J Antimicrob Chemother 2010; 65: 186 201

doi:10.1093/jac/dkp434 Advance publication 21 December 2009

In vitro pharmacodynamic models to determine the effect

of antibacterial drugs
Julia Gloede1, Christian Scheerans1,2, Hartmut Derendorf3 and Charlotte Kloft1,2*
1

Department of Clinical Pharmacy, Institute of Pharmacy, Martin-Luther-Universitaet Halle-Wittenberg, Halle, Germany;

2
Department of Clinical Pharmacy, Institute of Pharmacy, Freie Universitaet Berlin, Berlin, Germany;
3
Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL, USA

*Corresponding author. Department of Clinical Pharmacy, Institute of Pharmacy, Martin-Luther-Universitaet Halle-Wittenberg, Wolfgang-LangenbeckStr. 4, 06120 Halle, Germany. Tel: 49-345-5525190, Fax: 49-345-5527257; E-mail: charlotte.kloft@pharmazie.uni-halle.de

Keywords: in vitro models, antibiotics, dilution models, dialysis/diffusion models, static models

Introduction
The dosing regimens of antibiotics are often not optimal and the
doseresponse relationships not well known.1 One important
reason is that in the patient the pure antibiotic effect, i.e. the
pharmacodynamic (PD) characteristics,2 cannot clearly be separated from other factors determining the response to the antibacterial treatment. The effect also has to be regarded along with
pharmacokinetic (PK) properties,2 such as the ability of the
drug to reach its target. Thus, the PK, and the PD,2 are characteristics of an antibacterial agent and should be considered in the
development and prediction of the efficacy of the antibacterial
therapy. By linking the concentration time course (at the site
of action) to the drug effect (PK/PD), various dosing regimens
for different pathogens can be investigated in silico, enabling
the identification of potentially effective dosing regimens.
However, there is no standardized procedure for PK/PD evaluation for antibiotics, although the European Medicines Agency
(EMEA)1 and the FDA3 clearly recommend these investigations
for new compounds.
For characterizing the PD of an antibiotic, bacterial growth
and death under antibiotic exposure have to be investigated.
Since these are difficult to measure in human tissue, animal
and in vitro models have been developed. Animal models
provide similar growing conditions for bacteria, closely imitating
the characteristics of a human infection, and the endpoint of an

infection is clearly defined (cure or death) and comparable to

that in humans.4,5 A significant disadvantage of animal models
is differences in the PK,1,5 e.g. in metabolism, which limit or
necessitate sophisticated scaling methods for transferring data
from animals to humans.5
In contrast, in vitro models can mimic human PK1 and are
thus better suited for the investigation of antibiotic activity.6 In
addition, they allow resistance analyses,7,8 determination of
time kill behaviour, and the identification and optimization
of PK/PD indices and breakpoints.9 13 Although a large number
of models have been developed, in practice they are all variants
of 10 basic experimental set-ups. Rather than discuss all the
models reported in the literature, this article provides a generalized overview of the most frequently used and newly developed
in vitro PD models. The historical development of in vitro models
has been already reviewed by Grasso until 198514 and
others.15 17 Grasso divided in vitro models according to their
working principle into two basic groups: (i) models based on
dilution; and (ii) those based on diffusion or dialysis. MacGowan
et al. 18,19 described the information and conclusions obtained
from in vitro models. The impact of in vitro models has been discussed by Li and Zhu,17 and others.16 PK modelling of in vitro
models has been basically described by Blaser,20 Rowe and Morozowich,21 and Firsov et al.;15 detailed mathematical descriptions
for the interpretation of PK/PD analyses and PK/PD modelling
can be found in the work of Derendorf and Meibohm,22 Czock

# The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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In vitro pharmacodynamic (PD) models are used to obtain useful quantitative information on the effect of
either single drugs or drug combinations against bacteria. This review provides an overview of in vitro PD
models and their experimental implementation. Models are categorized on the basis of whether the drug
concentration remains constant or changes and whether there is a loss of bacteria from the system. Further
subdifferentiation is based on whether bacterial loss involves dilution of the medium or is associated with
dialysis or diffusion. For comprehension of the underlying principles, experimental settings are simplified and
schematically illustrated, including the simulations of various in vivo routes of administration. The different
model types are categorized and their (dis)advantages discussed. The application of in vitro models to
special organs, infections and pathogens is comprehensively presented. Finally, the relevance and perspectives
of in vitro investigations in drug discovery and clinical research are elucidated and discussed.

