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The gut microbiota is a highly specialized organ containing host-specific assemblages of microbes whereby metabolic activity directly impacts human health and disease.
In vitro gut fermentation models present an unmatched
opportunity of performing studies frequently challenged
in humans and animals owing to ethical concerns. Multidisciplinary systems biology analyses supported by omics platforms remain widely neglected in the field of
in vitro gut fermentation modeling but are key to advancing the significance of these models. Model-driven experimentation using a combination of in vitro gut
fermentation and in vitro human cell models represent
an advanced approach in identifying complex hostmicrobe interactions and niches central to gut fermentation
processes. The aim of this review is to highlight the
advances and challenges exhibited by in vitro human
gut fermentation modeling.
Introduction
For decades the contributions of gut microbiota to human
health and disease were widely underappreciated. Acknowledgement of the gut microbiota, described as an
independent organ with immunostimulatory properties,
was first noted in 1992 by Bocci [1]. A decade later Eckburg
et al. identified a suite of gut microbial contributions to the
human host such as providing nourishment, regulating
epithelial cell development and modulating innate immune responses, yet acknowledged that many basic functions of the gut microbiota remain unknown [2]. Since 2005
the majority of studies have focused on species identification and cataloguing, a process that has been hampered by
the inability to culture most gut microbes. Most species are
recognized solely by their 16S rRNA sequence and identification relies heavily on high-throughput technologies
such as second and third generation sequencing platforms
[3]. Success and advancement of these technologies has
demonstrated a link between various microbial species and
multiple pathologies such as obesity, Crohns disease and
irritable bowel syndrome [48].
16S rRNA-based cataloguing of gut microbiota via
high-throughput sequencing platforms does not, however,
provide information on the functionality of any species
identified. Unraveling the complexity of microbemicrobe
interactions and identification of niches central to gut
fermentation are, however, dependent on functional
studies. In vitro gut fermentation models represent an
0167-7799/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.06.011 Trends in Biotechnology, January 2012, Vol. 30, No. 1
17
(Figure_1)TD$IG][ Review
Batch
Key:
Nutrients
Concentration
Filling
Emptying
Metabolites
Planktonic
gut microbes
pH 5.6 5.9
Time (hours)
Inoculation
Continuous
pH 5.6 5.9
proximal
R2
pH 6.1 6.4
transverse
R3
R1
Concentration
R1
pH 6.6 6.9
distal
R2
R3
Steady-state
Time (days)
Inoculation
pH 5.6 5.9
proximal
R2
pH 6.1 6.4
transverse
R3
R1
Concentration
R1
pH 6.6 6.9
distal
R3
Steady-state
Inoculation
Released bacteria
(a)
R2
Time (weeks)
(b)
1-2 mm
1 m
TRENDS in Biotechnology
Figure 1. Types and process characteristics of in vitro colonic fermentation models simulating proximal (R1), transverse (R2) and distal (R3) colon regions, operated at
physiological section-specific constant pH [41], temperature (37 8C) and under strictly anaerobic conditions (e.g. through continuous CO2 or N2 flushing of the headspace).
Initial (R1) and steady-state (R1, R2 and R3) fermentation profiles. (a) Picture detail of a proximal colon reactor containing polysaccharide beads with immobilized fecal
microbiota. (b) Electron microscope image of microbes embedded and attached to the surface of an intestinal bead (the authors acknowledge support of Stefan Handschin
from EMEZ Electron Microscopy at ETH Zurich, Switzerland).
(Figure_2)TD$IG][ Review
In vitro gut
fermentation
modeling
In vitro human
intestinal cell
model
In vivo animal
studies
In vivo human
studies
Figure 2. Human gut systems biology approach for functional analysis of gut microbiota.
