Long term in vitro culture systems to study human microbiome

Abstract

Microbial communities are eubiotic ecosystems that interact dynamically and synergistically with the human body. Imbalances in these interactions may cause dysbiosis by enhancing the occurrence of inflammatory conditions, such as periodontal or inflammatory bowel diseases. However, the mechanisms that lie behind eubiosis-dysbiosis transitions are still unclear and constantly being redefined. While the societal impact of these diseases is steadily increasing, the lack of a clear understanding behind the onset of the inflammatory conditions prevents the proper clinical strategies from being formulated. Although preclinical and clinical models, and short-term planar in vitro cultures represent superb research tools, they are still lacking human relevance and long-term use. Bioreactors and organ-on-a-chips have attracted interest because of their ability to recreate and sustain the physical, structural, and mechanical features of the native environment, as well as to support long-term co-culture of mammalian cells and the microbiome through modulation of pH and oxygen gradients. Existing devices, however, are still under development to sustain microbiome-host co-culture over long period of time. In this scenario, to understand disease triggers and develop therapeutics, research efforts should command the development of three-dimensional constructs that would allow the investigation of processes underlying microbial community assembly, and how microorganisms influence host traits in both acute and chronic conditions.

Graphical Abstract

Overview of current experimental models to systematically study the human microbiome. Partially created with BioRender.com.

Summary:

The human body functions synergistically with a large population of symbiotic microbial cells forming the microbiome. This synergy has resulted in an intertwined and balanced relationship, whereby the microbiome contributes to host physiological processes in several organ systems, such as digestive, immune, and overall organ development. The host provides nutrition, various niches (i.e. oral, skin, lung, gut, or vagina), and the release of antimicrobial factors against exogenous species for microbial survival.^1–3 Within the body, in fact, the microbiome forms an ecosystem that is eubiotic and constantly evolving in response to host life events (i.e., lifestyle, age, nutrition, or diseases).⁴ This synergistic relationship begins with the recognition of microbial components, pathogen-associated molecular patterns (PAMPs), by the host that instructs the adaptive immune response and promotes pathogen elimination.⁵ Several factors could alter these balanced interactions leading the microbiome toward a dysbiotic state.¹ Indeed, alteration of host immune competence, diet, environmental factors, or antibiotic treatments may cause the microbiome composition to change or be altered or to translocate to a different site in the body.^4,6–7 Although the boundaries between eubiosis and dysbiosis are continuously redefined, one of the accepted hypotheses views low-abundance microbial pathogens prevailing and remodeling the microbiome toward a dysbiotic state by enhancing inflammatory conditions.^{1, 8–9}

Due to strong correlations of disease prevalence with an imbalanced microbiome, awareness in host-pathogen interactions has been recently growing. Currently, the National Institutes of Health is investing $215M in the Human Microbiome Project (HMP) with the intent of studying the role of microbial communities in human health and diseases,¹⁰ projecting a human microbiome market estimate of $1.6 billions by 2028.¹¹ Some of the pathologies currently investigated are periodontitis, atopic dermatitis, or asthma and inflammatory bowel disease.^2,12 Indeed, these chronic diseases are emerging as drivers of global socioeconomic status because of the associated cost of diagnosis and treatments;^13–14 for example, one-tenth of the worldwide adult population suffers from periodontitis, resulting in a cumulative financial burden of $3.49 billion in the United States alone.¹⁵ In addition, healthy human microbiome has been probed using pre/prosynbiotics in personalized medicine as modulators for obesity, liver disease, or environmental-induced perturbations.¹⁶ Current knowledge of these unbalanced states is hampered by the intrinsic biological complexity of microbiome systems, stemming from the large intra- and inter-individual variation in composition and prevalence.¹⁷ Advances in research have sought to develop frameworks that go beyond the single pathogen-disease correlation. Specifically, the HMP highlighted how some diseases are the results of microbial membership and metabolites combination that depend on host factors (i.e., race or ethnicity), but also on the type of microbial strain.^18–19 Combining multi-omics analysis⁷ with in vivo and in vitro studies may be a robust strategy to investigate microbial community structure, inter-individual variation, through modeling host-microbiome interactions associated with pathologies, and to develop successful therapeutic approaches.^1,19 It is still unclear whether dysbiosis precedes, and thus causes, the disease state or vice versa, or whether it initiates a vicious cycle; nevertheless, the role of the microbiome in the onset of the disease remains an underdeveloped field of research.