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Review

and Keller,23 and others.24 37 PK/PD parameters for antibiotics,

the PK/PD indices, are well defined in the literature from
Mouton et al. 10,11

Fundamentals
Characteristics of in vitro models

Classification
As a consequence of these definitions, all in vitro models for antibiotics can be classified according to the change of the drug concentration and whether or not there is bacterial loss (Table 1):
I models with a constant drug concentration and no bacterial
loss;
II models with changing drug concentration and bacterial
loss; and

The first models can be more accurately named static in vitro

models (Table 1, No. I). This term is already advised in the literature of microbiology and biotechnology for models with a constant environment, i.e. constant antibiotic exposure, with
unchanged medium;39,40 thus, no in- and outflow of medium
occurs in these systems.41,42 These models have been used
extensively;28,43 48 however, these models had either not been
named before or have been described as models investigating
constant concentrations of antibiotics.42
The other two groups of models are known as dynamic in vitro
models and are further differentiated on the basis of whether or
not bacterial loss occurs (Table 1, Nos II and III). Usually, bacterial loss38 (No. II) is not intended and causes bias, which can
be corrected,40,49,50 whereas the dilution of toxic waste cannot
be considered. To avoid bacterial loss, appropriate technical
arrangements have to be carried out (No. III), e.g. by a membrane or filter system.38 Models Nos II and III can be subclassified depending on whether the mechanism of drug loss involves
dilution (Nos II and IIIa) or dialysis/diffusion (No. IIIb). Dilution
models with bacterial loss (No. II) work by stepwise substitution
or continuous dilution of medium. Dilution models without bacterial loss (No. IIIa) operate by stepwise or continuous dilution, or
stepwise substitution of medium through a filter system. In the
special case of dilution models without bacterial loss (No. IIIa),
medium is added, with a resulting increase in volume, i.e. no
loss of bacteria, although their concentration is reduced due to
dilution (see the Stepwise simple dilution section below and
Table 2). Dialysis/diffusion models can be further classified by
the type of membrane used (either artificial or natural).
Our review shows that the current classification14 (dilution or
dialysis models) does not encompass all models (e.g. intracellular models), although they could still be integrated into the
classification scheme. We adapted and revised the existing
classification, and focused on in vitro models, which mimic PK
profiles in plasma and other biological matrices, and thus allow

Table 1. Revised classification of in vitro models

Bacterial loss
Drug concentration

yes (open systems)

Constant
Changing

II

dynamic dilution models

via stepwise substitution (without filters)
via continuous simple dilutionb (without filters)

no (closed systems)
I

static models

IIIa

dynamic dilution models

via stepwise simple dilutiona
via stepwise substitution (with filters)
via continuous dilution
without outleta
b
W with filters
dynamic dialysis/diffusion modelsb
with artificial membranes
with natural membranes
W

IIIb

With bacterial dilution.

Multicompartments possible.

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The two main characteristics of in vitro PD models are drug

exposure and bacterial concentration. The literature does not
comprise a uniform and complete definition of these main
characteristics. Hence, we characterize these terms as follows:
constant drug exposure is achieved by not replacing or changing
the medium, while changing drug concentrations are obtained in
systems with flowing medium. Consequently, we will focus on
the terms constant and changing to describe the drug exposure.
The bacterial concentration represents the magnitude of the PD
effect. A loss of bacteria due to the experimental setting, which
is observed in some models, may therefore have a substantial
influence on the results. Thus, to define the loss of bacteria in
in vitro models we suggest the terms open and closed:38 open
models allow the exchange of bacteria with the environment;
and closed models have no bacterial exchange. As a result, an
open model always has a flowing medium (i.e. changing drug
exposure), whereas closed models can have an unchanging or
flowing medium (i.e. constant or changing drug exposure).

III models with changing drug concentration and no bacterial

loss.

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188
Table 2. Special applications of in vitro models
Application and rationale
Models accounting for the immune system
realistic cell/fluid environment, examine the
penetration into immune cells,46 and
include the effect of immune cells

Type of PD in vitro model

Special features

Ref.

static model
dilution model with stepwise substitution
dilution model with continuous dilution
dialysis model

using immune cells and human serum

using human serum
using serum
using blood

46, 109
133
80, 81
93

static model
dialysis model

47, 135138
100, 132, 139

Intracellular models
PK and PD of intracellular bacteria different
from extracellular

static model
dialysis model

Site of infections/diseases
different PK profiles or bacterial growing
conditions41
bladder/bacterial cystitis

dilution model with continuous dilution without outlet

otitis media
endocarditis
chronic pneumonia
infected fibrin clots
dental infections
skin infections
tuberculosis
Multicompartmental models
simulate PK of drugs with numerous
theoretical compartments, no. of
compartments determines no. of (culture)
vessels

dialysis model
dialysis model
static model
dialysis model
static model
dialysis model
static model
dialysis model
dilution model with continuous dilution without and with
filters

