Name: Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform (Video) | JoVE Journal
Uploaded: 2013-11-07T19:45:00.000+00:00
Duration: 8 min 2 s
Description: Learn cell squeezing for vector-free intracellular delivery using a microfluidic device. This method enables efficient macromolecule delivery to challenging cell types for cancer therapy and immune cell manipulation.

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5 minutes

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. no denaturing agents), all system components should be fully compatible and will not degrade over time.
<ol start="2">
	<li>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.</li>
</ol>
2. Experiment Preparation
<ol>
	<li>Place containers (2) and (3) in an ultrasound bath for 5-10 min&#160;before each use. This helps remove any contaminating particles from previous experiments.</li>
	<li>Clean workspace in biosafety cabinet with 70% ethanol solution.</li>
	<li>Spray all materials (3 containers, and tweezers) with a 70% ethanol solution before placing them inside the biosafety cabinet.</li>
</ol>
3. Assembly
<ol>
	<li>Set down 2-3 low-lint wipes in the work area.</li>
	<li>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.</li>
	<li>Insert O-rings into their appropriate slot on the reservoirs.</li>
	<li>Remove the holder and chips from respective containers and allow ethanol to evaporate (~1-2 min).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Cell Preparation
<ol>
	<li>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.</li>
	<li>Place cells in suspension (in PBS or relevant media) and aim for an operating concentration of 1.0 x 106 cells/ml&#160;to 1.0 x 107 cells/ml. NOTE: We have not observed significant changes in delivery performance due to cell concentration.</li>
	<li>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 &#181;M 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.</li>
</ol>
5. Operation
<ol>
	<li>Pipette mixture of cells and delivery material into a reservoir (current design has a max 150 &#181;l 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.</li>
	<li>Attach pressure tubing to the filled reservoir and tighten the nut to ensure proper sealing (finger tight is often sufficient).</li>
	<li>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.</li>
</ol>
NOTE: In previous work, nitrogen or compressed air have worked equally well as the carrier gas.
<ol start="4">
	<li>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.</li>
	<li>Press the button to pressurize the reservoir and begin cell flow.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Disassembly
<ol>
	<li>Gently disconnect the reservoirs from the main holder by pushing aside the clip arms.</li>
	<li>Place each part in the appropriate storage container (detailed in section 1).</li>
	<li>Place used chips in a separate container for disposal.</li>
</ol>

