Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Podsumowanie

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Streszczenie

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Wprowadzenie

Delivery of macromolecules to the cell cytoplasm is a critical step in therapeutic and research applications. Nanoparticle mediated delivery, for example, has shown potential in gene therapy^1,2, while protein delivery is a promising means of affecting cellular function in both clinical³ and laboratory⁴ settings. Other materials, such as small molecule drugs, quantum dots, or gold nanoparticles, are of interest in applications ranging from cancer therapeutics^5,6 to intracellular labeling^7,8, and single molecule tracking⁹.

The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles^10,11, liposomes¹², or chemical modifications¹³ to facilitate membrane disruption or endocytotic delivery. In these methods, the delivery efficiency and cell viability are often dependent on the structure of the target molecule and the cell type. These methods can be efficient at the delivery of structurally uniform materials, such as nucleic acids, but are often ill-suited for the delivery of more structurally diverse materials, such as proteins^14,15 and nanomaterials⁷. Moreover, the endosome disruption mechanism that most of these methods rely on is often inefficient, hence leaving much material trapped in vesicle structures¹⁶. Finally, methods that are often developed for use with established cell lines do not translate well to primary cells.

Membrane poration methods, such as electroporation^17,18 and sonoporation¹⁹, are an attractive alternative in some applications; however, they are known to cause low cell viability and can be limited by the charge of the target delivery material.

Rapid mechanical deformation of cells, a microfluidic approach to delivery, has recently demonstrated its advantages over current techniques in the context of cell reprogramming²⁰ and nanomaterial delivery²¹. This method relies on mechanical disruption o the cell membrane to facilitate cytosolic delivery of materials present in the surrounding buffer. The system has demonstrated enabling potential in previously challenging cell types (e.g. primary immune cells and stem cells) and materials (e.g. antibodies and carbon nanotubes). Herein, the general procedure to use these devices for intracellular delivery of target macromolecules is described. The procedure is generalizable to most cell types and delivery materials; however, it is recommended that one conduct a brief optimization of conditions, as detailed in our previously reported design guidelines²⁰, for any previously unreported applications. To date, the system has been used successfully for the delivery of RNA, DNA, gold nanoparticles, quantum dots, carbon nanotubes, proteins, and dextran polymers^20,21.

Protokół

1. Storage

1. Store the reservoirs, holders, O-rings and microfluidic devices in 70% ethanol. Use a container (e.g. jar or beaker) that has a lid to prevent evaporation and contamination by dust or outside particles. Place the devices in one container (1), reservoirs and O-rings in a second container (2), and holders in the third (3).

Note: The use of 70% ethanol for storage is to maintain sterility. If the only components of the solution are ethanol and water (i.e. no denaturing agents), all system components should be fully compatible and will not degrade over time.

2. Change ethanol solution in containers (2) and (3) before each use to prevent cross-contamination across experiments and minimize the presence of unwanted particles that can cause clogging.

2. Experiment Preparation

1. Place containers (2) and (3) in an ultrasound bath for 5-10 min before each use. This helps remove any contaminating particles from previous experiments.
2. Clean workspace in biosafety cabinet with 70% ethanol solution.
3. Spray all materials (3 containers, and tweezers) with a 70% ethanol solution before placing them inside the biosafety cabinet.

3. Assembly

1. Set down 2-3 low-lint wipes in the work area.
2. Remove plastic reservoirs from their container with tweezers and set them on the wipes to facilitate evaporation of ethanol solution from inner surfaces. Gently tap the reservoirs on the surface or blow air through them to facilitate removal of the ethanol solution.
3. Insert O-rings into their appropriate slot on the reservoirs.
4. Remove the holder and chips from respective containers and allow ethanol to evaporate (~1-2 min).
5. Use tweezers to place the desired chip face-up (i.e. access holes up) in the holder. Raise the holder with the chip to eyelevel to make sure the chip is lying flat in the holder and adjust if necessary with the tweezers. IMPORTANT: If the device does not fit properly in its holder, there is a risk that it will break during the subsequent steps.
6. Next, gently place the reservoirs on the holder and align them with the clips. Be careful that O-rings do not fall out of their slots during this process.
7. Gently press down on the reservoirs until they click into place. Ensure that both sides of the reservoirs are secure and that the chip appears to be in the correct position.