, special interest: Mycobacterium 105,141

useful to describe conditions in emptying

compartments
disease-specific infectious bacteria used

140, 141
103108, 141

77, 142145

bacteria suspended in a fibrin clot

bacteria form biofilm on artificial teeth

92, 146
131
140
131, 147, 148
149
149151
152
105, 141

see also Model developments and Figure 2(c and f)

21, 40, 52, 84

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Biofilm models
bacteria in biofilms: properties distinct from
single organisms, e.g. increased
antimicrobial resistance134

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with artificial membranes

with natural membranes
dilution model with stepwise simple dilution
dialysis model

Production of biofilms by growing slime-producing bacteria, e.g. Staphylococcus aureus 47 and Pseudomonas aeruginosa 135 on various surfaces.
Host cells grown until a stable culture appears (continuous layer), bacterial suspension directly added, culture is incubated and investigations start when the infection is positive.
c
If antibiotics with different half-lives are simultaneously investigated in dynamic models, the flow of the medium, which decreases the drug, has to be adjusted to the shortest halflife41 or, more appropriately, the drug with the longer half-life has to be substituted to the reservoir.
b

Models for fungi and anaerobic organisms

dialysis model

static model
dilution model with continuous dilution

with filters
without filters
static model
dilution model with stepwise substitution (with filters)
dilution model with continuous dilution
Models for combination therapyc
synergistic effects

investigations on the relation/s of concentration- and timedependent bacterial growth. Specific organs and their special
conditions appear separately in Table 2 (Applications). The
new, extended classification (see Table 1) is based on both mentioned main characteristics of in vitro models, i.e. drug concentration and bacterial concentration, whereby all commonly
used models can be categorized.

Experimental settings of in vitro models

An in vitro model with its main componentthe culture vessel
has to fulfil special requirements (Table 3). Numerous different in
vitro models for antibiotics have been developed. Not all models
are applicable for all purposes, and some can mimic only
selected aspects and conditions, e.g. infections in specific compartments. The bacterial concentration (as the antibacterial
effect) in the in vitro system is monitored over time under different antibiotic exposures by different methods (Table 4). The bacterial concentration time courses (time kill curves) and derived
PK/PD indices, such as area under the time kill/bacterial curve,
allow for detailed analysis of bacterial growth and death following antibiotic exposure.18,19,33,34,41,51

Common working principles

In static in vitro models, bacteria should be suspended homogeneously in a culture vessel with constant antibiotic exposure in
the medium. All conditions remain the same over the entire
observation period. The bacterial growth without antibiotic can
be limited by nutrition, space, aeration and toxic metabolites.
The bacterial concentration changes in the vessel and can be
studied over time.41,42 The working principle of dynamic
models is more complex. The idea is to simulate the body clearance or half-life of the antibiotic and is realized in dynamic
models by changing drug concentrations (Figure 1).41 In dilution
models the drug concentration in the culture vessel changes via
substitution with fresh medium or by simple dilution. Substitution means to remove a defined volume from the in vitro
model and supplement the same volume of fresh medium. In
this case, both flow processes (i.e. in- and outflow) are controlled. The volume in the model remains constant all the time.
Simple dilution means to add a defined volume of medium to
the culture vessel. Either (i) medium is added to the input and
the outflow is uncontrolled via overflow (or does not exist) or
(ii) a pump removes medium from the culture vessel and fresh
medium is sucked in from a reservoir by low pressure. In both
cases, the drug concentration in the culture vessel will be
diluted. The input of medium in dilution models can happen continuously or stepwise, i.e. at intervals.42 Fresh medium is pumped
from a reservoir into the culture vessels and from there into the
waste. The input of the drug can mimic bolus, infusion or firstorder absorption. The experimental implementation of in vivo
administration routes is explained below.
Another method for achieving changing drug concentrations
is via drug diffusion across a membrane (dialysis), with the concentration gradient as the driving force. The dialysis models
consist of a central compartment, where the drug initially
appears after dosing, and a peripheral compartment, with bacteria. The central compartment and peripheral compartment

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without filters
with filter

161, 162
121
163
72
164
162, 165

48, 153
73
154
20, 155157
51, 117, 158160

Review

Review

Table 3. Requirements for in vitro models

Parameter
Growth medium
Temperature
Mixing

Rationale

Implementation

appropriate growing conditions

choice and control to mimic in vivo growing condition
quick homogeneous distribution and aeration of bacterial suspension

waterbath, incubator
shaking or stirring

Model developments
Static models (No. I)
Static models consist of a closed culture vessel (Figure 2a). These
vessels are available in a variety of shapes, such as tubes,46,47
flasks,43,54 cell culture flasks44 or spinner flasks,55 and may be
made of glass43 or polystyrene.47 The first time kill investigations in static models were established by Garrett et al. 43
in 1966.