(Figure_3)TD$IG][ Review
Transverse colon
1011-1012 cells g-1
Depletion of substrates
Reduction in bacterial activity
Decreasing SCFA production
pH 6.1 - 6.4
Proximal colon
1010-1011 cells g-1
Stomach
<103
cells g-1
Distal colon
1012 cells g-1
Small intestine
104-107 cells g-1
Protein fermentation
Low SCFA production
Carbohydrate fermentation
pH 6.1 - 6.9
TRENDS in Biotechnology
Figure 3. Schematic representation of the human gastrointestinal tract: the amount of bacteria per gram of intestinal contents, nutrient availability and bacterial
fermentative activity typically found in different sections of healthy individuals.
Advantages
Easy to set up, useful for fermentation studies and especially
substrate digestion assessment
Continuous culture
20
Limitations
Short-term fermentation
studies and weakness in
microbiological control
No host functionality and
experiments are time
limited (days or weeks)
No host functionality and
experiments are time
limited (days or weeks)
No host functionality
Refs
[59,61]
No immune and
neuroendocrine response
and experiments are
limited to few days time
[71,81]
[95,97]
[58,95]
[13,14]
Review
community and microbial metabolic modulation requires
continuous substrate replenishment as substrate depletion restricts the operational time of batch fermentations
to several hours and prevents the establishment of steadystate conditions in vitro.
Continuous fermentation models
Continuous culture fermentation models exist as either
single- or multistage systems and are necessary for performing long-term studies as substrate replenishment and
toxic product removal are facilitated. Single-stage continuous fermentation models are often used to elucidate
proximal colon function and metabolic activity as the
mixing of digesta from both the caecum and ascending
colon is well simulated in these models [17]. Single-stage
continuous fermentation models have been used to investigate the mechanism of Salmonella infection in children
as well as colonization of the infant gut [13,57].
Human colonic function occurs, however, over the span
of the ascending, transverse and descending colonic
regions and differences in metabolic activity and microbial
communities have been horizontally demonstrated for
each colonic region [62]. A major advancement of in vitro
gut fermentation models was the development of multistage continuous fermentation models which enable the
simulation of horizontal colon processes [9]. This design
facilitates the spatial, temporal, nutritional and physicochemical properties of the gut microbiota by combining
three chemostats connected in series, replicating the proximal (R1), transverse (R2) and distal (R3) colon regions
(Figure 1) [9]. Adaptation, survival and proliferation of an
in vivo acquired human gut microbiota to in vitro continuous fermentation models are dependent on environmental
parameters such as pH, retention time, temperature, anaerobiosis and flow rate of the medium. Strict control of
these factors facilitates establishment of steady-state conditions regarding both microbial composition and metabolic activity, with these steady-state conditions being
required to create a reproducible system (Figure 1). A wide
range of studies and novel results on gut microbial community modulation and metabolic function have been
obtained using the various multistage continuous fermentation model designs, evidence of the dynamic capacity of
this technology [9,10,14,58]. Probiotic and prebiotic modulation and impact investigations represent the most commonly performed experimentations using three-stage
continuous in vitro fermentation models [63].
Perhaps the major discriminating factor between the
different in vitro continuous fermentation systems is the
technique used for fecal inoculation. Operation of most in
vitro systems uses a liquid fecal suspension as inoculum,
resulting in several limitations due to the free-cell state of
the bacterial populations [57]. Systems with liquid fecal
inocula generally experience a rapid washout of less competitive bacteria and are consequently limited in operational time to less than 4 weeks [9,64,65]. These systems
also struggle in reproducing both the planktonic (free-cell)
and sessile (biofilm-associated) states of bacterial populations in the colon [17,66]. To address problems associated
with inoculum washout, a process for the immobilization of
fecal microbiota has been developed [10,14,57]. Here, fecal
Review
Box 1. Challenges and perspectives of in vitro fermentation
models
Permanent challenges
Enhance and evaluate microbial ecosystem stability.
Carefully select models depending on their advantages and
limitations related to the research question.
Make the right compromise between technical complexity and
biological significance.
Assess reproducibility and define repeatability needs.