4. Cell Preparation

1. For adherent cells (primary or established lines): Plate cells 1-2 days prior to the experiment such that they are no more than 80% confluent on the day of the experiment.
2. Place cells in suspension (in PBS or relevant media) and aim for an operating concentration of 1.0 x 10⁶ cells/ml to 1.0 x 10⁷ cells/ml. NOTE: We have not observed significant changes in delivery performance due to cell concentration.
3. Mix cells and the desired delivery material in a separate tube to obtain the desired material concentration for the experiment. IMPORTANT: Because the described delivery method relies on diffusion to facilitate delivery, a higher material concentration will yield higher delivery. If possible, it is recommended to use a 1 µM solution of the desired material for initial trials. This concentration can then be titrated down in future experiments as needed. The lowest reported concentration used with this device is 10 nM²¹.

5. Operation

1. Pipette mixture of cells and delivery material into a reservoir (current design has a max 150 µl capacity). NOTE: Most device designs are fully reversible therefore direction of flow through the channels does not matter. Samples may be loaded into either of the two reservoirs.
2. Attach pressure tubing to the filled reservoir and tighten the nut to ensure proper sealing (finger tight is often sufficient).
3. Adjust pressure to the desired level on the regulator. This controls the speed at which cells travel through the device. Note that cell flow does not start until button is pressed.

NOTE: In previous work, nitrogen or compressed air have worked equally well as the carrier gas.

4. Raise device to eye level and orient it so that the liquid in the reservoir is easily visible. NOTE: Track the liquid column to shut off the system before the reservoir is emptied.
5. Press the button to pressurize the reservoir and begin cell flow.
6. When the liquid level is approximately 2 mm from the bottom of the reservoir, quickly turn the regulator to 0 psi to stop flow. IMPORTANT: If one fails to stop the flow before the reservoir is emptied, one risks ejecting the sample from the collection reservoir. Also note that if the fluid column is not moving at an appreciable pace, or has slowed substantially relative to its initial flow rate (e.g. 3x slower), the mounted device is probably clogged and needs to be exchanged. Operating a clogged device can lead to higher cell death.
7. Collect the treated cells from the appropriate reservoir and place them in the desired collection tube/plate. IMPORTANT: Do not dilute the collected cells in any buffers at this stage as the porated cells will continue to uptake material for up to 10 min²⁰. After this window has passed, dilute the cells in the desired media/buffer.
8. To collect more treated cells or try alternative experimental conditions, repeat steps 5.1 - 5.7 as needed. Recall that the chips are reversible, therefore samples can be mounted in either reservoir. Be sure to exchange chips as needed if they clog. Discard clogged devices.

6. Disassembly

1. Gently disconnect the reservoirs from the main holder by pushing aside the clip arms.
2. Place each part in the appropriate storage container (detailed in section 1).
3. Place used chips in a separate container for disposal.

Wyniki

Figure 1 contains a descriptive schematic of the microfluidic delivery system. Figures 2a-b illustrate typical results from treating HeLa cells with different device designs in the presence of fluorescently conjugated 3 kDa dextran²⁰. If the procedure is followed correctly, system performance will be sensitive to device type and operating speed. Therefore, one should optimize these conditions for a given application before proceeding to more complex experiments. In the range o...

Dyskusje

Certain aspects of the described experimental procedure (i.e. factors other than chip design and operating speed) may need to be optimized depending on the cell type and delivery material the system is applied to. The discussion that follows addresses some of the most common factors to consider when designing experiments.

To improve the delivery signal for fluorescently labeled compounds, one needs to address sources of background fluorescence. Surface binding and endocytosis, for exa...

Materiały

Name	Company	Catalog Number	Comments
Device Holder & Plastic reservoir	SQZ Biotechnologies	Holder
LSRFortessa Analyzer	Becton Dickinson	N/A	Flow cytometry machine used at the Koch Institute Core Facilities
Microfluidic device	SQZ Biotechnologies	Cell Squeeze
O-Rings	McMaster	9452K311
Pressure system to operate device	SQZ Biotechnologies	Pressure System
Tweezers
Ultrasound bath