Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate
Jeffrey T. Borenstein, Malinda M. Tupper, Peter J. Mack, Eli J. Weinberg, Ahmad S. Khalil, James Hsiao, Guillermo Gárcia-Cardeña. “Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate”. Biomed Microdevices (2009)
Summary:
With rapid advancements in tissue engineering and therapeutic technologies, it is becoming increasingly important to develop functional endothelialized microvascular networks. Complex tissues require a vascularized system in order for oxygen and nutrient delivery while drug development for relevant vascular diseases require a sufficient and realistic in vitro model with a viable microcirculation. For the past decade, attempts to create such a model have been made utilizing the biocompatible material polydimethylsiloxane (PDMS). These microfluidic devices, however, are limited in terms of geometry, i.e., difficulty in fabricating cylindrical channels, and lack of characterization of the PDMS surface, making efforts to endothelialize the surface minimally successful. The authors have developed a method to microfabricate cylindrical channels, approximately 175-190 μm in diameter, with tissue-culture-grade polystyrene by thermally bonding two semi-circular sets of the channels together to create circular cross-sections. The choice in material is justified by fact that this type of polystyrene, used in standard well plate systems, is well understood in terms of maintaining a stable surface chemistry for cell attachment. The technique utilized allows for varied channel depths as well as smooth transitions at bifurcations in the channels.
Summary:
With rapid advancements in tissue engineering and therapeutic technologies, it is becoming increasingly important to develop functional endothelialized microvascular networks. Complex tissues require a vascularized system in order for oxygen and nutrient delivery while drug development for relevant vascular diseases require a sufficient and realistic in vitro model with a viable microcirculation. For the past decade, attempts to create such a model have been made utilizing the biocompatible material polydimethylsiloxane (PDMS). These microfluidic devices, however, are limited in terms of geometry, i.e., difficulty in fabricating cylindrical channels, and lack of characterization of the PDMS surface, making efforts to endothelialize the surface minimally successful. The authors have developed a method to microfabricate cylindrical channels, approximately 175-190 μm in diameter, with tissue-culture-grade polystyrene by thermally bonding two semi-circular sets of the channels together to create circular cross-sections. The choice in material is justified by fact that this type of polystyrene, used in standard well plate systems, is well understood in terms of maintaining a stable surface chemistry for cell attachment. The technique utilized allows for varied channel depths as well as smooth transitions at bifurcations in the channels.
Primary human umbilical vein endothelial cells (HUVEC) were seeded into these microchannels by plating them on 0.1% gelatin (Difco)-coated circular channels and then incubating them for the duration of the experiments performed. Viability of these cells was then tested by a calcein AM live cell stain about 24 hours after the initial plating of the cells. Their results demonstrated that cell attachment/monolayer formation and viability were independent of channel diameter and that the HUVECs attach, form a confluent monolayer, and remain viable for at least 24 hours after the initial seeding process.
Critique:
This paper addresses the issue of creating channels that can emulate the microvasculature with a predictable surface chemistry as well as more physiological geometries, i.e., cylindrical channels that can vary in diameter and have smooth bifurcation transitions. However, there are still certain limitations to their method. In terms of fabricating the channels, it appears that the dimensions of the semi-circular cross section are inconsistent: some are round while others are elliptical. This may cause problems during the alignment of the two sheets of molded polystyrene. The channel sizes are also fairly large (about 200 μm in diameter) and in order to successfully emulate the microvasculature environment, the channels should account for the capillaries, venules, and/or arterioles, vessels that range anywhere between 5–40 μm in diameter.
Though the researchers show that the HUVEC are still alive after the initial plating of the cells, the calcein AM live cell stain is not sufficient to prove that these cells are behaving as they should in such an environment. There is no evidence that shows these cells have not been altered in such a way that renders the model unreliable for further applications. Perhaps some experiments need to be performed to demonstrate that these cells still produce the appropriate response to certain stimuli, e.g., stimulating the endothelial cells with some cytokine that should cause a subsequent upregulation of a certain adhesion molecule. The paper also did not address the flow rate of fluid perfusion through the channels, which would affect the shear forces applied to the endothelial cells. These HUVECs were shown to be viable for only 24 hours, but in order for the device to be applicable for drug testing or tissue engineering purposes, there needs to be evidence that these cells can survive for more than just one day.
