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.
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.
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 therapy1,2, 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 therapeutics5,6 to intracellular labeling7,8, and single molecule tracking9.
The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles10,11, 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 proteins14,15 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 electroporation17,18 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 polymers20,21.
1. Storage
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. Experiment Preparation
3. Assembly
4. Cell Preparation
5. Operation
NOTE: In previous work, nitrogen or compressed air have worked equally well as the carrier gas.
6. Disassembly
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 of operating conditions illustrated in the figure, cell viability remains above 80%, however, one must note that suboptimal delivery parameters (e.g. inappropriate chip type) could lead to viabilities below 30%. Thus viability and delivery efficiency must be optimized simultaneously for any previously unreported cell type. If a device design is inappropriate for the target cell type, one will see results ranging from no delivery to high cell death (e.g. 10% viability). If the experimental conditions were inappropriate, for example the target material bound to the cell surface, one may not be able to measure the delivery accurately as the surface binding signal can mask the cytoplasmic signal.
Figures 2c-d illustrate our preferred method for measuring delivery efficiency within the live cell population. The control case in Figure 2c is meant to reflect all surface binding and endocytotic effects as the cells are exposed to the delivery material for at least as long as the treated cases. The delivered cell population is defined as the unshaded region such that only 1-5% of the control population falls within that region. Note that previous work has established that delivery in this approach is not mediated by endocytosis20. The double-peak distribution observed in the delivered example is not necessarily characteristic of the system as we have observed more and less uniform distributions for different experimental conditions.
Figure 1. Schematic of the full system and its interface to the microfluidic devices. Click here to view larger image.
Figure 2. Representative results with HeLa cells20. a) Delivery efficiency and b) cell viability of HeLa cells treated by 3 different device designs in the presence of cascade blue conjugated 3 kDa dextran (0.2 mg/mL). In these experiments, the cell solution was at a concentration of 106 cells per mL in PBS containing 3% fetal bovine serum and 1% F68 pluronics. Viability was measured by propidium iodide staining of dead cells and delivery results are shown for live cells only. All data points were run in triplicate, and error bars represent 2 SDs. c) A representative histogram for an experimental control where HeLa cells were exposed to cascade blue conjugated 3 kDa dextran for at least the same amount of time as a device treated case. d) The corresponding case where the cells were passed through the device to facilitate delivery. The fraction of the live cell population that is in the unshaded region of the histogram is defined as the ‘delivered’ population. Click here to view larger image.
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 example, can be addressed by washing cells 5-10 min after delivery to remove excess material before culture or further processing. If electing to forgo a wash step, it would be best to dilute the treated cells by 5-10x in the desired media to lower the concentration of target material in the surrounding solution and hence reduce endocytotic rates. In some cases, for example protein delivery, one may find it necessary to actively block the cell surface (e.g. using unlabeled bovine serum albumin) to minimize non-specific binding of the delivery material. In a given application, if the fluorescence intensity of the untreated case, as measured by flow cytometry, is over one decade lower than the endocytosis control then surface binding/endocytosis may be an issue. Note that issues with background signal are only relevant for direct fluorescent detection of the delivery material, functional assays (e.g. cell reprogramming) are usually unaffected as endocytosed or surface bound material are often inactive.
To improve the viability of cells after treatment, one needs to minimize the amount of processing post-delivery. It is recommended that one avoid excessive centrifugation or wash steps and transfer cells into their desired media in an incubator 5-10 min after delivery is complete. In adherent lines, cells are often fully adhered and dividing within 24 hr after treatment. As discussed previously20, we have not observed any long-term side-effects from the use of this technique.
The microfluidic devices used in this delivery system are prone to clogging over time. Clogs often form at the constriction as it is the narrowest feature on the device. Clogging can be caused by damaged cells or by environmental contaminants. The latter's effect can be minimized by ensuring a clean, dust-free environment for operating the devices (e.g. a well maintained biosafety cabinet). For cell lines, we recommend passaging cells 1-2 days prior to delivery so as to minimize the presence of cell debris in the resulting suspension. When operating the device, one must be mindful of the volumetric flow rate of the fluid in the reservoir. If the flow rate drops to under a third of its initial rate, the device may be causing excessive cell death and should be exchanged, or the direction of flow reversed. Potential clogging issues can also be mitigated by passing the cell suspension through a 40 μm cell strainer mesh. This process ensures a single cell suspension thereby preventing potential clogs from forming in the wider, low flow rate sections of the chip.
When designing experiments for different delivery materials, one must account for the size of the target material and its concentration. As this technology relies on the passive diffusion of material across the disrupted cell membrane, the molar concentration gradient and the hydrodynamic radius of the delivery material will govern the rate of mass transfer – assuming the membrane disruptions are large enough to accommodate the material. It is thus advisable to use high molar concentrations (e.g. 1 µM) of large target materials relative to their smaller counter parts. The technique has demonstrated effective delivery at concentrations as low as 10 nM in the case of quantum dot delivery21. Moreover, the technique is not thought to be limited by the nature of the target delivery material except in the context of material size and diffusivity (i.e. low diffusivity materials will have lower delivery efficiencies and material larger than the size of the disruptions probably cannot enter the cytoplasm).
Studies in cell reprogramming and quantum dot delivery have illustrated the strengths of the cell squeezing approach relative to electroporation. Based on the current understanding of the membrane disruption mechanisms in these two techniques, it would appear that cell squeezing has a significant advantage in applications involving proteins, small molecules, and nanomaterials20,21. The contrast between the two methods in applications involving nucleic acids (which are highly charged compared to the aforementioned materials) is unclear.
This microfluidic approach to delivery relies on mechanical disruption of the cell membrane to facilitate passive diffusion of material into the cell and has been shown to be applicable to a range of cell types20. The system is thus in principal appropriate for the cytosolic delivery of almost any material to almost any cell type. We believe the system's advantages are most pronounced in cases involving traditionally difficult to transfect primary cells (e.g. immune and stem cells) and conventionally challenging materials (e.g. proteins, quantum dots, carbon nanotubes, and RNA). The aforementioned potential in immune and stem cell applications is emphasized by the shortcomings of traditional delivery methods in addressing these cell types. However, the system may require further improvement to facilitate gene delivery by plasmid vectors as these applications require delivery to the nucleus. In summary, we believe the microfluidic deformation approach to delivery can potentially address many existing challenges to intracellular delivery. The system's independence from chemical vectors or electric fields enables its robust application to a range of studies. The instructions provided herein should enable any interested researchers to operate such a system independently and adapt it for their research purposes.
The authors have nothing to disclose.
We thank T. Shatova for helpful discussion on experimental design and data analysis. The assistance and expertise of G. Paradis, the personnel of the flow cytometry core at the Koch Institute, and the staff of the Microsystems Technology Laboratory at Massachusetts Institute of Technology are gratefully acknowledged. This work was supported by National Institutes of Health Grants RC1 EB011187-02, DE013023, DE016516, EB000351, and partially by National Cancer Institute Cancer Center Support (Core) Grants P30-CA14051 and MPP-09Call-Langer-60.
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 |