Perspectives
Assess functional stability of the in vitro microbiome.
Application of omics technologies to in vitro gut fermentation
modeling.
Modeling of diseased microbiota.
Combination of in vitro models.
Review
functionality of certain populations of gut microbiota or
assigned to a particular function of interest, might be elucidated by integrating metabolomic technologies [91,92].
Polyphenol metabolism by human gut microbiota is
just one example of the enormous promise top-down and
bottom-up systems biology approaches hold in discerning
the complexity of microbiomemetabolomic interactions
[80]. Given the recent advances in meta technologies, perhaps the greatest challenge rests with the biocomputational
and bioinformatic community in developing correctly annotated gene databases, facilitating faster, easier analysis of
large datasets arising from high-throughput sequencing
and metabolomic profiling [93].
Perspective applications of in vitro model systems
Elucidating the functionality of the coding capacity of
human gut microbiota modulated during in vitro gut fermentation modeling and the metabolic profiles generated
as a result requires the application of -omics based technologies. The use of a multidisciplinary systems biology
approach in combination with -omics platforms as outlined in Figure 2 will facilitate the most advanced system
for unraveling the complex microbial and host factors
governing human gut microbiota functionality. As mentioned in the previous section, the inability to sufficiently
simulate host responses in in vitro gut fermentation models remains a major limitation of this technology. However,
host response factors can still be assessed in vitro through
a combinatorial model system using both in vitro gut and in
vitro human intestinal cell models. A recent study assessing adhesion and cytokine expression of Caco-2 cells using
bacterial cultures obtained from a three-stage continuous
fermentation model demonstrates this concept [27]. In
another approach, an in vitro small intestinal model was
combined with Caco-2 cells [26]. However, small intestinal
in vitro models are devoid of intestinal microbes, designed
to replicate only digestion and absorption processes and
are thus incapable of providing information on intestinal
fermentation processes. Another type of combined model
approach utilized supernatants obtained from a batch
fermentation of inulin for application onto human colonic
biopsies [94]. The difficulty and ethical barriers involved in
regularly obtaining biopsies for experimentation makes
the use of in vitro cultivated human cells a more reliable
approach. Results using this combined in vitro approach
indicate that the application of fermentation effluents to in
vitro human intestinal cell models is a feasible platform for
assessing epithelial responses as a result of in vitro fermentative processes.
Future in vitro gut fermentation model systems include
but are not limited to the establishment and maintenance
of diseased microbiota. The development of such models
would allow investigations on the functional role of gut
microbiota in inflammatory bowel diseases such as Crohns
disease and ulcerative colitis. Investigation of the etiopathologies of these diseases has been, to date, heavily
centered on immune system function based studies owing
to the complexity and lack of knowledge regarding additional contributing factors [95,96]. Functional studies
performed in vitro could potentially advance the understanding of these diseases beyond the immune system.
Although the cause versus consequence of the gut microbiota in many of the aforementioned diseases remains
heavily debated, in vivo studies alone have, to date, been
unsuccessful at fully elucidating the mechanisms of these
diseases and make a strong case for using a combined
human gut systems biology approach.
Concluding remarks
In vitro fermentation models are an innovative technological platform where the greatest advantages are exhibited
by the virtually limitless experimental capacity as experimentation is not restricted by ethical concerns. Host intestinal function is only partially simulated in some model
designs (e.g. TIM-1 and TIM-2) and together with microbial population balancing remains a major challenge of in
vitro gut fermentation modeling. Host functionality, however, does not need to be completely ignored during such
experimentation. The use of a combined in vitro model
system comprising both in vitro gut and human intestinal
cell models has the ability to provide novel results on host
responses to in vitro fermentation processes. As in vivo
results remain the most coveted, an ultimate approach to
investigating gut microbiota functionality must include a
combination of complimentary in vitro and in vivo models,
each yielding complementary results which not only
strengthen the overall validity of each individual model
but also distinguish between the functionality of gut microbial and human processes.
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