How to stain cells cultured in a microfluidic chip for dynamic cell culture?

Dynamic cell culture

Forces are abundant on cells and tissues in the body but are largely absent from traditional cell culture setups, which are static. Microfluidic platforms enable the controlled addition of fluid flow and mechanical stress to cells grown in vitro. A dynamic culture environment promotes the continuous transport of nutrients, metabolites and waste, as well as the addition of shear stress, an important regulator of mechanosensitive cellular processes such as proliferation, alignment and remodelling.

Watch Lisa Muiznieks’s webinar about dynamic cell culture:

Advantages of this setup for cell biology:

In this application note we describe how to stain cells after dynamic cell culture. Stain with the OB1 pressure controller to maintain a precise flow rate of wash buffer and stains over your cells in the perfusion chamber. Automate the sequence to free up your time for multi-stain assays and replicate experiments. Suitable for temperature sensitive stains and small volumes. Read also our application notes about automated cell seeding and microfluidic perfusion for dynamic cell culture.

Some main applications of dynamic cell culture include:

• Live cell imaging (e.g. calcium imaging, FISH)
• Drug screening
• Shear stress
• Cell rolling-adhesion assay
  • Immune response
  • Cancer invasion and metastasis
• Models of physiology and disease
  • Organs on chip
  • Blood vessel formation & occlusion (atherosclerosis)
  • Bone homeostasis and disease (osteoporosis)
  • And many more!

List of components

Microfluidic chamber designed for perfusion
(IBIDI µSlide I Luer)

Cell and biology pack

Flow controller OB1 Mk3+

Mux-distributor

Microfluidic bubble trap

Flow sensor

MUX Wire & 1 3-way valve

Tubing, fittings and reservoirs

Setup diagram for cell staining

HARDWARE:

• Pressure & Flow controller: Impose a given pressure in order to create a stable and pulseless flow.
• Reservoirs: Contain your medium, buffer, stains, cell suspension or samples. Various cap sizes are available, from Eppendorf to bottles.
• Rotation valve (MUX-Distributor): Select the injected liquid.
• Flow Sensor: Monitor and control the flow rate in real time (MFS3 2.4-80 µL/min).
• Bubble trap: trap and remove air bubbles to avoid them entering your chip, disturbing flow and adhered cells.
• MUX Wire & a 3-way valve: On/off and selection valve for fluid control
• Kit starter pack luer lock + ¼”28 fittings packs
• Manifold: 9-port splitter for microfluidics
• Perfusion chamber or microfluidic chip: Contains your cells. Compatible with microscopy.
• Heated water bath
• CO2 incubator for incubation
• Microscope for observation
• Computer: Control all the parameters with our software and automate your experiment by creating injection sequences.

REAGENTS:

• Cells: HeLa cells (1×10⁶ cells per mL)
• Medium: DMEM high glucose, 10% FBS, Penicillin/ Streptomycin (100 U/mL; 100 µg/mL)
• Wash buffer: PBS
• Live/ dead cell stains: AM/ Propidium Iodide/ Hoechst 33342 (nuclear stain)

If the microfluidic chip used is non treated, and/or not sterile, here are two steps to prepare the culture chamber:

• Wash the microfluidic chip with aniosyme (1%, 30 min), rinse thoroughly with water and sterilize using an autoclave or a plasma gun (depending on the chip material).
• Treat the culture surface with cell adhesion coating (e.g. collagen I or fibronectin).

Protocol to perform automated cell staining in a microfluidic channel

Protocol summary

Follow two guides to seed and perfuse cells in a microfluidic chip.

Wash: Switch MUX-Distributor inlet to flow PBS to gently wash cells.

Add Stain 1: Switch MUX-Distributor inlet to flow stain 1 and gently fill the channel with stain.

Incubate: Stop the flow and hold while cells take up stain.

Wash: Switch MUX-Distributor inlet to flow PBS to gently wash out stain 1.

Additional Stains: Repeat steps “Wash”, “Add Stain” and “Incubate” until cells are stained as desired.

