(M0930-05-30) Dynamic In Vitro Cell Culture Model to Study Pathogenic Interactions in Periodontitis In Flow Using a Lab-on-a-Disc Platform

Purpose: Periodontitis is one of the most common chronic inflammatory diseases worldwide [1]. Recently, the overgrowth of human herpes viruses (HHVs), including herpes simplex virus type 1 (HSV-1), has been suggested as a possible trigger for oral dysbiosis resulting in the progression of periodontitis [2]. Thus, there is a demand for in vitro platforms to facilitate studies of the interactions between oral cells (e.g. epithelial cells and fibroblasts) and HHVs in conditions mimicking the oral cavity to enable a better understanding of fundamental biological processes and evaluate new treatment strategies. Traditionally, viral infections are studied in cells cultured in static conditions, e.g. well plates. However, different methods to study cells in perfusion have received increasing attention [3, 4, 5]. A recently established methodology to study cell growth under perfusion in vitro is based on the Lab-on-a-Disc (LoD) technology [6]. LoD is a disc-shaped microfluidic platform the size of a DVD, where centrifugal forces are used to manipulate fluids through channels and chambers [6]. Compared to conventional perfusion systems, LoD platforms are advantageous since there are no external pumps, valves or tubings. In this work, we aim to establish a physiologically relevant LoD model to study dynamic interactions between cells isolated from the oral cavity, e.g. periodontal ligament (PDL) cells, and HHVs as it may occur in periodontitis.

Methods: Lab-on-a-Disc platform for cell culturing: The LoD platform was designed and fabricated as previously described [6]. Briefly, the platform is composed of four poly(methyl methacrylate) (PMMA) layers bonded together by three double-sided pressure-sensitive adhesive (PSA) layers. It comprises an inlet (3 mL) and waste reservoir (5 mL) as well as a cell chamber (23 µL), which are all connected by microchannels (Fig. 1). Cell growth and HSV-1 infection studies in the LoD: PDL were cultured in alpha minimum essential medium (αMEM) containing 10% (v/v) fecal calf serum, 100 units/mL penicillin and 100 µg/mL streptomycin. Prior to cell seeding (7.7 x 10⁴ cells/cm²), the LoD was sterilized for 1 h with 0.5 M sodium hydroxide and the cell culture chamber was coated with 23 µL Matrigel^® Matrix (Corning^®, NY, USA) at 37°C for 1 h. The cells were allowed to adhere in static conditions overnight before rotation was initiated (48 rpm). At 48 h after seeding, HSV-1 mCherry (fluorescent tag), gifted from Professor Desai [7], was added to the medium in the inlet reservoir (multiplicity of infection (MOI) of 6) to infect the cells via flow. After 1.5 h, the cells were washed with PBS and fresh medium was added. The cells were imaged at 2, 24 and 48 h after infection (ZEISS Axio Vert.A1, ZEISS, Germany). RNA extraction and RT-PCR analysis: RNA extraction was performed using a Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany), and quantified using a microvolume spectrophotometer (SimpliNano™ Biochrom, Cambridge, UK). All samples were normalized to 1.7 ng/µL in the retro-transcriptase step, which was done independently of the PCR. RT-PCR using Power SYBR^® Green PCR Master Mix (Qiagen, Hilden, Germany) was applied to detect the following six target genes; infected cell protein (ICP) 0, 4 and 8 as well as interferon (IFN) α, β and λ. RT-PCR experiments were performed using QuantStudio™ 5 (Applied Biosystems™, Waltham, MA, USA) with 3.4 ng of cDNA (equivalent RNA) in a final volume of 20 µL, applying the following amplification conditions: 95°C, 10 min; (95°C, 15 sec; 60°C, 1 min) cycled 40 times. Each sample was run in technical triplicates and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control.

Results: The PDL cells successfully formed a confluent monolayer 48 h after seeding during culture in flow (0.15±0.05 µL/min, n=4). At 24 h after infection with HSV-1 mCherry, the infection was visible by fluorescent imaging. The infection increased in intensity after 48 h where most cells were infected (Fig. 2B). The increasing intensity of the infection with time was confirmed by normal light microscopy images of the cells compared to a control (Fig. 2A). From the RT-PCR analysis, an increasing level of ICP0, ICP4 and ICP8 was observed during the experiment (Fig. 3A), all at levels much higher than what has previously been observed in static cultures of the same cells with lover viral MOI [Chevalier and Ortis et al., unpublished]. At 48 h after the infection, the antiviral proteins IFNα, IFNβ and IFNλ were detected as a cellular response to the viral infection (Fig. 3B).

Conclusion: The results show that we have developed a flow-based in vitro infection model using a novel LoD platform to study dynamic interactions related to oral diseases, which is otherwise not available today. The higher levels of ICP in the LoD compared to static cell cultures illustrate the importance of studying viral infections in flow. Future work will include using the model to study the effect of antiviral drugs and IFNs on viral infections.

References: 1. Kinane DF, Stathopoulou PG and Papapanou PN. Periodontal diseases. Nat Rev Dis Prim., 3(1):17038 (2017).
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5. Rahimi C, Rahimi B, Padova D, Rooholghodos SA, Bienek R, Luo X, Kaufman G and Raub CB. Oral mucosa-on-a-chip to assess layer-specific responses to bacteria and dental materials. Biomicrofluidics, 12 (5): 054106 (2018).
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7. Etienne L, Joshi P, Dingle L, Huang E, Grzesik P and Desai P. Visualization of herpes simplex virus type 1 virions using fluorescent colors. J. Virol. Methods. Mar;241:46-51 (2017).

Acknowledgements:The authors would like to acknowledge the Independent Research Fund Denmark for funding of the project ‘Novel Microfluidic Platform for Investigation of Virus-Biofilm Interactions’ (Grant No. 2031-00008B) and the Novo Nordisk Foundation for funding of the project ‘Cell culture and On-line Monitoring Platform Anchored on Centrifugal microfluidics Technology’ (COMPACT) (Grant No. NNF21OC0069057).

Figure 1. Compartments in LoD for cell culturing in flow. It comprises an inlet reservoir (3 mL), a waste reservoir (5 mL), a cell culture chamber (23 µL) and a channel for cell seeding.

Figure 2. Representative images (10x) showing the growth of a) brightfield images of PDL cells without HSV-1 infection, and b) corresponding brightfield and fluorescent images of PDL cells infected with HSV-1 mCherry at 2, 24 and 48 h after initiation of HSV-1 infection.

Figure 3. RT-PCR quantification of a) ICP0, ICP4 and ICP8 and b) IFNα, IFNβ and IFNλ after extraction of RNA from PDL cells at 2, 24 and 48h after initiation of HSV-1 infection. All quantifications are relative to the corresponding controls (PDL cells without infection), which are set to 1 in the graphs. All samples were run in technical triplicates and normalized to GAPDH as endogenous control.