Team:WHU-China/Hardware
Nosocomial infections are fierce under the epidemic, and ‘The Negotiator’ has provided a feasible solution to deal with ventilator-associated pneumonia. To assist the biological designs, two hardwares were designed and constructed. To illustrate Quenching Module, we have designed a co-culture experimental platform that can facilitate controllable and quantifiable co-culture between engineered bacteria (EcN) and pathogenic bacteria (Pseudomonas aeruginosa) under limited contact conditions. To demonstrate Sensing Module and the whole scope of ‘The Negotiator’, we tried to use a lung small-airway microfluidic chip to replace animal models to simulate the environment of lower respiratory tract and investigate the prophylactic strategy at the organ level.
In the design, we need to detect the growth and metabolism status of engineered bacteria (EcN) and pathogenic bacteria (Pseudomonas aeruginosa) under the condition of limited material exchange and cell communication. Because the existing experimental equipment for such co-culture, such as U-tubes with bacterial filters, cannot fully meet our experimental requirements, we designed and constructed a co-culture equipment based on purchasable components, which can control the mixing time to detect the impact posed on both bacteria.
Considering that the growth of engineered bacteria and pathogenic bacteria are not synchronized in the actual treatment process, the co-culture platform is required to allow two bacteria to be cultured separately. They can also enter the co-culture status to exchange materials (e.g. AHLs) at any time. In order to study the materials exchange at different times, we designed a valve on the connecting pipe for sampling and cutting off the connection. To meet these requirements, we have designed a device, as shown in the figure below：
Fig. 1 Concept manuscript of co-culture device
1,2: Special needle tubular culture tube, separately cultivated "negotiation expert" and "pathogenic bacteria", with graduations, easy to estimate the volume (the culture tube and the bacteria filter can be detached)
3,4: Bacteria sampling tube
5,6: Bacteria filter
7,8: Sterile sampling tube
9,10: Mixed-isolation-connected bridge, with left and right arms, both of which are hollow tubes, with a cock in the center to control whether the left and right arms are connected
11: Left and right inflatable holes (the actual opening direction is diagonally upward, here is the opening downward for the convenience of drawing)
12: Inflatable hole plug (openable)
13: Inflatable needle tube: After opening the inflatable hole plug, it can be assembled with the inflatable hole
Considering that during the actual sampling process, the sampling port may remain part of the sample and medium, which may contaminate the sampling port during the next sampling, so we tried to improve the sampling port.
Fig. 2 Schematic diagram of improvement of sampling part (only part 1 and 3)
Initially, we planned to 3D print each part separately, and then stitch together to establish the co-culture platform. However, after research, considering the unaffordable cost of 3D printing materials, we decided to choose to DIY from purchasable components, under the premise of achieving the functions.
Fig. 3 Overview of Co-culture platform
Control element:
1-3: Three-way valve
4-9: Double pass valve
10-13: Pressure-Controlled needle (including pressure-controlled needle piston)
Sampling and ventilation elements:
14: Pressure regulating ventilation pipe
15, 18: Sterile sampling tube
16, 19: Bacteria sampling tube (consisting of rubber pipe, disposable bacterial filter, double-pass valve, joint and countersunk head of rubber pipe)
17, 20: Sampling mouth of bacterial liquid in the left culture bottle (consisting of rubber pipe, double-pass valve, joint and countersunk head of rubber pipe)
Other components:
21, 22: Culture bottle
23, 24: Multi-use bacterial filters
25: Completely closed bulkhead
26: Connecting bridge
Now we introduce how to use the device:
1. Assembly device and cultivation methods
(1) Assemble the instrument capable of autoclaving (excluding disposable parts);
(2) Rotate the three-way valve 1 to connect the ABC end, the three-way valve 2 to connect the DEF
end, and the three-way valve 3 to connect the GHI end;
(3) Unscrew the caps of the two culture bottles;
(4) Autoclave sterilization;
(Note: The pressure regulating ventilation tube 14 May not be assembled when it is used for only one
experiment in which both sides of the medium are mixed.)
(5) Complete the assembly of pressure control needle and disposable bacterial filter and other
components in the ultra-clean workbench after sterilization;
(6) Close the double-way valve 5, 6, 8, 9 and open the double-way valve 4 and 7; Screw three-way
valve 1 to the connected BC end, screw three-way valve 2 to the connected DEF end, and screw
three-way valve 3 to the connected GI end.
(7) Pull the maximum scale of pistons of all pressure control needles 10-13;
(8) Rotate the three-way valve 2 to the connected DE end;
(9) Open the caps of the left and right culture bottles (21 and 22) and transfer them into liquid
culture medium inoculated with different bacteria respectively. The total amount of liquid in each
bottle should be controlled within the range of 50ml-150ml, with 150mL being appropriate;
(10) The device can be taken out of the super clean table for cultivation.
