The inception of human skin-equivalent cultures occurred in the early 1980s. Since then, each rendition has added a layer of sophistication to achieve greater likeness to human skin. The lab of Dr. Jia Zhu, Associate Professor at the Fred Hutchinson Cancer Center in the Vaccine and Infectious Disease division, constructed a 3D skin-on-chip culture with channels of microvascular networks supporting the perfusion of immune cells or drugs to the interior layers of dermal fibroblasts for the study of herpes simplex virus (HSV) infection. The characterization of this modified skin culture and proof-of-principle experiments comparing human HSV infection in skin biopsies to the skin-on-chip platform were completed and published recently in Nature Communication.
Natural human HSV infections occur on the skin and mucosal surfaces resulting in lifelong, recurrent disease. The virus has both a latent (dormant) state and a lytic replicating (active) state that produces virus progeny and results in lesions of the skin. Various forms of stress are known inducers of HSV lytic replication and the formation of skin lesions and as a result, symptoms can occur sporadically. To learn more about HSV infections, biopsies of patient HSV lesions can be used to provide valuable insights. However, the spontaneous nature of virus lytic replication poses a challenge in studying early events during virus reactivation. One strategy to overcome this challenge is to use a cultured skin-equivalent system to study HSV infection and reactivation, but current systems are imperfect. Dr. Zhu explained the significance of her lab’s newly designed model: “our skin-on-chip model…provides a new 3D skin-mimetic that constitutes a functional vascular system for immune-cell and drug perfusion to allow one to study viral infection in skin, investigate both innate and adaptive immune responses and to evaluate antiviral drug efficacy in vitro with human cells.”
The initial construction of skin-like cultures included only two components, dermal and epidermal cell co-culture, and later grew in complexity to include fat, immune, neuronal, and other cell types. Yet, unsuccessful incorporation of vasculature and incompatibility with small molecules represented critical limitations of these systems prior to the skin-on-chip constructed in Dr. Zhu’s lab. As part of the solution, a collagen gel was used as the structural matrix in which human dermal fibroblasts were embedded to form the dermal compartment. Next, a hollow microchannel network was made within the gel by using two molding techniques, soft lithography and injection molding. The human microvascular endothelial cells were seeded into these open microchannels to form the vascular membrane of vessels. Lastly, on top of the gel matrix, human epidermal keratinocytes were seeded, grown to confluence, and then exposed to the air for cell differentiation of external skin barrier. Importantly, while the top layer of the differentiated keratinocytes was exposed to air, the lower dermal layers and vasculature remained submerged in the liquid cell culture media.
To characterize this 3D skin-on-chip system, the researchers first determined the resilience of differentiated keratinocytes to HSV infection. To test this, the top layer of keratinocytes was infected at different time points following exposure to the air which initiates cell differentiation. Once exposed to air, the percentage of infected keratinocytes decreased by half and 3 days later, infection rates were negligible. This observation is consistent with our current understanding of skin being the first line of defense against invading viruses. The researchers next used a biopsy tool to disrupt the keratinocyte layer as during tissue microinjury to allow entry of HSV to the interior epidermis layer of the skin culture. Following injury, HSV infected the epidermal cells efficiently and damaged cells displayed multi-nucleation, enlarged nuclei and changes in chromatin distribution, features which are observed in HSV lesions. The researchers also visualized neutrophil migration from the microvasculature into the epidermal-like compartment and quantified heightened pro-inflammatory cytokines in the media for HSV infected conditions as compared to uninfected. Again, these observed phenotypes were consistent with human biopsy samples of HSV lesions. As a final line of investigation, the researchers tested the ability of this system to evaluate the efficacy of antiviral drugs to HSV infection. Acyclovir is an antiviral drug routinely used to limit HSV disease severity. In the skin-on-chip system, addition of acyclovir via the inlet at the same time as HSV infection significantly limited HSV infection, validating the use of this system in drug efficacy screening for HSV infection.
The researchers demonstrated the utility of this skin-on-chip system to study immune responses and small molecule treatment of HSV infected cells. “Furthermore, the epidermis, dermis, and perfusable vasculature in the skin-on-chip were in direct contact with collagen and other extracellular matrix components, further promoting cell-cell and cell-matrix crosstalk among keratinocytes, fibroblasts, endothelial cells, and the surrounding microenvironment,” explained the researchers, which is necessary for studying the immune response. Another key feature of this system is the absence of polydimethylsiloxane (PDMS), a material often used in microfluidic system construction, that unfortunately absorbs small molecule drugs. This has been a significant barrier in studying the effect of drugs on the skin-equivalent systems. “This tool opens up new possibilities to study viral infection and host interactions, particularly in the human system,” stated Dr. Zhu. “It enables one to fine tune various parameters and test its consequences either individually or in combination.” Dr. Zhu continued by expressing her vision for the future, “we hope this technology will contribute to precision medicine when we generate patient-specific [skin-on-chip] devices.”
The spotlighted research was funded by the Cellular Imaging Shared Resource (CISR) of the Fred Hutch/University of Washington Cancer Consortium grant and the National Institutes of Health.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Jia Zhu contributed to this work.
Sun S, Jin L, Zheng Y, Zhu J. 2022. Modeling human HSV infection via a vascularized immune-competent skin-on-chip platform. Nat Commun. 13(1):5481.