II F La vocation (el estamento y profesión ordenados por Dios)

Here, I review the details of the microchemostat relevant to the design of the Sortostat, however more detailed descriptions are available elsewhere [82, 83]. The

microchemostat was fabricated from silicone elastomer polydmethylsiloxane (PDM multi-layer soft lithography [92]

run in parallel (Figure 4-1), and are made up of well established microfluidic components such as channels, valves (Figure 4

an integrated system consisting of a growth chamber loop

11.5mm in circumference), micromechanical valves and channels for moving fluids

throughout the reactor, and an integrated peristaltic pump for mixing cells and media (Figure 4-3). The growth chamber loop is made up of 16 addressable segments.

Figure 4-2 Cutaway view of a “push down” PDMS mic

The blue channel represents a fluid line that would contain cells and media in the microchemostat. The red channel represents a “push

water and that can be pressurized or de

control channel is unpressurized the valve is “open” and cells and media can flow freely. (B) When the control valve is pressurized the

line and fluid line and seals the fluid line resulti

flow. “Push-up” valves work by a similar principle except they

lines (grey layer in figure). When they are pressurized they deform the PDMS upwards and seal the fluid line by the same mechanism shown here. Image courtesy of Ty Thomson

89

ed from silicone elastomer polydmethylsiloxane (PDM

[92]. Each PDMS chip contained six microchemostats that are 1), and are made up of well established microfluidic components such as channels, valves (Figure 4-2), and peristaltic pumps [59, 92]. The microchemostat is an integrated system consisting of a growth chamber loop (10µm high, 140µm

, micromechanical valves and channels for moving fluids

the reactor, and an integrated peristaltic pump for mixing cells and media (Figure 3). The growth chamber loop is made up of 16 addressable segments.

Cutaway view of a “push down” PDMS microfluidic valve

The blue channel represents a fluid line that would contain cells and media in the

microchemostat. The red channel represents a “push-down” control line, it is filled with water and that can be pressurized or de-pressurized via computer control. (A) When the control channel is unpressurized the valve is “open” and cells and media can flow freely. (B) When the control valve is pressurized the pressure deforms the PDMS between the control line and fluid line and seals the fluid line resulting in a “closed” valve that prevents fluid

up” valves work by a similar principle except they are located below the fluid . When they are pressurized they deform the PDMS upwards and

mechanism shown here. Image courtesy of Ty Thomson ed from silicone elastomer polydmethylsiloxane (PDMS) using

. Each PDMS chip contained six microchemostats that are 1), and are made up of well established microfluidic components

. The microchemostat is 10µm high, 140µm wide, and , micromechanical valves and channels for moving fluids

the reactor, and an integrated peristaltic pump for mixing cells and media (Figure

The blue channel represents a fluid line that would contain cells and media in the

down” control line, it is filled with ntrol. (A) When the control channel is unpressurized the valve is “open” and cells and media can flow freely. (B)

PDMS between the control ng in a “closed” valve that prevents fluid

are located below the fluid . When they are pressurized they deform the PDMS upwards and

90

Figure 4-3 Microchemostat image with channels colored by food dye.

The growth chamber loop is colored with yellow food dye. The supply channels that bring fresh media to the reactor during cleaning events are colored in blue. The control lines for actuating the microfluidic valves throughout the chip are colored in red and green.

Microchemostat image by Frederick Balagadde [83].

Each of these 16 segments of the growth chamber loop can be individually isolated from the rest of the reactor and the cells and media within the chamber can be evacuated through a waste outlet and replaced with fresh media without cells. In the microchemostat the emptying of one of these segments serves two purposes: (1) it is the mechanism of dilution of cells associated with establishing the continuous culture environment and (2) it

91

prevents the formation of biofilms by flowing fresh media though the segment to sheer cells from the wall or by flowing lysis buffer to actively kill any cells that have adhered to the walls. The microchemostat has two modes of operation (Figure 4-4): (1) “continuous circulation” of the cells and media in the reactor to maintain a well-mixed environment and (2) a “cleaning event” where one of the 16 reactor chambers is isolated, cleaned, and replaced with fresh media. During an experimental run of the microchemostat the reactor spends most of its time in continuous circulation mode punctuated by scheduled cleaning events that clean each of the 16 reactor chambers.

Figure 4-4 Two modes of operation of the microchemostat.

(A) During a “cleaning event” one of the 16 segments of the reactor is isolated and lysis buffer is flowed through the segment to eliminate any biofilm growth. The lysis buffer is represented in pink flowing from the inlet through the ‘Nth dilution compartment’ and out the waste outlet. Following the lysis buffer, fresh media will be flowed into the segment and then the microchemostat will move to (B) “continuous circulation” mode. In continuous circulation mode, the cells and media are mixed continuously around the growth chamber loop by the inline peristaltic pump. This mixing ensures that the fresh media added during cleaning events is mixed throughout the culture. Image by Frederick Balagadde [83]

92