et al. 58 demonstrated that bacteria might adhere to the vessel

wall, forming a biofilm, which protects the bacteria from
(out)flow and from antibiotics. Hence, only released bacteria
can be counted in the medium and higher bacterial concentrations can be found. Nevertheless, due to its simplicity the
Grasso model was adapted by several groups.59 68 In a similar
model, air pressure instead of peristaltic pumps has been
used.69 Bergan et al. 70 introduced a second peristaltic pump
for the out-flowing medium. Later, a computer was added to
control three pump sets in parallel.71
Murakawa et al. 40 developed a two-compartment model
based on the Grasso model (Figure 2c). At the beginning, the
drug is administered as a bolus into the first (central) compartment containing the bacteria, with the second (peripheral) compartment remaining drug free. Fresh medium is pumped from
the reservoir into the first compartment. A second pump
exchanges the medium between both compartments. Bacterial
exchange is not prevented and bacteria are eliminated into the
waste; mathematical corrections have been applied.

Dynamic dilution models without bacterial loss (No. IIIa)

Dynamic dilution models with bacterial loss (No. II)
Stepwise substitution (without filters)
Nishida et al. 56 described a dilution model with stepwise substitution of the medium (Figure 2b). In this model, a tube contains
the bacteria in medium. Fresh medium is periodically added and
at the same time the same volume of used medium is discarded,
leading to a stepwise decline of the drug and a removal of the
bacteria.

Stepwise simple dilution

In a stepwise simple dilution model the medium is not removed
from the system. Fresh medium is added periodically and the
drug concentration declines over time, in relation to the increase
in the volume of the medium (Figure 2d).72 Simultaneously, bacteria will be diluted; hence, bacterial concentrations have to be
corrected.

Continuous simple dilution (without filters)

Stepwise substitution (with filters)

Models with continuous simple dilution reflect the in vivo conditions of a drug much more closely than a stepwise decline of
the drug. The decisive improvement in this field was made by
Grasso et al. 57 The Grasso model consists of a flask containing
the bacteria (culture vessel), a reservoir and a waste container
(Figure 2b). Fresh medium is continuously pumped from the
reservoir into the flask and used medium leaves the culture
vessel by the pressure of the incoming medium. Drug and bacterial samples can be taken from the vessel. A magnetic stirrer
ensures a homogeneous distribution of the drug and bacteria.
In the Grasso model, the bacteria are diluted by the incoming
medium and flow out with the outgoing medium, which
demands a mathematical correction for bacterial counts. In
theory, flow rates that are faster than the bacterial growth
rates would lead to a complete loss of bacteria; however, Haag

Nolting et al. 45 developed a model (syringe model) where the

drug concentration is decreased by stepwise substitution, but
the bacterial loss is prevented by a filter. A syringe needle is
stuck into a cell culture flask containing bacteria and medium
(Figure 2e). The needle is connected with a filter unit and a
syringe. Used medium is withdrawn at regular intervals from
the cell culture flask (in contrast to the stepwise simple dilution
model) and replaced by fresh medium.45,73
Another stepwise substitution model was introduced by
Haller,65 and comprises a Teflon-coated ultrafiltration unit filled
with medium and bacteria. After adding the drug, air pressure
is applied. Thus, medium is continuously eluted and discarded.
Fresh medium, however, is replaced at intervals (Figure 2e).65,74
The elution can also be performed by centrifugation of the filtration unit.75

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are separated by a semi-permeable membrane, i.e. permeable

for drug and medium but not for bacteria. Fresh medium is continuously pumped from a reservoir into the central compartment
and then into the waste. Thus, the medium in the peripheral
compartment is continuously renewed by diffusion (from the
central compartment), while the drug and bacteria can interact,
but the bacteria cannot leave this compartment (Figure 1). The
circulation of medium in the peripheral compartmentas counterflow towards the central compartmentcan help to optimize
the diffusion.52,53

Review

Table 4. Quantification methods for bacteria

Properties

Method

Principle

online
measurementa

direct
measurementb

detection of live
cells only

differentiation
between live
and dead cells

Comment

Viable cell counts

incubation of bacterial samples on agar,

followed by counting

NA

Turbidimetry

measurement of optical density of

bacteria in medium (correlates with
bacterial concentration)
measurement of impedance of bacterial
cells (correlates with bacterial
concentration)
determination of ATP content of
bacterial cells released (correlates
with bacterial concentration)
determination of bacteria by a phase
contrast microscopy

43

126

NA

has to be carried out within 126

one generation time of
bacteria to avoid doubling
127

NA

Impedance

Bioluminescence

Microscope

Fluorescence
quantification
RNA profiling

determination of release of fluorogenic

substances from a substrate by
bacterial phosphatases
quantitative PCR of RNA (correlates with
bacterial concentration)

most frequently used,

avoidance of antibiotic
carry-over effect
necessary123 125,c
discussed as not reliable79

Ref.
43, 56, 57, 97

76, 77, 84

128, 129

NA, not applicable.

