A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications

Hydrogel materials show a number of properties which make them interesting candidates to be utilized to mimic the extracellular matrix (ECM). Therefore, these materials are attractive for use in biological applications such as tissue engineering, cell culture 3D bioprinting and more.

Are you planning to use hydrogels for the first time in your research?

Then have a look at the insightful article “A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen.

In their article the authors describe and evaluate the different technologies that are most commonly used to assess hydrogel microarchitecture.

Francisco Drusso Martinez-Garcia et al. explain the working principle of the various methods and also discuss the merits and limitations of each of them in view of their usefulness for the characterization of hydrogels.

They introduce and explore the pros and cons of the following methods: Scanning Electron Microscopy (SEM), Cryogenic Scanning Electron Microscopy (Cryo-SEM), Environmental Scanning Electron Microscopy (ESEM), Micro-Computed Tomography (µ-CT), Confocal Laser Scanning Microscopy (CLSM), Second Harmonic Generation and Atomic Force Microscopy (AFM).*

Atomic force microscopy (AFM) can be used to investigate the hydrogel surface topology as well as a hydrogel’s mechanical properties. The latter can be achieved through mathematical modelling of force-distance curves.

When using the AFM to characterize the elasticity of a hydrogel sample it is essential to take the stiffness of the investigated material into account when choosing what kind of AFM probe to use for these experiments.

If an AFM cantilever used for probing a soft sample is too stiff (if the force constant/spring constant is too high) this might result in a poor signal-to-noise ratio.

If a soft AFM probe (an AFM probe with an AFM cantilever with a low force constant) is chosen to investigate a soft material this should lead to a better signal-to-noise ratio. On the other hand, if an AFM cantilever is too soft (if the force constant is too low) then it might not be stiff enough to indent the investigated material.

Another critical factor is the shape and the size of the AFM tip.

Spheroidal AFM probes might stick to the material, resulting in artefacts, disrupted force–distance curves, or even damaged AFM cantilevers. If the AFM tip is much smaller than the pore size of the hydrogel, it might get stuck in the fibrous network microarchitecture.

On the other hand, if the spherical AFM tip, e.g. as in colloidal AFM probes (a sphere glued to end of a tipless AFM cantilever), is too large, the weight of the sphere can have a negative influence on the spring characteristics of the AFM cantilever.

All these factors and more as described in the cited article have to be carefully weighed before deciding on the settings of the atomic force microscope and choosing an AFM probe for the investigation of a specific hydrogel.

NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article.*

Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022: Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article. NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article.
Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022:
Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article.

 

NanoWorld tipless Arrow-TL2 AFM probe array with two tipless AFM cantilevers
NanoWorld® Arrow™ TL2 AFM probes are tipless AFM cantilevers for special applications. They can for example be used for attaching spheres and other objects to the free end of the AFM cantilever, or for functionalizing and sensing applications.
The Arrow™ TL2 probes are optionally available with a sample facing side gold coating (Arrow™ TL2Au).

*Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen
A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications
Gels 2022, 8(9), 535
DOI: https://doi.org/10.3390/gels8090535

The article “A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Carbon nanotube porin diffusion in mixed composition supported lipid bilayers

Lipid membranes play a key role in living systems by providing a structural barrier that separates cellular compartments. Bilayer fluidity in the lateral plane is a key property of lipid membranes, that allows the membrane to have sufficient flexibility to accommodate dynamic stresses, shape changes and rearrangements accompanying the cellular lifecycle.*

In the article “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy describe how they used high-speed atomic force microscopy (HS-AFM) and all-atom molecular dynamics (MD) simulations to study the behavior of CNTPs in a mixed lipid membrane consisting of DOPC lipid with a variable percentage of DMPC lipid added to it. HS-AFM data reveal that the CNTPs undergo diffusive motion in the bilayer plane.*

Motion trajectories extracted from the HS-AFM movies indicate that CNTPs exhibit diffusion coefficient values broadly similar to values reported for membrane proteins in supported lipid bilayers. The data also indicate that increasing the percentage of DMPC leads to a marked slowing of CNTP diffusion. MD simulations reveal a CNTP-lipid assembly that diffuses in the membrane and show trends that are consistent with the experimental observations. *