Wash: Switch MUX-Distributor inlet to flow PBS to gently wash cells.

Image: Image cells on-chip by microscopy.

Cell staining detailed guide

INSTRUMENT CONNECTION FOR AUTOMATED CELL STAINS

• Connect your OB1 pressure controller to an external pressure supply using pneumatic tubing, and to a computer using a USB cable. For detailed instructions on OB1 pressure controller setup, please read the “OB1 user guide”.
• Connect the flow sensor to the OB1 and the 3-way valve to the MUX wire.
• Connect the MUX Distribution and MUX Wire to your computer.
• Turn on the OB1 by pressing the power switch.
• Launch the Elveflow software. The Elveflow Smart Interface’s main features and options are covered in the “ESI user guide”. Please refer to those guides for a detailed description.
• Press Add instrument \ choose OB1 \ set as MK3+, set pressure channels if needed, give a name to the instrument and press OK to save changes. Your OB1 should now be in the list of recognized devices.
• OB1 calibration is required for the first use. Please refer to the “OB1 user guide”. 
• Add flow sensor: press Add sensor \ select flow sensor \ analog or digital  (choose the working range of flow rate for the sensor if you have an analog one), give a name to the sensor, select to which device and channel the sensor is connected and press OK to save the changes. Your flow sensor should be in the list of recognized devices. For details refer to “MFS user guide”.
• Add MUX Distribution: press Add instrument \ choose MUX Distrib/Inj/Rec \ select your instrument, give it a name \ select the MUX version (MUX Distribution 10 here) and press OK to save changes. Your MUX should be in the list of recognized devices.
• Add MUX wire: press Add instrument \ choose MUX Wire \ select your instrument, give it a name and press OK to save changes. Your MUX should be in the list of recognized devices.
• Open the OB1, the MUX Distribution and the MUX wire windows.

PREPARATION OF THE SOLUTIONS FOR CELL STAINING

TIP: The idea is to keep everything as STERILE as possible. Wipe down the reservoirs with aniosyme, then water and air dry in the BSC. An additional step can be added by flushing aniosyme through the tubings that will be used for the experiment.

• Prepare the warmed medium reservoir and connect the supplied 1/16” OD tubing and the coil tubing 6mm OD to the reservoir cap. For more details, refer to the video “Connector for the OB1”. Once connected, place the medium into a heated water bath.
• Repeat step 2 for all the aqueous solutions you will be using, namely PBS buffer and stain(s).

• Connect all the coil tubings 6mm OD from the solution reservoirs to the outer ports of the manifold using 1/4’’-28 thread to 3/32’’ OD barb fittings (black fittings). To the central port of the manifold, connect a coil tubing 6mm OD. Connect the other end of this tubing to the OB1 pressure controller outlet.

TIP: If you are not using some ports of the manifold, close them using microfluidic plugs PFA 1/4’’-28 flat-bottom fittings.

TUBING LENGTH AND VOLUME WORKSHEET

Depending on the installation used with the CO2 incubator and the heated water bath, length of tubing can vary from laboratory to laboratory. Following is a worksheet to record tubing lengths, calculate the total volume of tubing from the reservoir to the microfluidic chip, and the time it will take to fill the microfluidic chip at the desired flow rate. This worksheet will also provide a guide to the total volume of each reagent needed for an experiment.

TIP: If light-sensitive reagents are used, aluminium foil can be wrapped around transparent tubing to minimize light-exposure during the experiment.

TIP: A low total volume of tubing is desirable to save time and minimize consumption and dilution of reagent, most critically for the distance between the valve and the microfluidic chip. Use a tubing with a small inner diameter such as PTFE tubing OD 1/32 between the valve and the microfluidic chip.

MICROFLUIDIC SYSTEM PREPARATION TO AUTOMATICALLY STAIN CELLS

• Connect the reservoirs containing the solutions to the MUX Distribution inlets by using the supplied 1/16” OD tubing and the 1/4“-28 fittings and add a dead-end block to one of the inlets of the MUX Distribution. For more details, please refer to “User guide MUX distribution 12/1”.

