Cell-cell interactions on chip

Introduction

The Interaction Chip (Fluidic 688) enables the co-culture of different cell types in separate but interconnected chambers. The co-culture of different cell types is an important step towards increased physiological relevance for in vitro models of physiology and disease. Vascular networks connect all organ systems, transporting biomolecules from one location to another, mediating signalling cascades, gene expression and regulation, tissue homeostasis, immune response and disease progression.

In this Protocol we describe how to fill the separate chambers of a Fluidic 688 Interaction Chip with different cell types, and to apply a controlled flow rate across the chip for a dynamic cell culture environment. We pre-stain cells to illustrate patterns of cell partitioning in the two chambers during seeding.

Cell-cell interactions on chip possible applications

Co-culture different cell types in interconnected chambers to study:

• Soluble factor effects
  • condition medium in chamber B with factors secreted by cells in chamber A
  • effect of cell “secretome” – growth factors, cytokines, chemokines
  • tumour vascularization models
  • molecular gradients for chemotaxis and cell migration assays
  • immune response and wound healing
• Multi-organ effects 
  • multi organ-on-chip models
  • pharmacokinetic/ pharmacodynamic drug testing (ADMET – drug absorption, distribution, metabolism, excretion, toxicity responses)
• Physiologically relevant length scales
  • cell signalling and cell-cell communication over distance

Cell-cell interactions microfluidic setup

Hardware

1. Flow controller OB1 MK3+ with at least 1 channel 0/2000 mbar
2. 1 x flow sensor MFS3 2.4-80 µL/min
3. 1 x Bubble trap
4. Kit starter pack Luer Lock
5. 1 x 15 mL Falcon reservoirs
6. Microfluidic chip (Fluidic 688 with 1 pack of Mini Luer connectors, 1 pack of Mini Luer plugs and silicone tubing from microfluidic ChipShop)
7. Heated water bath
8. CO₂ incubator for incubation
9. Microscope for observation

Chemicals

1. Cell type 1: MCF7 (3×10⁶ cells.ml^-1)
2. Cell type 2: HEK293 (3×10⁶ cells.ml^-1)
3. Medium: DMEM high glucose (with fetal bovine serum, 10%; penicillin/ streptomycin, 100 U/ml; 100 µg/ml)
4. Buffers / stains: PBS / Cell stains (Calcein AM, 2 µM; Hoechst 33342, 10 µg.ml^-1)

Design of the microfluidic chip for cell-cell interactions

The Interaction Chip enables the flow of different liquids via its set of double interfaces on each end of the chip (interfaces 2 & 3 and 4 & 5).
Flow control options for multiple liquids include (i) to duplicate the configuration described below, (ii) to add a MUX-Distributor to control switching between up to n x 12 different liquids, and (iii) to add a valve to inject small volumes of reagent.

Interaction chip

• Geometric features: two chambers of volume 37.8 µl; six mini-luer interfaces, three on either end, including two connected to long channels for initial filling/ seeding (#1 and #6) and four connected to short channels for flowing medium or injecting reagents.

Chip preparation

• Surface treatment: a “hydrophilized” aids cell attachment to the chip surface (similar to “tissue-culture treated” cell culture equipment). No additional surface treatment is required for cells to adhere, although extra coating may help, depending on cell type.
• Chip handling: the chip may be secured flat in a petri dish to help with stability when tubing is connected.
• Connectors: Mini luer interfaces fit microfluidic ChipShop‘s Mini Luer connectors and plugs. Tubing (1/16” outer diameter) can be attached to Mini Luer connectors via a short length of silicon sleeve.

Microfluidic setup quick start guide

CELL PREPARATION

• Following standard protocols: Trypsinize cells from culture flask. Spin down and reconstitute to remove trypsin. Count carefully and prepare a stock suspension of 3×10⁶ cells.ml^-1 for seeding.

TIP: Aim for >70% confluency at time of seeding and a confluent monolayer before perfusion.

GENERAL LIQUID FILLING PROCEDURE

• To fill chamber A, close all interfaces securely at the opposite end of the chip (i.e. interfaces 4–6) with Mini Luer plugs. This acts like blocking the end of an empty straw with a finger before inserting it into a drink. The air will stay in place and keep chamber B from filling with fluid.
• Fill chamber A from interface 1 with a pipette (option: consider using a Mini Luer pipette adapter) or with a pressure-driven flow controller. Both interfaces 2 and 3 can be left open. If using a pipette tip make sure to insert it perpendicular into the Mini Luer interface.
• Once filled, gently open interface 6, then close interfaces 1–3 with Mini Luer plugs.
• To fill chamber B, carefully remove plugs from interfaces 4 and 5.

TIP: It is important to insert and remove Mini Luer plugs very carefully to minimally disturb the fluid in the opposite chamber.

• Fill chamber B from inlet 6.