<ol><li>Schaffert, D., Wagner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+therapy+progress+and+prospects%3A+synthetic+polymer-based+systems%5BTitle%5D)+AND+%22Gene.+Ther%22%5BJournal%5D)">Gene therapy progress and prospects: synthetic polymer-based systems.</a> Gene. Ther. 15, 1131-1138 (2008).</li>
<li>Whitehead, K. A., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Knocking+down+barriers%3A+advances+in+siRNA+delivery%5BTitle%5D)+AND+%22Nat.+Rev.+Drug+Discov%22%5BJournal%5D)">Knocking down barriers: advances in siRNA delivery.</a> Nat. Rev. Drug Discov. 8, 129-138 (2009).</li>
<li>Leader, B., Baca, Q. J., Golan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+therapeutics%3A+a+summary+and+pharmacological+classification%5BTitle%5D)+AND+%22Nat.+Rev.+Drug+Discov%22%5BJournal%5D)">Protein therapeutics: a summary and pharmacological classification.</a> Nat. Rev. Drug Discov. 7, 21-39 (2008).</li>
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<li>Dhar, S., Daniel, W. L., Giljohann, D. A., Mirkin, C. A., Lippard, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyvalent+Oligonucleotide+Gold+Nanoparticle+Conjugates+as+Delivery+Vehicles+for+Platinum%28IV%29+Warheads%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Polyvalent Oligonucleotide Gold Nanoparticle Conjugates as Delivery Vehicles for Platinum(IV) Warheads.</a> J. Am. Chem. Soc. 131, 14652-14653 (2009).</li>
<li>Jiang, Z. -X., Zhang, Z. -Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+PTPs+with+small+molecule+inhibitors+in+cancer+treatment%5BTitle%5D)+AND+%22Cancer+and+Metastasis+Reviews%22%5BJournal%5D)">Targeting PTPs with small molecule inhibitors in cancer treatment.</a> Cancer and Metastasis Reviews. 27, 263-272 (2008).</li>
<li>Derfus, A. M., Chan, W. C. W., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+Delivery+of+Quantum+Dots+for+Live+Cell+Labeling+and+Organelle+Tracking%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking.</a> Adv. Mater. 16, 961-966 (2004).</li>
<li>Michalet, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantum+Dots+for+Live+Cells+in+Vivo+Imaging%2C+and+Diagnostics%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Quantum Dots for Live Cells in Vivo Imaging, and Diagnostics.</a> Science. 307, 538-544 (2005).</li>
<li>Dahan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusion+Dynamics+of+Glycine+Receptors+Revealed+by+Single-Quantum+Dot+Tracking%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Diffusion Dynamics of Glycine Receptors Revealed by Single-Quantum Dot Tracking.</a> Science. 302, 442-445 (2003).</li>
<li>Slowing, I. I., Trewyn, B. G., Lin, V. S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesoporous+Silica+Nanoparticles+for+Intracellular+Delivery+of+Membrane-Impermeable+Proteins%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Mesoporous Silica Nanoparticles for Intracellular Delivery of Membrane-Impermeable Proteins.</a> J. Am. Chem. Soc. 129, 8845-8849 (2007).</li>
<li>Pack, D. W., Hoffman, A. S., Pun, S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+development+of+polymers+for+gene+delivery%5BTitle%5D)+AND+%22Nat.+Rev.+Drug+Discov%22%5BJournal%5D)">Design and development of polymers for gene delivery.</a> Nat. Rev. Drug Discov. 4, 581-593 (2005).</li>
<li>Joseph, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cationic+lipids+used+in+gene+transfer%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Cationic lipids used in gene transfer.</a> Advanced Drug Delivery Reviews. 27 (97), 17-28 (1997).</li>
<li>Verma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface-structure-regulated+cell-membrane+penetration+by+monolayer-protected+nanoparticles%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles.</a> Nat. Mater. 7, 588-595 (2008).</li>
<li>Yan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+intracellular+protein+delivery+platform+based+on+single-protein+nanocapsules%5BTitle%5D)+AND+%22Nat.+Nano%22%5BJournal%5D)">A novel intracellular protein delivery platform based on single-protein nanocapsules.</a> Nat. Nano. 5, 48-53 (2010).</li>
<li>Shi Kam, N. W., Jessop, T. C., Wender, P. A., Dai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanotube+Molecular+Transporters%3A+Internalization+of+Carbon+Nanotube-Protein+Conjugates+into+Mammalian+Cells%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Nanotube Molecular Transporters: Internalization of Carbon Nanotube-Protein Conjugates into Mammalian Cells.</a> J. Am. Chem. Soc. 126, 6850-6851 (2004).</li>
<li>Varkouhi, A. K., Scholte, M., Storm, G., Haisma, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endosomal+escape+pathways+for+delivery+of+biologicals%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Endosomal escape pathways for delivery of biologicals.</a> Journal of Controlled Release. 151, 220-228 (2011).</li>
<li>li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroporation+Gene+Therapy%3A+New+Developments+In+Vivo+and+In+Vitro%5BTitle%5D)+AND+%22Current+Gene+Therapy%22%5BJournal%5D)">Electroporation Gene Therapy: New Developments In Vivo and In Vitro.</a> Current Gene Therapy. 4, 309-316 (2004).</li>
<li>Fox, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electroporation+of+cells+in+microfluidic+devices%3A+a+review%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Electroporation of cells in microfluidic devices: a review.</a> Anal. Bioanal. Chem. 385, 474-485 (2006).</li>
<li>Miller, D. L., Pislaru, S. V., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonoporation%3A+Mechanical+DNA+Delivery+by+Ultrasonic+Cavitation%5BTitle%5D)+AND+%22Somatic+Cell+and+Molecular+Genetics%22%5BJournal%5D)">Sonoporation: Mechanical DNA Delivery by Ultrasonic Cavitation.</a> Somatic Cell and Molecular Genetics. 27, 115-134 (2002).</li>
<li>Sharei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+vector-free+microfluidic+platform+for+intracellular+delivery%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">A vector-free microfluidic platform for intracellular delivery.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 2082-2087 (2013).</li>
<li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonendocytic+Delivery+of+Functional+Engineered+Nanoparticles+into+the+Cytoplasm+of+Live+Cells+Using+a+Novel%2C+High-Throughput+Microfluidic+Device%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Nonendocytic Delivery of Functional Engineered Nanoparticles into the Cytoplasm of Live Cells Using a Novel, High-Throughput Microfluidic Device.</a> Nano Lett. 12, 6322-6327 (2012).</li>
</ol>