Within 10 min.
b
In the culture vessel.
c
Special caution for carry-over effect should be paid for quinolones.
a

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(Figure 2f). Several vessels are connected in series and the

number of vessels depends on the number of compartments
of the underlying mathematical model. The model
operates as the Grasso model. Bacterial loss is prevented by
a special filtration unit, which is placed between each vessel.

Dynamic models

Working principle

Dynamic dialysis/diffusion models (No. IIIb)

Dilution models

Dialysis/
diffusion models

Prevention of bacterial loss

Yes

Simple
dilution

Adding and
removing
of medium

Substitution

Membrane material

Artificial
barrier

Natural
barrier

Figure 1. Detailed overview on dynamic in vitro models.

Artificial membranes
Continuous dilution
Continuous dilution models operating without outlet, resulting in
increasing volumes, were described by Sanfilippo and Morvillo,76
and OGrady and Pennington.77 They mostly reflect only selected
aspects of the in vivo situation. Pumps transport the medium
from a reservoir into the culture flask (Figure 2d). Since there is
no outlet, the volume of the second flask continuously increases,
the drug concentration changes and the bacteria will be
diluted.76 The increasing volume does not necessarily allow
exact exponential decline of the drug concentrations (see also
bladder/bacterial cystitis in Table 2).77
Continuous dilution without bacterial loss can also be achieved
with filters. The model by Greenwood and Tupper78 consists of a
vessel separated by a filter membrane in two chambers, with
the bacteria and drug being added to the upper one
(Figure 2e).78 However, this model has not been further used.
Instead, for the already mentioned decisive Grasso model
(dilution, but bacterial loss),57 different modifications have been
suggested to improve the accuracy. Filters are inserted between
the culture vessel and the waste, and the outlet has moved
from the side panel to the bottom of the flask.79 81 A practical solution to prevent the bacterial loss in the Grasso model was found
by Lowdin et al. 55 (Figure 2e). The base of a spinner flask is modified, including an outlet and a perforated metal support, on which
a filter membrane and a pre-filter are adjusted. Above the membrane, a magnetic stirrer is placed to prevent membrane blockage.
Fresh medium is pumped in via one side arm in the spinner flask.
The other arm is prepared with a silicon membrane for repeated
sampling.55,82,83
A multicompartment model based on the Grasso model
with retention of bacteria was presented by Navashin et al. 84

192

In dialysis models with artificial membranes, different bacteria

vessels were used, such as tubes,85 (square) vessels,86 separating
funnels,87,88 artificial kidneys,89 91 a plexiglass chamber with
changeable membrane filters,92,93 a hollow t-tube94,95 and
hollow fibres.96 These models can have adjacent peripheral
and central compartments or compartments embedded in
each other.
In the adjacent setting, as in the model of Drugeon et al.,87
the upper and lower part of a separating funnel are part of an
entire loop (peripheral compartment), which also runs through
one part of the dialysis unit. The other part of the dialysis unit
forms the central compartment; the dialysis unit enables the
exchange of the drug and the medium. Continuous dilution of
the central compartment decreases the drug concentration
(Figure 2g).87,88 Toothaker et al. 86 horizontally separated a
vessel by a haemodialysis membrane in two parts. One part contains fresh medium and the antibiotic, and the other part
includes the bacteria (Figure 2g).
In the embedded setting, Guggenbichler et al. 89 work with an
artificial kidney, the inner part of which is connected to the bacteria vessel (Figure 2h). Shah utilizes a plexiglass chamber with
changeable membrane filters at both ends as the bacteria compartment (peripheral compartment).93 This chamber is placed in
an outer chamber with medium (central compartment;
Figure 2h). The same model was adapted by Garrison et al.,94
who modified the inner chamber to a hollow t-tube. In the
hollow fibre model by Zinner et al.,96 the tubing of the central
compartment includes a bundle of artificial capillaries. These
capillaries consist of polysulphone fibres, permeable for drug
and medium, and are continuously flushed with medium. The
fibres pass through a vessel with bacteria (peripheral

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Direct adding or
direct removing
of medium

Yes

No

In the majority of dialysis/diffusion models (for simplification,

further called dialysis models), the setting is as follows: fresh
medium is pumped from a reservoir into the central compartment and then into the waste (except for the model by
Al-Asadi et al. 85), thereby decreasing the drug concentration.
The hallmark of these models is that the drug (and medium)
has to diffuse through a membrane to reach the bacteria in
the peripheral compartment. In consequence, bacterial growth
and fresh medium flow happen in two compartments. Two
different settings of the central and peripheral compartment
have been employed: adjacent and embedded.
Dialysis models can be subclassified by the nature of their
membrane, i.e. models with artificial and natural membranes
(Table 5). In this way, the classification presented here includes
models that previously have not been named dialysis models.
At first glance this might seem unusual, but since the working
principle of these models is diffusion across a membrane, this
classification seems meaningful.

