Microfluidic cell culture: How to do fast medium and drugs change in a microfluidic device?

Ability to switch drugs in seconds or less in microfluidic chips allows to study cell response at cells timescale. There are a lot of applications in microfluidic cell culture where the use of pulseless syringe pumps is relevant. However, these flow control devices are generally not suited for fast flow switch in microfluidic devices due to system compliance. There are several types of microfluidic devices that enable fast flow change (<1 sec) of cell culture media, starting from a single straight PDMS channel to more complex methods like hydrodynamic focusing. In the following tutorial, we will describe the advantages and limitations of each method.

Basic microfluidic cell culture chip with narrow microchannels

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In this configuration, cells are injected/pumped using high pressure or manual syringe inside tight channels with sizes a little bit smaller than cells. Once cells are trapped, medium or drugs can be perfused at lower pressure through the microchannels. This type of device can be used for classical cell culture in your microfluidic chip with medium renewal (setup 1). To quickly change cell culture medium, you can add valves upstream the microchip (e.g. rheodyne valve).
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For basic cell culture with medium renewal you can use a pressure pump or a push syringe, rigid tubing and a liquid tank containing the medium or the drug. Eventually, a flow sensor can be added to adapt the pressure depending of the required flow rate.

On the left-hand picture, the microchip has two inlets. Cells are trapped with the same method as above..

In this case, when drug is injected on one side, the other side is corked with an electromechanical valve. It enables faster drug change with no backflow within inlet capillaries.

Advantages of basic microfluidic cell culture chip with tight microchannels

  • Easy fabrication of the single layer PDMS microfluidic device
  • Such microchips are compliant with non adherent cells such as worms, bacteria or yeasts
  • This kind of cell culture microfluidic device can be used with basic flow control setup

Limitations of basic microfluidic cell culture chip with tight microchannels

  • Not compliant with adherent cells
  • Requires precise flow control to reduce the medium switch time without flushing cells
  • Injection of cells within microchannels can be stressful for cells
  • Medium switch time is between 1 and 10 seconds, depending on the required flow rate. This time can be reduced down to 100 ms using relevant fluidic connections.
  • The drug treatment on cells can be non uniform; it depends on the cells density in each microchannel
  • Flow rate during flow change can induce mechanical stress on cells

In order to reduce the flow switch time, ideally, for one drug the device should have one inlet, for four drugs the device needs four inlets, etc… Pressure controller will be faster than push-syringe as well. For faster than seconds flow switches and flow sequencing, special attention should be paid on tubing, fluidic connection and bubbles.

Eventually, if you use a pressure controller, a flow sensor can be added on the outlet to adapt the pressure depending on the required flow rate.

For multiple flow switch using two drugs, for example, you can use a one outlet pressure pump coupled to two liquid tanks containing the drugs and two valves. If you need to inject a higher number of drugs like four, you will need 4 inlets on your device, 4 tanks and 4 valves.

Multiple inlets microfluidic cell culture chip using laminar flow

In microfluidic devices, flow streams are laminar so there are no turbulences between them.
It is then possible to use laminar properties of microfluidic flow to perform medium switch..
This kind of method requires microchips with multiple inlets like Y-patterned channels..
By changing pressure at the inlet of the device, the laminar flow boundary moves in the main stream channel and change the type of medium in contact with cells. Since all inlets are already pressurized and capillaries are filled with drugs the reactivity of the system is enhanced and then allows to perform very fast medium/drug changes (around 100ms).

For laminar flow switch, the easiest method is to use a pressure pump with two inlets and two liquid tanks containing the media or the drugs. Pressure patterns (rectangular, sinus ect..) will ease the set-up and flow rates implementation. Eventually, a flow sensor can be added to adapt the pressure depending on the required flow rate.

Two push-syringes can eventually be used but the correlated flow rate settings and especially their coordinated precise modifications may be tricky to apply.

Using three inlets allows to confine the central flow stream between the two side flows. Depending on the flow rate conditions, the central flow stream can be down to 50 nanometers wide. This method, called hydrodynamic focusing, also allows to treat only a given portion of a cell with drugs (PARTCELL method, [1]).

For hydrodynamic flow focusing you can use one pressure controller with three oultets and three liquid tanks containing the media or the drugs. Eventually, a flow sensor can be added to adapt the pressure depending on the required flow rate.

