab on a chip, 13(16), 3246–3252. https://doi.org/doi:10.1039/c3lc50493j
  9. ↑ 9.0 9.1 Nguyen, D. H., Stapleton, S. C., Yang, M. T., Cha, S. S., Choi, C. K., Galie, P. A., & Chen, C. S. (2013). Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proceedings of the National Academy of Sciences of the United States of America, 110(17), 6712-7. https://doi.org/10.1073/pnas.1221526110
  10. ↑ 10.0 10.1 Kramer, B.; Corallo, C.; van den Heuvel, A.; Crawford, J.; Olivier, T.; Elstak, E.; Giordano, N.; Vulto, P.; Lanz, H. L.; Janssen, R. A. J.; Tessari, M. A. High-Throughput 3D Microvessel-on-a-Chip Model to Study Defective Angiogenesis in Systemic Sclerosis. Sci Rep 2022, 12 (1), 16930. https://doi.org/10.1038/s41598-022-21468-x.
  11. ↑ Fares, J.; Fares, M. Y.; Khachfe, H. H.; Salhab, H. A.; Fares, Y. Molecular Principles of Metastasis: A Hallmark of Cancer Revisited. Sig Transduct Target Ther 2020, 5 (1), 1–17. https://doi.org/10.1038/s41392-020-0134-x.
  12. ↑ Zhang, Q.-Z.; Zhu, Y.-P.; Rahat, M. A.; Kzhyshkowska, J. Editorial: Angiogenesis and Tumor Metastasis. Front. Oncol. 2023, 12. https://doi.org/10.3389/fonc.2022.1129736.
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