2. Open the terminal ports of the HF cartridge and check that
medium is flowing through the cartridge and CR via the
duet pump.
3. Thread the input and output tubing lines through the peristaltic pump drives and secure in place over the rollers (see Note 8).
4. Prime the rest of the tubing system by turning on the peristaltic
pump and opening the vertical port of the 3-way stopcock valve
above the CR to allow flow to begin from the media tank.
5. Monitor medium flow through the system to check for leaks or
blockages (see Note 9).
3.3 Establishing
Experimental PK
Parameters
The flow rate required to simulate a particular compound half-life
(t 1/2 ) is calculated using the following formula: Clearance
rate ¼ ln2 Â V/t 1/2 , where V ¼ volume of the afferent section of
the system from the medium tank outlet up to and including the
CR. The amount of drug (μg/mL) required to achieve a maximum
circulating concentration (C max) ) ¼ C max  V/1000.
1. Run simulations without bacteria in an HFS with specifications
are identical to those to be used in the BSL3 facility (see Note 10).
2. Take two sets of PK samples from the same system; the first set
at day 0, and the second after running the system continuously
for several days (see Note 11). For small HFCs (ECS volumes
less than 5 mL), samples need only to be taken from the CR via
the stopcock port but for larger HFCs, matching PK samples
should also be taken directly from an ECS port to check for
congruency between sample sites (see Note 12).
3.4 Compound
Addition
Drug infusion is traditionally performed using a syringe pump and
tubing connected to a valve that feeds directly into the central
reservoir (CR—see Fig. 1).
1. Inside the BSC, load a syringe with an appropriate amount of
drug solubilized in a volume of medium sufficient to deliver the
required dose in the chosen infusion time, plus at least enough
volume to occupy the void volume of the input tubing.
2. Attach the syringe to the input line and prime the tubing
between the syringe and CR stopcock (see Note 2).
3. If running a PK experiment, take a baseline (t ¼ 0) sample from
the CR via the microclaved port on the second stopcock (see
Subheading 3.5).
4. Turn off the peristaltic pump to prevent input from the
medium tank and turn the CR stopcock valve to block the
medium line and open flow from the syringe pump. Turn on
the syringe pump and begin timing the PK experiment from
the moment that drug begins to enter the CR (see Note 13).
Mycobacterium Tuberculosis in a Hollow Fiber System
721
medium is flowing through the cartridge and CR via the
duet pump.
3. Thread the input and output tubing lines through the peristaltic pump drives and secure in place over the rollers (see Note 8).
4. Prime the rest of the tubing system by turning on the peristaltic
pump and opening the vertical port of the 3-way stopcock valve
above the CR to allow flow to begin from the media tank.
5. Monitor medium flow through the system to check for leaks or
blockages (see Note 9).
3.3 Establishing
Experimental PK
Parameters
The flow rate required to simulate a particular compound half-life
(t 1/2 ) is calculated using the following formula: Clearance
rate ¼ ln2 Â V/t 1/2 , where V ¼ volume of the afferent section of
the system from the medium tank outlet up to and including the
CR. The amount of drug (μg/mL) required to achieve a maximum
circulating concentration (C max) ) ¼ C max  V/1000.
1. Run simulations without bacteria in an HFS with specifications
are identical to those to be used in the BSL3 facility (see Note 10).
2. Take two sets of PK samples from the same system; the first set
at day 0, and the second after running the system continuously
for several days (see Note 11). For small HFCs (ECS volumes
less than 5 mL), samples need only to be taken from the CR via
the stopcock port but for larger HFCs, matching PK samples
should also be taken directly from an ECS port to check for
congruency between sample sites (see Note 12).
3.4 Compound
Addition
Drug infusion is traditionally performed using a syringe pump and
tubing connected to a valve that feeds directly into the central
reservoir (CR—see Fig. 1).
1. Inside the BSC, load a syringe with an appropriate amount of
drug solubilized in a volume of medium sufficient to deliver the
required dose in the chosen infusion time, plus at least enough
volume to occupy the void volume of the input tubing.
2. Attach the syringe to the input line and prime the tubing
between the syringe and CR stopcock (see Note 2).
3. If running a PK experiment, take a baseline (t ¼ 0) sample from
the CR via the microclaved port on the second stopcock (see
Subheading 3.5).
4. Turn off the peristaltic pump to prevent input from the
medium tank and turn the CR stopcock valve to block the
medium line and open flow from the syringe pump. Turn on
the syringe pump and begin timing the PK experiment from
the moment that drug begins to enter the CR (see Note 13).
Mycobacterium Tuberculosis in a Hollow Fiber System
721