As before, push-syringe can be used for hydrodynamic focusing but its modification will be more difficult to implement and much less fast and precise.
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Advantages of multiple inlets microfluidic cell culture chip using laminar flow

  • Single layer PDMS microfluidic device: Easy fabrication
  • Those devices are compliant with adherent cells
  • Small setup required for flow control
  • Medium switch time is around 100 msec

Limitations of multiple inlets microfluidic cell culture chip using laminar flow

  • Does not work with non-adherent cells
  • Requires precise flow control to maintain laminar frontiers
  • Deposition and positioning of cells in channels can be difficult
  • Flow on cells can induce mechanical stress

Y-channel microfluidic cell culture chip using laminar flow and narrow microchannels

For non adherent cells, it is also possible to mix laminar flow and cell trapping. In this case, cells are injected using high pressure to trap them in the narrow microchannels.

Then both entries are set at a different pressure.

Varying the pressure will change the liquid interface and submit trapped cells to the new drug..
Nevertheless, the presence of cells trapped in microchannels will change their fluidic resistivity and will perturb the predictability of the laminar interface position.

For laminar flow switch you can use two pressure pumps and two liquid tanks containing the media or the drugs. Eventually, a flow sensor can be added to adapt the pressure depending on the required flow rate.

As before, push-syringes can eventually be used but the correlated flow rate settings and especially their coordinated precise modifications may be tricky to apply.

Advantages of Y-channel microfluidic cell culture chip containing trapped cells and using laminar flow

  • Single layer PDMS microfluidic device: Easy fabrication
  • Compliant with non adherent cells such as worm, bacteria or yeast
  • Small setup required for flow control
  • Medium switch time is around 100 ms

Limitations of Y-channel microfluidic cell culture chip containing trapped cells and using laminar flow

  • Not compliant with adherent cells
  • Requires precise flow control to control the medium switch time without cell flushing and to position precisely the laminar interface
  • Injection of cells within microchannels can be stressfull for cells
  • The drug treatment on cells can be non uniform; it depends on the cells density in each microchannel
  • Flow rate during flow change can induce mechanical stress on cells

Drug treatment using diffusion

Here the cells are first injected in microchannels or chambers.


Then outlets of the cell chambers are closed and media or drugs are injected in the main channel.


Then, the drugs diffuse into the cell chamber. In this configuration the drug change speed depends on the diffusion time between drug channel and cell room. This time depends on the molecular size of the drug and the lenght of the gap separating cells and mainstream channel.

Another kind type of device also allows this kind of medium change (see below).

For diffusion-based flow switch you can use a single pressure pump, liquid tanks containing the media or drugs and valves. When changing drug, the intlets of the cell chambers have to be blocked using plugs or valves. Eventually, a flow sensor can be added to adapt the pressure depending on the required flow rate.

Advantages of drug treatment using diffusion

  • Easy fabrication
  • Compliant with non adherent cells such as worm, bacteria or yeast
  • No mechanical stress on cells by flow rate
  • Medium switch time is around 1 s (depending on the diffusion length)

Limitations of drug treatment using diffusion

  • Injection of cells in channels can be stressfull for cells
  • Medium change speed may be difficult to adjust.

Drug treatment using diffusion and narrow microchannels

Injection of drugs on one side :

In this case the channel containing cells are thinner ( i.e : 5µm) than the channel used for fluid circulation (i.e. : 50µm). Cells are first injected using high pressure.

After outlets blocking, medium is perfused at low pressure. In this case , user perfuses medium only on one side and block the other side outlets.

Injection of drugs on both sides:

In this case, user perfuses through both sides. It reduces the diffusion time in cells channels but it is harder to control undesired flows through cells channels.

For diffusion-based flow switch, you can use a single pressure pump and a liquid tank containing the medium or drugs. When changing drug, outlets of the cell chamber have to be blocked using plugs or valves.

To inject drugs on both sides of the device, we recommend two pressure pumps rather than push-syringe to finely tune the pressures. Eventually, a flow sensor can be added to adapt the pressure depending on the required flow rate.

Advantages of Drug treatment using diffusion and narrow microchannels

  • Compliant with non adherent cells such as worm, bacterias or yeasts
  • With diffusion by both sides, medium switch can be 4 times faster than by one side
  • Medium switch time is around 1 s (Depending on the diffusion length)
  • No mechanical stress on cells by flow rate

Limitations of Drug treatment using diffusion and narrow microchannels

  • Injection of cells within channels can be stressfull for cells
  • Requires quite precise flow control to limit medium convection in cells channels leading to cells movements
Cells are injected using high pressure or manual syringe inside tight channel with a size a little bit smaller than cells. Then, medium or drug can be perfused at lower pressure through those channel.

For more tutorial about microfluidics, please visit our other tutorials here: «Microfluidics tutorials». The photos in this article come from the Elveflow® data bank, Wikipedia or elsewhere if precised. Article written by Guilhem Velvé Casquillas and Timothée Houssin and revised by Lauren Durieux.

References:

1. Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE, Whitesides GM. Selective chemical treatment of cellular microdomains using multiple laminar streams. Chem Biol. 2003;10(2):123‑30

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