Current experimental strategies to investigate host-microbiome interactions consist of preclinical and clinical models (Figure 1). Due to the complexity of these models, it is difficult to independently control experimental variables, to identify disease triggers, and to target responses. In addition, animal models exhibit differences in microbiome composition and organization, that limit their clinical translation.^{20–21,22–24–25–26} In light of this, a successful strategy could be the implementation of a humanized in vitro model, that would recreate the physical, structural and mechanical conditions of the native environment in a long-term high throughput-controlled system. Patient-derived 2D organoid monolayer models (stomach, intestine, gallbladder) have been used to study infection processes, epithelium integrity, inflammatory response and are commonly employed as high-throughput platforms for drug screening.^27–28–29 These approaches would provide a short-term simplified, although controlled, experimental model to investigate host-microbiome interactions.^23,30–31 However, 3D models have been proved to better recapitulate the native tissue architecture of the host-microbiome interface, but most importantly the physical, structural, and metabolic conditions to support the co-culture of aerobic and anaerobic bacteria species in the microbiome by modulating physiological mechanical cues, oxygen, and pH gradients.^32–34 Accordingly, static 3D-tissue models (e.g. scaffolds, hydrogels, organoids) have been also developed; pre-clinical models include collagen type I hydrogels to study pathogens infiltration in an oral mucosa-like construct,³⁵ or intestinal epithelial cells seeded in a silk-based scaffold colonized by Lactobacillus rhamnosus GG.³⁶ Likewise, intestinal organoids colonized by non-pathogenic E. Coli were used to assess epithelium barrier function.³⁷ In addition to the 3D architecture,³⁸ a major factor in promoting long-term cellular survival as well as bacterial colonization is represented by the introduction of a dynamic culture regime.^39–40

Figure 1 – Current experimental models.

Overview of current experimental strategies and design criteria to investigate underlying microbiome assembly and prevalence, and how microorganisms influence host responses. Partially created with BioRender.com.

Bioreactors and organ-on-a-chips are emerging technologies that aim to recreate long term structural and functional features of native environments coupled with dynamic fluxes (Figure 1).^41–44 Several humanized intestine systems, or gut-on-a-chip, have been developed to analyze the role of microbiome in physiological and inflammatory processes.^45–46 These devices have been successfully implemented for drug and therapeutic screenings, supporting the co-culture of human cells with artificial or patient-derived microbiome for up to 24h.⁴⁷ A recent successful example is an anaerobic intestine-on-a-chip device that sustained the co-culture of mammalian cell lines and infant microbiota samples for up to 3 days.³⁹ Additionally, other devices have been developed to study the oral mucosal response to selected bacterial populations^48–49 or to recreate the alveolar-capillary interface of the human lung and simulate bacteria pulmonary infection.⁵⁰

Despite the recent progress, the long-term survival of human-derived microbiome with mammalian cells and the formation of oxygen and pH gradients are some of the main challenges in existing technologies, most of which have failed a day after bacteria inoculum.^{51–49–48, 52} Furthermore, the struggle in adequately balancing richness and diversity in the microbiome has contributed to both the use of bacterial lines, which that partially resemble the overall microbiome,⁵³ due to the invasiveness of sampling methods, or gnotobiotic mice.^{39 54} Moreover, difficulties have been encountered on incorporating immune cells due to their short lifespan as well as the negative impact of microgravity on their functions.^39,55–56 Finally, the integration of sophisticated biorecognition sensors for pathogens or biomolecules analysis to encompass lifestyle-related changes have been adopted for acute studies although not cost-effective for large scale screenings.⁵⁷ Thus, there is a compelling need to improve current culture technologies to provide sustained in vivo like environments to investigated host-pathogen interactions in acute and chronic conditions.

As the field advances, emphasis should be given to engineer three-dimensional constructs and culture devices that would support the formation of physiological oxygen gradients, while maintaining a neutral pH range, and recreating native mechanical cues. The acting of these elements in concert would support the maintenance of the native microbiome in composition and spatial organization, as well as promote phenotypic tissue traits.^48,58 Importantly, the inclusion of dynamic culture regimes that buffer bacterial population might help to extend the culture viability to study the host-microbiome cross-talk over time.⁴⁵ Such bioreactors would be instrumental to investigate the complexity of the inter-individual variation of the microbiome using mapping tools to assess the microbiome spatial organization and to identify both host-microbiome and microbe-microbe relationships at single-cell resolution under non-physiological perturbations.^{59,60–61,62} In addition, these devices should be implemented with sensors capable of detecting fluctuations of physical conditions (i.e. oxygen content, pH) that may be associated with health to disease transitions, as previously reported.^63,64 Finally, the conjunction of human cells with the microbiome will be instrumental for the investigation of processes underlying microbial community assembly, and how microorganisms influence host traits in both acute and chronic conditions.

Glossary/Definitions:

Host

a larger organism, inhabited by other organisms (i.e., bacteria, virus, fungus), able to provide a suitable environment to live and nutrients to support the growth of such smaller organisms.

Microbiome

multiple communities of microorganisms (i.e., bacteria, virus, fungus) that colonize a defined niche in the human body and live synergistically with the host by contributing to the maintenance of its health and well-being.

Eubiosis

a physiological and balance state characterized by richness and abundance in species forming the microbiome.

Dysbiosis

a non-physiological state characterized by imbalanced interactions among bacteria within the communities or between host and microbiome. Dysbiosis can be detrimental to both the host and the microbiome leading to a shift in microbiome composition toward virulent species and activation of the immune response by the host.

Tissue model

two or three-dimensional in vitro environments, designed to mimic the native tissue cytoarchitecture and functions to study cellular interplay in both physiological and pathological conditions.

Bioreactor

a manufactured dynamic apparatus used in tissue engineering and regenerative medicine to sustain in vitro long-term viability and function of human tissue systems by providing physiological and controlled mechanical and chemical stimuli, while providing adequate mass transfer, nutrients supply and waste products management.