The authors Armon Sharei, Robert Langer, and Klavs F. Jensen are shareholders of SQZ Biotechnologies Company that produces the microfluidic devices used in this article.

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><tbody><tr><td>Device Holder &amp;amp; Plastic reservoir</td><td>SQZ Biotechnologies</td><td>Holder</td><td></td></tr><tr><td>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><td>Microfluidic device</td><td>SQZ Biotechnologies</td><td>Cell Squeeze</td><td></td></tr><tr><td>O-Rings</td><td>McMaster</td><td>9452K311</td><td></td></tr><tr><td>Pressure system to operate device</td><td>SQZ Biotechnologies</td><td>Pressure System</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Ultrasound bath</td><td></td><td></td><td></td></tr></tbody></table>

cell squeezing as a robust, microfluidic intracellular delivery platform

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...

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 ...

The overall goal of the falling experiment is to deliver macromolecules vector free to target cells using mechanical disruption. This is achieved by first placing the target cell type in suspension with the desired delivery material and loading into the reservoir of a specially designed microfluidic device. As a second step, the microfluidic device is pressurized and the cells are driven through an array of microfluidic channels that squeeze the cells to facilitate transient membrane disruption.Next, the cells are allowed to incubate in the presence of the target delivery material to facilitate diffusive transport of the material into the cell cytoplasm. While the cell membrane is repaired. Results are obtained that show effective delivery of macromolecules through the use of flow cytometry.The main advantage of this technique over existing methods like ation and lipo perfection, is that it's capable of addressing challenging cell types and materials that cannot be effectively delivered by other methods. This method can answer key questions in the biological field, such as how to improve cell re programming. The implications of this technique extend towards the therapy and diagnosis of cancer because the system enables manipulation of immune cells to enhance their anti-tumor efficacy.To begin, fabricate or purchase the microfluidic device, device holder, plastic fluid reservoirs and O-rings shown here and listed with details in the accompanying text protocol, then fill three sealable containers partway with 70%ethanol. Place the devices in the first container, the reservoirs and O-rings in the second container and holders in the third. Then fill the containers the rest of the way with 70%ethanol.Next place, the container containing the reservoirs and O-rings as well as the one containing the holders in an ultrasound bath for five to 10 minutes before use. This will help to remove any contaminating particles from previous experiments while the component sonicate wipe down the workspace in a biosafety cabinet with 70%ethanol. Then sterilize all the required materials in including the three containers and a pair of tweezers by wiping them down with 70%ethanol and place them inside the biosafety cabinet to begin assembly.First, set down two to three low lint wipes in the work area. Then remove the plastic reservoirs from the ethanol container with tweezers and set them on the wipes to facilitate wicking and evaporation of ethanol from it inner surface. Gently tap the reservoirs to assist in the removal of the ethanol solution.If necessary, use pressurized gas to fully purge the reservoir. Once the reservoirs have dried, insert the O-rings into the appropriate slots on the reservoirs. Then use tweezers to place the chip face up in the device holder.Make sure the chip is lying flat in the holder and adjust if necessary using the tweezers. If the device does not fit properly in its holder, there is a risk that it will break during the subsequent steps. Next, gently place the reservoirs on the holder and align them with the clips.Make sure the O-rings don't fall outta their slots. During this process, 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.Do not press down on the reservoirs with excessive force. As the pressure could break the chips for primary or adherence. Cell lines, place cells one to two days prior to the experiment so that they are no more than 80%confluent.On the day of the experiment. Immediately before the experiment, harvest the cells and resus, suspend them in media or other desired buffer at between one and 10 million cells per milliliter. Then carefully pass the cell suspension through a 40 micron cell strainer mesh to prevent clumps of cells and matrix from clogging the device.Once strained, mixed 300 microliters of the cell suspension with the desired concentration of delivery material in a separate tube. For this demonstration, two microliters of dextrin at five milligrams per milliliter and two microliters of Alexa. 