(Note: Confirm that the double-way valves 5, 6, 8 and 9 remain closed, the double-way valves 4 and 7
remain open, and the three-way valve 1 rotates to the connected BC end, the three-way valve 2
rotates to the connected DE end, and the three-way valve 3 rotates to the connected GI end during
the culture process)
2. Sampling method:
(1) The initial state shall be the state during the cultivation process: double-way valves 5, 6, 8
and 9 shall remain closed, double-way valves 4 and 7 shall remain open, three-way valve 1 shall
rotate to the connected BC end, three-way valve 2 shall rotate to the connected DE end, and
three-way valve 3 shall rotate to the connected GI end; All 10-13 pistons are on the maximum
scale.
(2) Close the double-way valve of (4 or 7), open the double-way valve (16 or 19) or (17 or 20) of
the sampling port of single-side sterile culture medium or culture bottle bacteria solution as
required, and only one double-way valve can be opened at a time;
(3) Prepare the EP tube to collect the sample liquid, and align the sampling port to the EP tube. It
is recommended to operate beside the alcohol lamp and keep the sampling port beside the alcohol
lamp; Slowly push the piston of the pressure-controlled needle (10 or 11) on the same side of the
sampling mouth to be sampled and directly connected with the culture bottle, and the sample liquid
will flow out slowly.
(4) After the sampling is completed, pull back the piston of the pressure control needle pipe. After
the sample liquid returns in the pipe, close the double-pass valve of the sampling port and reopen
the double-pass valve 4 and 7.
In order to introduce the use of this hardware more intuitively, we have recorded a demonstration
video. The following is a demonstration of sampling.
The method of mixing the liquid culture medium on both sides without changing the bacterial
concentration and liquid volume in the culture bottle on both sides:
(1) The initial state should be the state during the culture process: the two-way valves 5, 6, 8,
and 9 are kept closed, the two-way valves 4 and 7 are kept open, the three-way valve 1 is rotated to
the connecting BC end, and the three-way valve 2 is rotated.
To the connecting DE end, turn the three-way valve 3 to the connecting GI end; all the pressure
control needle tubes 10-13 pistons are at the maximum scale.
(2) Rotate the three-way valve 1 to the AB end, keep the three-way valve 2 connected to the DE end,
and rotate the three-way valve 3 to the GH end;
(3) Close the two-way valve 7 of the aseptic vent tube 18 of the left culture bottle;
(4) Slowly push the piston of the pressure control needle tube 11 to the bottom of the pressure
control needle tube, so that the liquid culture medium in the right culture bottle 22 is compressed,
and enters the left culture bottle 21 through the connecting bridge 26. The bacteria in the culture
medium will be in the culture liquid.
Separate from the liquid culture medium when passing through the multiple-use bacterial filter 24;
after the liquid volume in the left culture bottle 21 increases by about 50 mL, close the two-way
valve 4 of the aseptic vent pipe 15 of the right culture bottle;
(5) Open the two-way valve 7 of the aseptic vent tube 18 of the left culture bottle;
(6) Slowly push the piston of the pressure control needle tube 10 to allow the mixed liquid medium
to flow from the left culture bottle 21 back to the right culture bottle 22 through a part of the
communication bridge 26. After the liquid levels in the left and right culture bottles are basically
level,
Open the two-way valve 4 of the sterile vent tube 15 of the right culture bottle;
(7) Steps (2)-(5) can be repeated several times to fully mix the liquid medium;
(8) After mixing, turn the three-way valve 3 to the GI end (keep the two-way valves 4 and 7 open,
the three-way valve 2 keeps the DE terminal connected, and the three-way valve 1 keeps the AB
terminal connected);
(9) Push the piston of the pressure control needle tube 13 to the bottom of the needle tube. In this
step, the air in the pressure control needle tube 13 can be used to make the liquid culture medium
in the left half of the connecting bridge 26 return to the left culture flask 21.
The air bubbles blown into the left culture flask 21 during the process also played a role in
re-mixing the bacterial liquid; after completing this step, the three-way valve 1 is immediately
turned to the connection BC end
(10) Rotate the three-way valve 3 to the GH end;
(11) Push the piston of the pressure control needle tube 12 to the bottom of the needle tube. In
this step, the air in the pressure control needle tube 12 can be used to make the liquid culture
medium in the right half of the rubber tube of the connecting bridge 26 return to the right culture
flask 22.