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compartment; Figure 2i). The hollow fibre model was further

developed by Blaser et al.,52,97 who added a second and more
bacteria vessels.20,52,97 101 Al-Asadi et al. 85 use two tubes
clamped together, separated by a membrane, with drug in one
(central compartment) and bacteria in the other tube (peripheral
compartment). After a finite time of drug diffusion from the drug
to the bacteria tube, fresh medium is pumped into the bacteria
tube and leads to a decrease of the drug concentration. In contrary to all other dialysis models, here the bacteria compartment
itself is flushed (Figure 2j).

Natural membranes
The principle of dialysis models with natural membranes is
almost the same as for those with artificial membranes. Bacteria

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(a)

are captured behind a barrier and the drug has to pass the
barrier to reach the bacteria. Haller102 described a tissue
culture model, where tissue cells are grown on a dialysis ultrafilter until a continuous layer is formed. The membrane, consisting
of the filter and the tissue cell layer, is placed on a cylinder
(central compartment). Another cylinder located above serves
as the peripheral compartment with bacteria. The antibiotic is
administered in the lower part of the chamber by syringes and
diffuses through the cells to the upper part (Figure 2g). The
model was suggested to investigate the penetration of the
drug through intercellular spaces and was later used by numerous groups to investigate drug bacteria effects.103 107 An intracellular model implementing tissue cultures in PD in vitro models
is presented by Hulten et al. 103 Tissue cells are grown in inserts
in a glass chamber, similar to a closed Petri dish (central

Static Model (No. I)

B

(b)

Dynamic dilution models (No. II)

stepwise substitution (1.)
continuous simple dilution (2.)
1.
or
2.

continuous simple dilution,

multi compartments

(c)

W
B

(d)

Dynamic dilution models (No. IIIa)

stepwise simple dilution (1.)
continuous dilution without outlet (2.)
1.
or
2.

stepwise substitution with filters, e.g.

models by Nolting (1. + 3.) or Haller
(1. + 4.)
continuous dilution with filters, model
by Lowdin (2. + 4.)

(e)

1.
or
2.

3.
or
4.

continuous dilution with filters,

multi compartments

(f)

Figure 2. Schematic depiction of settings of in vitro models at the beginning of an experiment.

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Dynamic dialysis/diffusion models (No. IIIb)

(g) Models with adjacent peripheral and central
compartments with
artificial membranes, models by e.g.
Drugeon, Toothaker
natural membranes, e.g. tissue culture
model by Haller

(h) Models with embedded peripheral

compartments in central compartments with
artificial membranes, e.g.
Guggenbichler, Shah
natural membranes, e.g. intracellular
model by Hulten, fibrin clot model
by McGrath

B Peripheral
Central

Central

(i) Hollow fibre model with artificial membrane

Peripheral

(j) Special case:

Model by Al Asadi with artificial membrane

Peripheral

Central

B
Peripheral

W
Central

Caption:

B
R
W

culture vessel with bacteria

reservoir
waste
flow direction
stepwise medium flow
continuous medium flow
filter
semi-permeable membrane, i.e. permeable for drug and medium, not for bacteria

Figure 2. Continued

Table 5. Types of membranes in dialysis models (alphabetical order)

Artificial membranes
material
cellulose acetate
haemodialysis membranes
polycarbonate
polysulphone
regenerated cellulose
synthetic regenerated cellulose
ester

Natural membranes
ref.
85
86
52, 94, 97
96
89
95

material

ref.

agarose gel
130
cells
102
cell membranes 102105
fibrin
131
slime
47, 132

compartment). The cells had previously been infected with

intracellular-growing bacteria (peripheral compartment). A
metal rack for permeable cell culture inserts facilitates the
tissue cell growth in the glass chamber. The cell membranes
operate as dialysis membranes and the cells are continuously
flushed with fresh medium (Figure 2h). The drug has to pass
the cell membrane to reach the bacteria. The bacteria can be
counted after destruction of the cells.103 109