4 88 isotope control antibody are added per 50 microliters of cell suspension.Next, gently mix 100 microliters of the cells by pipetting. Then using gel loading tips, pipette the cells and delivery material into either one of the reservoirs, attach pressure tubing to the filled reservoir and finger tighten the knot to ensure proper ceiling. Then adjust the air pressure to 70 PSI on the regulator to control the speed at which cells travel through the device.The pressure should be optimized for the specific cell and microfluidic chip combination used in each experiment. With everything prepared, raise the device to eye level and orient it so that the liquid in the reservoir is easily visible. Then press the push button valve to pressurize the reservoir and begin cell flow.Watch the speed at which the fluid is flowing from the reservoir. If the fluid slows substantially relative to its initial flow rate, the mounted device is likely clogged and needs to be exchanged. The increased shear caused by the blockage will cause greater than normal cell death.When the liquid level is approximately two millimeters from the bottom of the reservoir, quickly turn the regulator to zero PSI to stop the flow. It is important to stop the flow of air before the reservoir is emptied, or the sample may be ejected from the collection reservoir. Then collect the treated cells from the outflow reservoir and place them into a micro centrifuge tube.At room temperature, the rated cells will continue to uptake material for up to 10 minutes. After 10 minutes, plate the cells with additional media or continue on to use the cells according to experimental needs. Repeat these steps to collect as many treated cells as are needed and exchange the chips as needed if they clog and discard the C clogged devices for each subsequent sample.Change the flow direction to minimize clogging effects. When finished collecting cells, gently disconnect the reservoirs from the main holder by pushing aside the clip arms. Then place each part to be reused in the appropriate storage container and fill them with fresh 70%ethanol.Finally place all the used chips in a separate container for proper disposal. The The delivery efficiency and cell viability of hela cells were measured. Following a series of experiments, three different microfluidic chips were tested in a range of speeds up to 600 millimeters per second.These tests showed that increased flow speeds improved delivery of fluorescently conjugated, three kilodalton dextrin into the cells for each of the chips tested. Additionally, increased speeds also caused the cell viability to drop by about 20%in the tested devices. This amount of cell death is relatively low and is often much higher in inappropriately designed devices.The uptake of Pacific blue conjugated dextrin into the control cell population shown on the left is the result of limited surface binding and endocytic effects. As the cells are exposed to the delivery material for the same amount of time as the treated cells. On the right is the population of live cells that pass through the microfluidic device while exposed to the same concentration of dextrin as the control cells.The cells in the shaded region are considered undelivered, and the cells to the right of the line are defined as delivered. Once mastered, this technique can be completed in under one hour if it is performed properly. Following this procedure, other methods, life post optometry can be performed in order to answer additional questions like delivery efficiency and material functionality After its development.This technique paved the way for researchers in the field of regenerative medicine to explore cell programming in adult primary cells using direct protein delivery. After watching this video, you should have a good understanding of how to set up and operate the self squeezing system for your desired application.

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A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

Learn cell squeezing for vector-free intracellular delivery using a microfluidic device. This method enables efficient macromolecule delivery to challenging cell types for cancer therapy and immune cell manipulation.

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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