Although their choice in material allows for a well characterized surface that is conducive to HUVEC attachment and proliferation, the stiffness of polystyrene may not be suitable for scaffolds or other applications in tissue engineering. Future research into other biomaterials may be needed in order for this technology to be applicable for clinical use.
This paper addresses the issue of creating channels that can emulate the microvasculature with a predictable surface chemistry as well as more physiological geometries, i.e., cylindrical channels that can vary in diameter and have smooth bifurcation transitions. However, there are still certain limitations to their method. In terms of fabricating the channels, it appears that the dimensions of the semi-circular cross section are inconsistent: some are round while others are elliptical. This may cause problems during the alignment of the two sheets of molded polystyrene. The channel sizes are also fairly large (about 200 μm in diameter) and in order to successfully emulate the microvasculature environment, the channels should account for the capillaries, venules, and/or arterioles, vessels that range anywhere between 5–40 μm in diameter.
Though the researchers show that the HUVEC are still alive after the initial plating of the cells, the calcein AM live cell stain is not sufficient to prove that these cells are behaving as they should in such an environment. There is no evidence that shows these cells have not been altered in such a way that renders the model unreliable for further applications. Perhaps some experiments need to be performed to demonstrate that these cells still produce the appropriate response to certain stimuli, e.g., stimulating the endothelial cells with some cytokine that should cause a subsequent upregulation of a certain adhesion molecule. The paper also did not address the flow rate of fluid perfusion through the channels, which would affect the shear forces applied to the endothelial cells. These HUVECs were shown to be viable for only 24 hours, but in order for the device to be applicable for drug testing or tissue engineering purposes, there needs to be evidence that these cells can survive for more than just one day.
Although their choice in material allows for a well characterized surface that is conducive to HUVEC attachment and proliferation, the stiffness of polystyrene may not be suitable for scaffolds or other applications in tissue engineering. Future research into other biomaterials may be needed in order for this technology to be applicable for clinical use.
5 comments:
How precise is their method of bonding the two halves? It seems like a lot of work to make sure everything fits exactly right without having leaks or other complications.
Do the authors mention anything else about the fabrication method of the endothialized microvascular network? Did they use a pulsating flow model in order to simulate the pulsation of the heart during rhythmic beating? The speed and pattern at which the liquid within the developing vasculature flows affects the way the cells orient and develop.
After reading your post, my biggest question is in regards to the clinical applications of this technique. While it is scientifically interesting to create a TCPS substrate for endothelial cell growth, it seems to me like an adaptation of currently well-established technology with very little relevance. As you and the authors have stated, TCPS is a well-characterized surface that is commonly used to support cell adhesion and growth. It should not come as a surprise that endothelial cells are able to adhere and remain viable after 24hr. For this paper to really reach an interesting conclusion, I feel that the authors would need to show that endothelial cells not only grow, but, as you mentioned, remain fully functional or even start exhibiting in vivo properties. Additionally, the choice of TCPS as a vascular substrate material is puzzling, since the modulus of TCPS would make it useless for nearly every biological application.
Fergus: I agree that it seems the bonding and aligning process seems like a lot of work for something so basic. They used an optical aligner to align the two halves and then evaluated the alignment under a microscope. No mention of the precision was made, at least no quantitative measures were mentioned.
Brian: The authors basically just explained how they fabricated the device (more details in the paper itself). There is no mention of a pulsating or varied flow model. I think that's a good idea for future work. I think in this paper, they just wanted to show that the cells could survive in such an environment (though I agree that the flow is important in creating a physiologic environment).
Steven: The paper doesn't show ground-breaking technology, however, despite the fact that TCPS is a very well characterized material, the fact that the endothelial cells will adhere and survive in such a 3-D environment is novel. Other than that, I agree with your comments: TCPS is not a very physiological biomaterial and a lot more needs to be done to show the functionality of such a model.
That's an interesting method to create circular microfluidic channels.
One method that I know of is to simply place a thin wire in a petri dish, then pour PDMS over it until it surrounds the tube. You can then either pull the tube out, or depending on the material, dissolve it away using a particular solvent. Is there a reason they didn't use this technique to fabricate the circular microchannels? Also, what were their motivations behind specifically using PDMS, instead of making an initial PDMS mold and generating something like a plastic mold from that?
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