During the process, the air bubbles blown into the culture bottle 22 on the right also played the
role of re-mixing the bacterial solution; after completing this step, the three-way valve 3 was
immediately turned to the GI end;
If you only need to mix once, complete the above steps. If you need to mix again after completing
the above steps, you should turn the three-way valve 2 to connect to DEF after ensuring that the
pressure regulating vent tube 14 is assembled on the instrument.
Then pull the pistons of the pressure control needle tube 12 and the pressure control needle tube 13
to the maximum scale; turn the three-way valve 2 to the EF end.
At this time, the state of the container is restored to the initial state, and can be performed
again from step (1).
Fig. 4 OD600 value versus time in our co-culture hardware (experiment group) and conical flask (control group).
To confirm that the growth of our probiotics will not be interfered by Pseudomonas aeruginosa and to
test the basic function of our hardware, we designed a simple experiment: First, we added 2ml
bacteria suspensions (which was cultured overnight) to 120ml fresh LB medium (two flasks for E.coli
Nissle 1917 and one for P.aeruginosa PAO1) and cultured them in a shaker (37℃, 200rpm) for 2 hours
to obtain an initial bacteria density. And then we poured one flask of E.coli Nissle 1917 and P.
aeruginosa PAO1 in the left and right bottle separately. And we used the hardware to mix the medium
(without mixing the bacteria!) of the two bottles and took 2ml samples per 45 minutes to measure the
OD600 value in a spectrophotometer as the quantification of bacteria density since then. The same
procedure was also used on the other flask of our probiotics which was not mixed with the medium of
P. aeruginosa as the control group. And we used Matlab to draw the growth curve of the bacteria in
the three bottles. The mixing point was regarded as the 0 point at x axis.
From Figure 1 we can see that the growth of our probiotics in the hardware is similar to that in the
control group. Although maybe due to the medium we used, the density of P. aeruginosa is not so
high, both the pathogens and probiotics are still growing. This partially proves that our probiotics
can be cultured with P. aeruginosa normally, which provides evidence for the possibility of using
our probiotics as chassis to fight against the P. aeruginosa in our lungs. Also the function of our
hardware is verified again.
In the study of new coronavirus research and treatment options, many researchers have used lung simulation chips to achieve immune research at the organ or tissue level. Research on lung simulation chips to study bacterial infections in the lungs has also been obtained in the past effective results [1]. So we plan to use a small airway microfluidic chip to simulate the lower respiratory tract, so we reconstructed the existing chip [2], and 3D printed the chip holder.
Fig. 5 Overall assembly drawing
The microfluidic chip inside the red box in the picture, the chip holder outside the red box
Fig. 6 Microfluidic design drawing
1.Top component
2.PDMS-polycarbonate semipermeable membrane
3.Bottom component
Fig. 7 Channel profile
profile view of the channel, the picture below is the
red frame of the upper picture zoom
1-1mm*1mm Air channel
2-PDMS-Polycarbonate semipermeable membrane
3-1mm*0.2mm Blood channel
The Design of the Chip:
Fig. 8 Top component && PDMS-polycarbonate semipermeable membrane && Bottom component
1-Air channel connection port
2-Air channel
3-Blood channel connection port
4-Preserve holes: not covered with polycarbonate membrane, used to bond upper and lower
components
5-Polycarbonate membrane
6-Blood channel
After completing the design, we contacted Emulatebio, a company that can provide a complete construction of the chip, and its agents in China. They’re willing to provide us with a complete set of services from computer simulation to chip manufacturing and completion.
In order to complete our microfluidic chip, we designed a holder with reference to the literature [2]. The holder can clamp the PDSM-polycarbonate semipermeable membrane tightly between the top component and the bottom component to seal the channel. We successfully made the holder using 3D printing technology, with its project files shared here.
Fig. 9 Design drawing of the holder
1-Top connector
2-Vertical support
3-Pressure clamping plane
4-bottom plane
Fig. 10 Holder parts drawing
A-bottom plane
B-Pressure clamping plane
C-vertical support
D-top connector
Finished Product Display:
Fig. 11 Finished drawing of the holder
In summary, we successfully built a DIY co-culture experiment platform to help wet lab to explore the relationships between engineered bacteria and pathogenic bacteria. We also envisioned to rebuild a small airway microfluidic chip to illustrate the effectiveness of the therapy, and manufactured it with the help of emulatebio and Prof. Chen Pu. Also, a chip holder was 3D printed this year to complete the microfluidic chip.
1. Si L,et al. Human organ chip-enabled pipeline to rapidly repurpose therapeutics during viral
pandemics. bioRxiv 2020.04.13.039917.
2. Benam K H , Villenave R , Lucchesi C , et al. Small airway-on-a-chip enables analysis of human
lung inflammation and drug responses in vitro. Nature Methods, 2016.