CHIP SEEDING WITH CELLS

• Following the General Liquid Filling Procedure: fill chamber A with cell type A (3 x10⁶ cells.ml^-1 stock solution ~113 400 cells per chamber) and chamber B with cell type B (3 x10⁶ cells.ml^-1 stock solution). After each addition of cells, verify the flow of cells into the channel using a microscope.

TIP: Incubate 30-60 min between seeding chambers A and B to reduce the chance of cross-chamber mixing, i.e. seed chamber A and incubate 30-60 min, then seed chamber B.

• Place your microfluidic chip in the CO₂ incubator for 6 to 24 h for cells to attach under static conditions.

TIP: To stop the channel from drying, gently close all interfaces to minimise evaporation, place the chip in a petri dish along with a small reservoir of water, e.g. a 50 mL tube cap, and cover.

• Gently exchange medium manually with a pipette 1 to 2 times per 24 h in chambers A and B individually, following “General Liquid Filling Procedure”.

TIP: It is normal for a small volume of solution / cell suspension to flow across the connection bridge into the cone of the opposite chamber, or along the chamber walls (that are lined with a narrow trough) when changing the position of plugs during sequential filling of separate chambers.

• Option: To improve cell partitioning: once cells in chamber A are well attached, very gently flush chamber A with medium through the seeding inlet before switching plugs to seed chamber B. Most of the inter-chamber movement occurs with the pressure of inserting or removing plugs. Consider incubating up to 12 h between seeding chambers A and B, depending on experimental application.

INSTRUMENT CONNECTION FOR CELL-CELL INTERACTIONS

• 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 sensors to the OB1. For details refer to “MFS user guide”.
• 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 the guide 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 on the list of recognized devices.
• OB1 calibration is required for the first use. Please refer to the “OB1 user guide”.
• Add the flow sensors: 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 on the list of recognized devices. For details refer to “MFS user guide”.
• Open the OB1 Window

SYSTEM PURGING

• Fill the sample reservoir with medium and connect the supplied 1/16” OD tubing and the 4 mm OD coil tubing to the tank. For more details, refer to the video “connector for the OB1”.
• Plug the tank to the corresponding OB1 pressure controller outlet. For more details, refer to “Elveflow microfluidic reservoirs assembly instructions”.
• Connect the supplied 1/16” OD tubing using the 1/4”-28 fitting from the medium reservoir to the bubble trap.
• For flow measurement, connect the flow sensor between the bubble trap and the chip. For more details, refer to the “MFS user guide”.
• To fine-tune the system and to obtain the best performance in terms of flow rate control, add a resistance tubing (15 cm length of a 100 µm internal diameter) to the outlet of the flow sensor. For more details, please refer to the “Flow control tuning” document.

TIP: The resistance should always be placed downstream of the MFS (between the MFS and the chip) to ensure a stable measurement.

• Place the open end of the tubing coming from the flow sensor over a waste collection vessel.
• Pressurize the liquid reservoir and flow until liquid begins to drip from the exit.

PERFUSION INSIDE THE INTERACTION MICROFLUIDIC CHIP

• Once cells are well attached under static conditions, select interfaces to use for flow, e.g. interface 3 as liquid inlet and interface 4 as liquid outlet. Remove plugs from these interfaces and ensure the remaining interfaces are firmly plugged.
• Ensure that the chip is completely filled and that a large droplet of liquid is emerging from the inlet and outlet.
• Add a silicon sleeve and Mini Luer connector to the tubing coming from the flow sensor and to a piece of tubing going to a collection vessel.
• Set a low pressure (or flow rate) from the liquid reservoir until there is a droplet of liquid at the exit of the Mini Luer connector.

TIP: Hold the connector upwards to ensure all air is purged.

• Firmly connect tubing to the microfluidic chip inlet (i.e. interface 3).
• Firmly connect the exit tubing to the microfluidic chip outlet (i.e. interface 4).
• Set a pressure (or a flow rate) to start perfusing the medium into the interconnected chambers and start the cell-cell interactions.

TIP: Flow profile can be steady, pulsatile or custom. For more details, refer to “ESI User Guide”.

Partition of Cells into culture chambers A and B

Chamber A: MCF7 seeded (pre-stained with Calcein AM)
Chamber B: HEK293 seeded (pre-stained with Hoechst 33342)

Seeding chamber A

Figure 1: Chamber A, seeded with MCF7 cells (calcein stain, green). A. Infiltration of HEK293 (Hoechst 33342, blue) cells 3mm into chamber A from the center cone (scale 500 µm). B. Bright field image of cells in chamber A merged with  fluorescent image of HEK293 cells (blue) at furthest point of penetration into chamber A (inset from panel A; scale 100 µm). C. Image B merged with fluorescent image of MCF7 cells (calcein AM, green; scale 100 µm).

Seeding chamber B

Figure 2: Chamber B, seeded with HEK293 cells (Hoechst stain, blue). A. All cells in position (i) of the chip (red inset) are HEK293. MCF7 cells infiltrated chamber B along the lower wall (~20% of cells at position (ii) in chip).