TIP: Connect the solutions in the order of injection to optimize the rotation of the valve.

• Rename the reservoirs in the MUX Distribution window of the ESI.

• At the outlet of the MUX Distribution, connect one end of the supplied 1/16” OD tubing using the 1/4”-28 fitting to a waste reservoir.
• On the MUX Distribution window, select port #1 (PBS) and set a pressure to fill the tubing with the liquid. Once liquid gets out to the waste, air is completely removed from the system for the medium line. 
• Repeat step 4 for all the different solutions connected to the MUX Distribution by selecting the corresponding port on the MUX Distribution window.
• Once all the air is removed from the different solution tubing from the MUX Distribution, Connect the tubing from the outlet of the MUX Distribution to the bubble trap. From the outlet of the bubble trap, connect the supplied 1/16” OD tubing using the 1/4”-28 fitting to the inlet of the flow sensor and a tubing from the outlet of the flow sensor to the inlet of the 3-way valve.

TIP: To fine tune the system and to obtain the best performance in terms of flow rate control, a resistance tubing can be added to the system. For more details, please refer to “Flow control tuning”.

• Connect 1/16” OD tubing using the ¼”-28 fitting from the NO (normally open) port to a waste container (this is in order to fill the tubing and remove air before connecting the microfluidic chip containing cells).  At the NC (normally closed) port of the valve, connect the supplied 1/16” OD tubing to a waste container.

• On the MUX Wire window, select the configuration NO for the 3-way valve. On the MUX Distribution window, select port #1 (PBS) and set a pressure to fill all the tubing with the liquid. Once liquid gets out to the waste, air is completely removed from the system for the medium line and you can set pressure to zero.
• You can now carefully connect the microfluidic chip containing cells to the filled tubing coming from the NO port of the valve.

TIP: It is critical to not introduce any air bubbles in the microfluidic chip, especially if the chip contains small channel features as air bubbles tend to get trapped in the chip.

ON-CHIP CELL STAINING

• Select port #1 (PBS buffer) on the MUX distributor and switch the valve to NO configuration (to chip) on the MUX wire. Set a low pressure (or flow rate) to flow the buffer inside the microfluidic chip to wash the cells (e.g., one chip volume).
• Select port #2 (stain #1) on the MUX distributor and switch the valve to NC configuration (to waste) on the MUX wire. Set a high pressure (or flow rate) to push the staining solution up until the valve. Set a low pressure (or flow rate) and switch the valve to NO configuration (to chip) on the MUX wire. Fill the microfluidic chip with staining solution.

TIP: It is not necessary to fill the entire system with a precious staining solution. After a short injection, PBS (or buffer of choice) can be used to push the staining solution from the MUX distributor to the chip. It is important to calculate or measure the filling time of the system in order to optimize the timing steps in order to minimize waste of reagents. 

• Once the microfluidic chip is filled, stop the flow by switching to port #4 (dead-end channel) on the MUX Distribution window and set a zero pressure (or flow rate) on the OB1 window.

TIP: Switching to a dead-end channel rather than turning off the pressure prevents back flow, in order to ensure that the staining solution remains inside the microfluidic chip.

• Incubate for the required amount of time.
• Repeat the previous four steps for all the different stains needed for the experiment.
• Select port #1 (PBS buffer) and set a pressure (or flow rate) to flow the buffer inside the microfluidic chip to wash the cells.
• Select port #4 (dead-end channel) on the MUX Distribution window and set a zero pressure (or flow rate) on the OB1 window.
• The cells inside the microfluidic chip are ready to be imaged under the microscope.

GENERAL CLEANING OF CELL STAIN SETUP

• Flush lines and modules with water, then aniosyme (1%). Rinse thoroughly with water. Do a final flush with ethanol and air dry (flush air from an empty reservoir).
• Change fluidic tubing between experiments and use sterile reservoirs for new experiments. Periodically replace the Bubble Trap membrane.

AUTOMATED CELL STAINING

The following steps can be implemented to automatise the flow control and liquid switching of the cell staining.