194

Experimental implementation of in vivo routes

of administration
In vitro models can be used to simulate different routes of drug
administration in patients. Generally, in dilution models the drug
can be added directly to the culture vessel or into an additional
vessel between the reservoir and the culture vessel, simulating
no (i.e. bolus administration) or first-order absorption (i.e. extravascular administration), respectively. From the additional vessel,
the drug is transported with the medium into the culture vessel
and into the waste. Zero-order absorption (i.e. infusion) of the
drug can be achieved by adding the drug to the reservoir. Drugcontaining medium is transported to the culture vessel and from
there into the waste. The end of absorption in this case can be
realized by exchange of the drug-containing reservoir to a drugfree reservoir (Figure 3a).
Simulation of in vivo routes of drug administration in dialysis
models is the same as in dilution models. The drug is transported
with the flowing medium to the central compartment (Figure 3b).
From there it diffuses to the peripheral compartment. In all scenarios the drug concentrations are suggested to follow in vivo
absorption/PK.21,57,62 Determinations of drug concentrations in
samples from the culture vessel should support this assumption.

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Review

(a) In dilution models with the example of a continuous simple dilution model
Bolus administration
Extravascular
administration
No absorption
0-order absorption 1st-order absorption
Infusion

A
R

or

Infusion

Extravascular
administration

Bolus administration

0-order absorption 1st-order absorption

Downloaded from http://jac.oxfordjournals.org/ by guest on April 27, 2014

(b) In dialysis/diffusion models with the example of an embedded peripheral

compartment

No absorption

D
Central

A
R

D
R
A
B
W

or

B
Peripheral

drug
reservoir
additional vessel, mimicking absorption (optional)
culture vessel with bacteria
waste

Figure 3. Schematic depiction of in vitro implementation of the different in vivo routes of administration.

Applications
A substantial number of in vitro PD models have been developed
to simulate specific conditions. Even if not all of these models
can imitate the designated PK profiles, they are useful tools for
specific conditions. In Table 2, the models are grouped by their
main aspects and may appear in different categories.

Relevance and perspectives

For the approval and rational use of antibiotics in pharmacotherapy, pre-clinical investigations will have to focus more on PK/PD
investigations in the future. In this respect, in vitro models
might present a valuable predictive tool.1 A standardized methodology for use in pre-clinical research would provide a valuable
tool for the optimization of dosing strategies.
Generally, in vitro models have several advantages compared
with in vivo animal studies: they are more flexible and adaptable

to different conditions, and are less cost- and resource-intensive.

Additionally, the relatively high inocula and volumes in in vitro
models allow better studies of resistance, because of the higher
mutation frequency than in animals.110 The PK properties of the
drug of interest can be applied in vitro and the time course of
an antimicrobial agent can be monitored exactly. On the
other hand, in vitro models need special conditions, such as
a temperature-controlled environment, and the risk of contamination of the culture vessel with external bacteria increases the
longer the experiment lasts.111 Since in vitro models cannot
mimic all in vivo conditions,112 such as immunological factors
(e.g. host defence mechanisms), the pathology of the infection,
and the virulence and metabolic behaviour of a pathogen,1 the
derived PD parameters cannot directly be transferred to the
in vivo situation. The in vivo growth environment is different from
the in vitro one. This may lead to phenotypic differences
between bacteria grown in vitro and in vivo.113 In general, in vitro
bacterial growth is much faster than that in vivo.38,114,115 Hence,

195

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196

static models. They do not offer a continuous dilution and, therewith, not the same exposition profile for bacteria as in vivo.
Nevertheless, it is possible to achieve more realistic results
than with static models.
Dialysis models are extensively used, as well. Their main
advantage is the closed system, whereby bacteria cannot
escape and no further filters need to be installed. Dialysis
models enable simultaneous investigations of different bacterial
strains in separated vessels, but in one model.97 However, bacteria accumulate at the membrane, which might become
blocked (as in the case of dilution models).52,85 Furthermore,
the changing drug concentration in the bacteria compartment
does not necessarily follow the designated PK profile,14 since
the drug has to pass a barrier. Diffusion of a drug is a first-order
process. The extent of drug transfer across the barrier depends
on the site of membrane permeation and varies with time.
This means there is a specific concentration gradient between
the drug concentration in the central compartment and peripheral compartment at each timepoint. Unfortunately, only a few
groups have determined the drug concentration in the bacteria
compartment,52,53,85,86,89,92,94,97 99 where the concentration
gradient was confirmed. The gradient can be improved by
higher circulation (addition of pumps) and contraflow of the
medium in the central compartment and peripheral compartment.52,53,97 The early dialysis models with artificial membranes
as well as most with natural membranes suffered from a small
surface area of the membrane to volume ratio. This led to diminished diffusion or membrane blockage.85,87,93 Later, the exact
ratio between the membrane surface area and the volume of
the peripheral compartment was estimated and changed, e.g.
by Vance-Bryan et al.,92,122 for the Shah model.93 In intracellular
models, determination of the drug concentration is even more
problematic as the site of action is inside the cells. Here, the
flow is not directed for an optimal exchange between extraand intracellular fluid, which may lead to different PK profiles
that the bacteria will be exposed to. Only with special equipment
and procedures such as fluorescence microscopy is it possible to
measure the drug concentration inside the cells. So, the effect is
often related to the drug decline in the central compartment,
because of easier determination.
In summary, in spite of their simplicity, static models will still
play an important role for antibacterial PK/PD studies in the
future, but should be regarded as a starting point. For more
complex PK designs, dynamic models will be more important
and their use will hopefully increase. The dilution models,
such as the Grasso model,57 have existed since the 1970s
and have been intensively diversified. Almost at the same
time, dialysis models have been introduced and, later,
improved. Currently, the ratio of using dialysis or dilution
models is balanced. Both types of dynamic models have been
further developed in the past and are presented in the
current literature. New developments combine the ideas of a
one-compartment dilution model with filters and a twocompartment dialysis model, resulting in a computer-controlled
semi-automated in vitro model for industrial purposes.111 In
future, this trend of combining models for different purposes,
as well as automation, might lead to more frequent use and,
eventually, they might become an inherent part of drug discovery and development. Comprehensive understanding of the PD
of antibiotics should facilitate the development of rational