The above-mentioned study confirms that CNTPs mimic the major features of the diffusive movement of biological pores in lipid membranes and shows how the increase in bilayer viscosity leads to a corresponding slowdown in protein motion. It should be possible to extend this approach to studies of other membrane protein dynamics in supported lipid bilayers. The authors note that those studies, however, will need to be mindful of the challenge of unambiguous visualization of the membrane components, especially in systems that incorporate smaller proteins, such as antimicrobial peptides. Another challenge that could complicate these studies would be microscopic phase separation of the lipid matrix that could lead to complicated pore dynamics in the membrane. *

NanoWorld Ultra-Short AFM cantilevers with high-density carbon/diamond-like carbon (HDC/DLC) AFM tips of the USC-F1.2-k0.15 type were used for the high-speed atomic force microscopy described in the article. *

Figure 2 from “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan et al.:

CNTP motion in supported lipid bilayers. (a) Representative frames (with times in seconds indicated on each image) from an HS-AFM movie showing a CNTP diffusing in a supported lipid bilayer with 80:20 DOPC-DMPC ratio (see also Supplementary Movie 2). (b) A representative trajectory for CNTP diffusion in the bilayer. The time step between each datapoint is 0.5 s. NanoWorld Ultra-Short AFM Cantilvers USC-F1.2-k0.15 were used for the HS-AFM imaging
Figure 2 a and b from “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan et al.:

CNTP motion in supported lipid bilayers. (a) Representative frames (with times in seconds indicated on each image) from an HS-AFM movie showing a CNTP diffusing in a supported lipid bilayer with 80:20 DOPC-DMPC ratio (see also Supplementary Movie 2). (b) A representative trajectory for CNTP diffusion in the bilayer. The time step between each datapoint is 0.5 s.
Please refer to the full article cited below for the full figure.

*Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy
Carbon nanotube porin diffusion in mixed composition supported lipid bilayers
Nature Scientific Reports volume 10, Article number: 11908 (2020)
DOI: https://doi.org/10.1038/s41598-020-68059-2

Please follow this external link to read the full article: https://rdcu.be/b69wj

Open Access : The article “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels

Representing a central element of the innate immune system, neutrophils are recruited from the blood stream to a site of inflammation. The recruitment process follows a well-defined sequence of events including adhesion to the blood vessel walls, migration, and chemotaxis to reach the inflammatory focus. A common feature of the underlying signalling pathways is the utilization of Ca2+ ions as intracellular second messengers. However, the required Ca2+ influx channels are not yet fully characterized.*

In the article “Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels” Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab report a novel role for TRPC6, a member of the transient receptor potential (TRPC) channel family, in the CXCL1-dependent recruitment of murine neutrophil granulocytes.*

The authors describe how they tested whether TRPC6 channels are central elements of the signalling cascade underlying CXCR2-mediated neutrophil recruitment. They combined intravital microscopy, single-cell force spectroscopy with atomic force microscopy, Ca2+ imaging, and microfluidic flow chamber assays to investigate the role of TRPC6 channels in murine neutrophils for their recruitment in renal ischemia-reperfusion and cremaster models as well as in in vitro assays.*

The study reveals that TRPC6 channels in neutrophils are crucial signalling modules in their recruitment from the blood stream in response to CXCL1.*

The single-cell force spectroscopy experiments were performed by using atomic force microscopy (AFM) with NanoWorld Arrow-TL1 tipless cantilevers which were incubated prior to experiments for 30 min in Cell-Tak to make the AFM cantilever sticky for neutrophils.*

NanoWorld Arrow-TL1 Tipless AFM cantilever, single cantilever beam on a silicon support chip
NanoWorld Arrow-TL1
Tipless cantilever,
single cantilever beam on a silicon support chip

*Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab
Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels
Journal of Molecular Medicine volume 98, pages349–360(2020)
DOI: https://doi.org/10.1007/s00109-020-01872-4

Please follow this external link to read the full article: https://link.springer.com/article/10.1007/s00109-020-01872-4

Open Access The article “ Intravascular adhesion and recruitment of neutrophils in response to CXCL1 depends on their TRPC6 channels “ by Otto Lindemann, Jan Rossaint, Karolina Najder, Sandra Schimmelpfennig, Verena Hofschröer, Mike Wälte, Benedikt Fels, Hans Oberleithner, Alexander Zarbock and Albrecht Schwab is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.