Cell-based in vitro assays provide a step-up approach in the evaluation of specific functionalities of food ingredients, that could be related to in vivo effects. Our cell culture models target gut and immune health, and in particular the ability of ingredients to affect resistance to infection. Several of these models can be used in combination with exposure to specific pathogens or pathogen, which will give you information on the potential efficacy of the ingredient to protect humans against infection with this or similar pathogens.
Specific cell assays that we offer include:

• Pathogen adhesion to intestinal epithelial cells
  (anti-adhesion assay)
• Probiotic adhesion to intestinal epithelial cells
• Gut barrier integrity or enforcement (TEER assay)
• Intestinal epithelial signalling (cytokine/chemokine
  production)
• Pathogen co-aggregation
• Pathogen viability/antimicrobial activity
• Immune assay (using RAW264.7 murine macrophage cell
  line or human PBMCs). The pathogens that we mostly work with are E. coli strains (e.g. a typical diarrhoea strain like ETEC H10407) or Salmonella strains. Our unique human E. coli challenge model uses an attenuated E.coli strain (E. coli strain E1392/75-2A) to induce mild and transient gastrointestinal symptoms in healthy volunteers, which is why we prefer to use this, or similar strains, also in our in vitro assays, enhancing the validity of the in vitro – in vivo translation.
  Pro- or anti-inflammatory activity can be analysed through a range of cytokines and chemokines produced upon cell stimulation (e.g. IL-8, IP-10, IL-10, TNF-alpha), the panel of which is determined based on the type of assay and the stimulus used in the assay.

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The NIZO SIMPHYD platform (SIMulation of PHYsiological Digestion) has been developed to study gastric behaviour and gastrointestinal digestion under simulated human digestion conditions. The behaviour of food during gastrointestinal passage may affect the dynamics of overall digestion, gastric emptying, nutrient absorption and subsequent nutrient use, for example in muscle protein synthesis. The platform allows you to compare food ingredients or formulations with respect to textural properties during gastrointestinal passage, structure formation and breakdown, digestibility and digestion rate. SIMPHYD includes different in vitro model systems, focusing on elucidation of different characteristics:

•  Apparent viscosity of a protein solution under
  gastric conditions (including particle size
  distribution).
• Aggregation of proteins under gastric conditions (to
  be combined with protein degradation measures).
• Protein breakdown over time, under both gastric and
  small intestinal conditions.

All of the model systems can be used with either adult or infant conditions. Depending on the specific research question, mixing with artificial saliva may be the first step, before simulated gastric acidification is initiated and simulated gastric secretions (main component: pepsin) are added. To simulate duodenal conditions, pH is neutralized and simulated intestinal secretion (including pancreatin and bile acids) is added. Protein breakdown can be measured by various analytical techniques, e.g. OPA, SDS-PAGE, HPLC, LC-MS.

The human large intestine is not easily accessible for in vivo investigation of the interactions of food ingredients, pathogens or pharmaceutical components with gut microbiota. Therefore, there is a high demand for in vitro models for assessment of pre- and probiotic or antimicrobial effects in a system mimicking the in vivo gut microbiome. NIZO’s in vitro high-throughput fermentation microcolon technology allows for anaerobic screening of food, pathogenic and pharmaceutical components to assess their modulating effects on the gut microbiome.

Our microcolon in vitro model is a 96-well plate system, in which food ingredients are added to different concentrations of human (infant or adult) or animal faecal slurry. Different culture media are used, specific for the adult,infant or animal gut microbiome setting. The system enables monitoring of the effect of a wide range of food and pharmaceutical ingredients,(potential) probiotics and pathogens on live and active bacteria, in terms of composition as well as activity.
Bacterial or gene targets can be quantified by qPCR, whereas shifts in composition and functionality within the gut microbiome can be monitored by 16S profiling (Illumina platform), and shotgun metagenomics, respectively. Metabolite production (e.g. short chain fatty acids, specific gases) can be analysed by HPLC, NMR, LC-GCMS.

Applications of the microcolon system includes:
• Comparison of novel infant formula ingredients with the gold standard (human breast milk), for their stimulating effect on beneficial Bifidobacteria strains, or the effect on the production of short chain fatty acids
• Testing of relevant strategies for pathogen inhibition and their effect on gut bacterial communities.
• Identifying prebiotic compounds to be added to animal feed on formation of malodorous volatiles/gases.

Recent developments and progress in skin microbiome research have enabled fast characterization of microbial communities and identification of relevant skin health microbiome modulators in the personal care and pharma industries. Next to the state-of-the-art high throughput robotized assays there is a high demand for in vitro studies that can assist in claim substantiation or selection of new candidates.

The MicroSkin model is a unique in vitro high-throughput screening technology that enables mimicking the growth and inhibition of important members of the skin microbiome upon addition of specific prebiotic or antimicrobial components. The technology makes uses of a human skin mimicking growth medium based on human stratum corneum (callus) and serves as the main nutrient source for commensal skin bacteria. MicroSkin is a 96-well-plate technology, in which components are tested for their ability to reduce or stimulate bacterial growth. Large numbers of test compounds/microbiome modulators such as antimicrobial agents or prebiotics can be tested simultaneously , whichsupports the biological relevance of the outcomes.
Bacterial growth can be determined by using automated CFU counting and monitoring the pH during the course of the experiment. Metabolic activity of the bacteria can be assessed by continuous pH monitoring and detection of bacterial metabolites by targeted or untargeted metabolomics. Assessment of bacterial composition or functionality can be assessed by 16S rRNA gene-based Illumina sequencing or shotgun metagenomics of the MicroSkin-collected samples, respectively.

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