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a stronger competition for nutrients can lead to a higher production of antimicrobial drug targets, resulting in a higher susceptibility in vitro.43,114 In spite of this, in vitro models allow good
prediction of bacterial growth in vivo and comparison of different
dosing regimens of one drug as well as comparison between different drugs. Finally, they contribute to dose optimization.18,26,100,112
The choice of a specific in vitro model is determined by the
objectives of a PK/PD study as well as the advantages and disadvantages of in vitro models. Static in vitro models are extensively
used as they are easy to handle and well investigated. They
provide basic information on the interaction between the antibiotic and bacteria. In contrast to these favourable economic
aspects is the unrealistic nature of the unchanged drug and
medium in static models,41 which fail to mirror two important
aspects of in vivo conditions, namely exchange of nutrients
and dilution of the drug. They are not useful for prolonged treatment studies, because nutrient depletion, space limitations and
toxic metabolites lead to growth restrictions.38 In our opinion,
static models should be used as the starting point for PD
studies of the effect over time. The quick-and-easy findings
from static models are useful preliminary knowledge for
dynamic investigations.
Dynamic in vitro models represent the in vivo conditions with
respect to the changing drug and medium much more
closely.14,41 Beside this, dynamic models also enable prolonged
treatment studies (up to 5,116,117 10118,119 and also 15 days120)
with multiple dosing.52,71 Associated potential problems include
the Haag factor58 and membrane blockage. On the other
hand, they require large volumes of growth medium for changing
the drug concentration according to the half-lives of the drug.
Hence, antibiotics with a long half-life have very low flow rates,
a low volume of medium replacement and thus nutrient
depletion, and an increase of toxic metabolites. The two main
principles for changing drug concentrations have been developed
and are both in use: dilution models and dialysis models.
Dilution models can imitate virtually all in vivo PK profiles. The
drug and bacteria are in one compartment, so the bacteria are
directly exposed to the designated drug concentration. Hence,
these models apply the designated PK drug profile to the bacteria, but they should be monitored. The early dilution models
were designed without bacterial retention as open models. The
bacterial loss always has to be corrected.49,50 In the Grasso
model,57 the bacteria leave the culture vessel with the outgoing
medium and toxic waste is diluted, but not considered. Additionally, bacterial aggregation and adherence to the vessel wall was
found, where the bacterial populations should not be detectable.58 In the model by Murakawa et al. 40 the bacteria are distributed into the second compartment and also eliminated into
the waste. This complicates corrections for accurate bacterial
concentrations and, therefore, reliable predictions of the antibacterial effect. The inserted bacteria filter causes new problems:
the filter often becomes blocked with bacteria the longer the
experiment lasts.85,121 The implementation of pre-filters or stirrers has provided potential solutions,55,82 but also open models
have been reused. This has meant a step back regarding the
loss of bacteria.121 In the later developed closed dilution
models, the bacterial backgrowth into the reservoir presented
another problem. Dilution models with stepwise substitution
(and filters), such as the syringe model by Nolting et al.,45 are
easily practicable, but need even more laborious effort than

Review

dosing schedules for patients, resulting in improved therapy and

lower mortality. We hope that in the future in vitro models will
increasingly be used to define the PK/PD characteristics of antibiotics and will serve to complement the data from clinical
trials.

Funding
This work was partially supported by a grant from the Dr. August und
Dr. Anni Lesmueller-Stiftung, Germany.

Transparency declarations
The authors do not have any financial, commercial or proprietary interest
in any drug, device or equipment mentioned in this paper.

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