Abstract
The avian respiratory tract is a common entry route for many pathogens and an important delivery route for vaccination in the poultry industry. Immune responses in the avian lung have mostly been studied in vivo due to the lack of robust, relevant in vitro and ex vivo models mimicking the microenvironment. Precision-cut lung slices (PCLS) have the major advantages of maintaining the 3-dimensional architecture of the lung and includes heterogeneous cell populations. PCLS have been obtained from a number of mammalian species and from chicken embryos. However, as the embryonic lung is physiologically undifferentiated and immunologically immature, it is less suitable to examine complex host–pathogen interactions including antimicrobial responses. Here we prepared PCLS from immunologically mature chicken lungs, tested different culture conditions, and found that serum supplementation has a detrimental effect on the quality of PCLS. Viable cells in PCLS remained present for ≥ 40 days, as determined by viability assays and sustained motility of fluorescent mononuclear phagocytic cells. The PCLS were responsive to lipopolysaccharide stimulation, which induced the release of nitric oxide, IL-1β, type I interferons and IL-10. Mononuclear phagocytes within the tissue maintained phagocytic activity, with live cell imaging capturing interactions with latex beads and an avian pathogenic Escherichia coli strain. Finally, the PCLS were also shown to be permissive to infection with low pathogenic avian influenza viruses. Taken together, immunologically mature chicken PCLS provide a suitable model to simulate live organ responsiveness and cell dynamics, which can be readily exploited to examine host–pathogen interactions and inflammatory responses.
Highlights
Organotypic tissue slices have been prepared from a number of organs for decades, with the advent of automated slicers allowing reproducible thin sections to Bryson et al Vet Res (2020) 51:2 by generating a large number of replicates, and reducing the number of animals required to test a hypothesis, meeting the 3Rs principle
Precision-cut lung slices (PCLS) were cultured with Dulbecco’s Modified Eagles Media (DMEM), DMEM/F12 or DMEM/F12/fetal calf serum (FCS), with DMEM/F12 providing a broader range of inorganic salts, amino acids and vitamins than DMEM
PCLS cultured with DMEM, in the absence of FCS had a mean 30% greater cell viability, than those cultured in the nutrient and growth factor rich DMEM/F12/FCS at 3 days post slice (Figure 1A)
Summary
Organotypic tissue slices have been prepared from a number of organs for decades, with the advent of automated slicers allowing reproducible thin sections to Bryson et al Vet Res (2020) 51:2 by generating a large number of replicates, and reducing the number of animals required to test a hypothesis, meeting the 3Rs principle. As PCLS can remain viable in culture for a number of weeks [2, 4], this allows dynamic time course studies to be carried out in the same tissue. As viable immune cells, including macrophages, neutrophils, DCs and T cells, have been identified in mammalian PCLS [8], PCLS have been used for studies on host–pathogen interactions, including viral infection and inflammatory responses [7, 9,10,11], and viral/bacterial coinfection [5]. PCLS are applicable to live, dynamic imaging of immune interactions [8, 12]. Applying bio-imaging tools to PCLS renders capturing the initial and very early immune events including host–pathogen interactions much more accessible than would otherwise be possible in vivo
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