• To create a sequence, click on the top middle button “Create Sequence” on the main window of the ESI software: a new window will appear.
• Back to the OB1 window: set up the desired flow rate or pressure for your experiment and save the configuration by clicking on “Config”. Repeat for all flow rates or pressure values that will be used throughout the experiment. Remember to save a “0” pressure or flow rate configuration. For more details, refer to the “ESI User Guide”.
• Similarly, in the MUX wire window: save configurations for each valve position, NC (normally closed) and NO (normally open) by clicking on “Config”.

TIP: Automated cell seeding and staining should be performed using microfluidics valves to avoid loss of reagents and clogging of the system.

• Back on the sequence window: on the left side of the window, click on the green “OB1” box (a line “OB1: Select instrument” appears in the middle part), on the right side, select the instrument (your OB1 pressure controller should appear when clicking on “Instrument”) and “load a configuration” saved previously.
• On the left side of the sequence window, click on the purple “DIST” box (a new line “DIST: Select Instrument” appears): select your instrument and write in the box “Valve position” the desired port’s number corresponding to the needed solution.

TIP: You can also choose the type of rotation of the MUX Distribution for your convenience (shortest, clockwise or counterclockwise).

• On the left side of the sequence window, click on the purple “MUX” box (a new line “MUX: Select Instrument” appears): select your instrument and load your valve configuration corresponding to the needed solution (normally closed or normally open).
• On the left side of the sequence window, click on the “Clock” box (a new line with a time frame appears): set a desired duration.
• Create your fluid injection sequence based on the procedures outlined in step 4-7.
• Option: on the left side of the sequence window, click on the “GO” box. This step will allow you to repeat your sequence: choose the “target step” (2 if you want to repeat the all sequence without changing the OB1 configuration) and “time” for how many times you want this to repeat.
• On the left side of the sequence window, click on the blue “END” box (a new line “END” appears). This means that your sequence is over.
• Following is an example of sequences for the cell staining with two stains:

a) Rinse cells with PBS.

b) Fill chip with stain #1 and incubate for 30 min

c) Repeat step (a) to rinse the cell with PBS.

d) Fill chip with stain #2 and incubate for 30 min.

e) Repeat step (a) to rinse the cell with PBS.

f) Chip is ready for imaging.

Results

LIVE/DEAD staining of HeLa cells (Calcein AM and propidium iodide, merged image)

This protocol can be adapted with MCF7 cells

Live cell staining of MCF7 cells (calcein AM/ Hoechst 33342, merged image)

LIVE/DEAD staining of MCF7 cells (Hoechst 33342 and propodium iodide, merged image)

Troubleshooting

I’m having trouble keeping the flow rate steady.
Check the height of the OB1, reservoir chip and exit (waste container). Adjust the Flow Sensor feedback to increase the pressure required to maintain constant flow rate. Adding more resistance tubing will also provide more tolerance for small changes and differences in height.

How do I know when the stain has reached my chip? Options to speed up the flow of stain to my chip?
Measure and cut each piece of tubing in a precise round number length. Calculate volume of lines (𝜋r2h) and set a constant flow rate, then use a timer, or program the ESI sequence scheduler.

TIP: (1) When switching between small volumes (e.g. PBS, stain1, PBS) you don’t have to wait until one reagent has filled the chip before switching to another. Move reagents in blocks of chip volumes. (2) Speed matters! Connect a 3/2 valve and waste vessel just before the chip to move stains quickly to the chip inlet without disturbing your cultured cells (liquid will flow into waste and not over cells).

Hints & tips

Application note written by Lisa MUIZNIEKS, Emma THOMEE, Subia BANO, Camila BETTERELLI GIULIANO and Jessica AYACHE.

Acknowledgement: This work was done thanks to the funding of European Union’s Horizon 2020 research and innovation programme (PANBioRA project, grant agreement No 760921; MECH-LoC project, MSCA grant agreement No 793749; MTOAC project, MSCA grant agreement No 795754; Protomet project, MSCA grant agreement No 813873; MaMi project, MSCA grant agreement No 766007).