complex range of diverse environments in the host that cannot be
recapitulated in the assay. Modified HF models have been used to
mitigate this shortcoming by including specific host-derived factors
such as infected macrophages in the ECS [3] or additional tissuecontaining “compartments” in the flow path to simultaneously
assess drug-induced toxicity [4]. A second shortcoming is the
relatively low number of HF-based assays that can be executed at
any particular time due to the space requirement and intricacy of
the experimental equipment setup. Nonetheless, HF modeling can
be of considerable value when used either prospectively, to fine tune
drug regimens for maximum efficacy prior to animal studies, or
retrospectively, to investigate a drug’s unexpected behavior in animal testing or even human clinical trials.
Here, we describe the standard procedure we use for studying
actively growing M. tuberculosis and its interaction with applied
compounds.
2 Materials
All media and solutions are prepared using milliQ purified water
and analytical grade reagents. Store prepared media at 4
C unless
specified otherwise and hold for not longer than 1 month before
use.
1. Liquid growth medium: Prepare the standard medium using
Middlebrook 7H9 broth base (MilliporeSigma) supplemented
with glycerol to 0.1% (vol/vol), 0.05% (vol/vol) Tween
80, and 10% (vol/vol) Middlebrook OADC (oleic acid, albumin, dextrose, and catalase) growth supplement (BectonD).
Sterilize by passing it through a 0.2μm membrane filter.
2. Mycobacterium tuberculosis H37Rv or M. tuberculosis H37Ra.
3. 7H11 agar plates: Prepare Middlebrook 7H11 agar base (MilliporeSigma) according to manufacturer’s instructions and sterilize by autoclaving. After the agar has cooled to about 60
C,
add
100
mL/L
Middlebrook
OADC
growth
supplement (BD).
4. Autoclavable Erlenmeyer flasks with 38–430 mm closures, e.g.,
TriForest, from Thermo Fisher Scientific.
5. Sterile Nalgene PETG bottles with 38–430 mm closures.
6. Hollow fiber cartridge, e.g., a high flux polysulfone cartridge
C2025D with 3.2 mL ECS volume (FiberCell Systems Inc.).
7. Duet pumps and cap assemblies (38–430 mm closure) for
medium bottles, CR, and waste bottles (FiberCell Systems).
8. Peristaltic pumps (Cole-Parmer Instrument Co).
Mycobacterium Tuberculosis in a Hollow Fiber System
717
recapitulated in the assay. Modified HF models have been used to
mitigate this shortcoming by including specific host-derived factors
such as infected macrophages in the ECS [3] or additional tissuecontaining “compartments” in the flow path to simultaneously
assess drug-induced toxicity [4]. A second shortcoming is the
relatively low number of HF-based assays that can be executed at
any particular time due to the space requirement and intricacy of
the experimental equipment setup. Nonetheless, HF modeling can
be of considerable value when used either prospectively, to fine tune
drug regimens for maximum efficacy prior to animal studies, or
retrospectively, to investigate a drug’s unexpected behavior in animal testing or even human clinical trials.
Here, we describe the standard procedure we use for studying
actively growing M. tuberculosis and its interaction with applied
compounds.
2 Materials
All media and solutions are prepared using milliQ purified water
and analytical grade reagents. Store prepared media at 4
C unless
specified otherwise and hold for not longer than 1 month before
use.
1. Liquid growth medium: Prepare the standard medium using
Middlebrook 7H9 broth base (MilliporeSigma) supplemented
with glycerol to 0.1% (vol/vol), 0.05% (vol/vol) Tween
80, and 10% (vol/vol) Middlebrook OADC (oleic acid, albumin, dextrose, and catalase) growth supplement (BectonD).
Sterilize by passing it through a 0.2μm membrane filter.
2. Mycobacterium tuberculosis H37Rv or M. tuberculosis H37Ra.
3. 7H11 agar plates: Prepare Middlebrook 7H11 agar base (MilliporeSigma) according to manufacturer’s instructions and sterilize by autoclaving. After the agar has cooled to about 60
C,
add
100
mL/L
Middlebrook
OADC
growth
supplement (BD).
4. Autoclavable Erlenmeyer flasks with 38–430 mm closures, e.g.,
TriForest, from Thermo Fisher Scientific.
5. Sterile Nalgene PETG bottles with 38–430 mm closures.
6. Hollow fiber cartridge, e.g., a high flux polysulfone cartridge
C2025D with 3.2 mL ECS volume (FiberCell Systems Inc.).
7. Duet pumps and cap assemblies (38–430 mm closure) for
medium bottles, CR, and waste bottles (FiberCell Systems).
8. Peristaltic pumps (Cole-Parmer Instrument Co).
Mycobacterium Tuberculosis in a Hollow Fiber System
717