What in vitro models does NIZO offer?
We have set up, optimized or developed a range of in vitro modelswhich are especially useful in the screening phase of product development, when comparison of a larger number of ingredients may provide relevant information and requires a high or medium throughput model system.
Our in vitro models include the following:
Cell culture models
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
- Probiotic adhesion to intestinal epithelial cells
- Gut barrier integrity or enforcement (TEER assay)
- Intestinal epithelial signalling (cytokine/chemokine
- 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.
SIMPHYD: simulation of digestion
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
- 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.
New potential probiotics and prebiotics are identified on a daily basis and it is practically impossible to test all these bacteria and compounds in clinical trials. To pre-select the bacteria and compounds with the highest potential of exerting a probiotic or prebiotic function we have developed the MicroColon model. The MicroColon technology allows you to screen early in the development process what type of gut bacteria are stimulated by specific microbiome modulators, in a high-throughput manner.
Our in vitro MicroColon model is a 96-well plate system, in which different concentrations of food ingredients are added to faecal slurries derived from infants, adults, or animals. When desired, MicroColon can be spiked with specific pathogens of interest. Different optimized culture media are being used, specific for the adult, infant or animal gut microbiota setting, and conditions mimicking the human gastrointestinal tract are employed. The technology enables monitoring of the effect of a wide range of food and pharmaceutical ingredients on live bacteria, in terms of composition as well as activity.
We routinely monitor shifts in composition and functionality within the gut microbiome by 16S profiling or shotgun metagenomics (Illumina platforms). In addition, specific bacterial or gene targets can be quantified by qPCR. Metabolite production, in particular of short chain fatty acids (SCFAs), is routinely analysed by our HPLC method. Sampling also allows for custom metabolomics analyses, including specific gases, by LC-MS, GC-MS or NMR.
These technologies let our customers rapidly benchmark their new ingredient, or select the most promising ingredients from large panels to rapidly move into a preclinical phase with a limited number of ingredients.
Would you like to screen for the prebiotic or probiotic with the highest potential? Contact Ioana Iorga.
Examples of applications of the MicroColon model include:
- Comparison of novel infant formula ingredients with the gold standard, for their stimulating effect on beneficial Bifidobacteria species, and/or the effect on the production of short chain fatty acids (SCFA).
- 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.