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.

1. Storage
<ol>
	<li>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).</li>
</ol>
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...

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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...

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.

<table border="0" cellpadding="0" cellspacing="0" width="777">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell squeezing as a robust, microfluidic intracellular delivery platform

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.

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...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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 ...

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Bioengineering

12.6K visualizações.  Massachusetts Institute of Technology.  Deformação mecânica rápida de células surgiu como um método promissor, livre do vector para a entrega intracelular de macromoléculas e nanomateriais. Este protocolo fornece etapas detalhadas sobre como usar o sistema para uma ampla gama de aplicações. 