Name: Title Cell Encapsulation by Droplets (Video) | JoVE Journal
Uploaded: 2007-10-01T13:09:54.000+00:00
Duration: 13 min 10 s
Description: Learn cell encapsulation by droplets for 3D bioprinting using a solenoid valve system. This method enables precise cell ejection and supports tissue engineering applications.

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5 minutes

NIH3T3 cells preparation:
A. Cells for Ejection
<ol>
<li>Trypsinize cells, then dilute 1:1 with cell media, and transfer from a T75 flask to a 15 mL Falcon tube</li>
<li>Spin down cells into a pellet by centrifuging, aspirate supernatant and wash cells with DPBS</li>
<li>Spin down cells into a pellet again, and aspirate supernatant</li>
<li>Resuspend cells in media</li>
<li>Determine cell density with hemocytometer (~200 X 104 cells/mL per T75 flask)</li>
<li>Centrifuge cell solution, aspirate supernatant, and resuspend in appropriate amount of media for varying cell concentrations</li>
</ol>
B. Cell ejection
<ol>
<li>Vortex cells before using for ejection</li>
<li>Transfer 200 &micro;L of cell solution into syringe</li>
<li>Set appropriate mode on pulse generator<ol>
<li>For ejecting single droplets and multiple droplets (bursts), set pulse generator to "E. BUR" mode</li>
<li>For continuous droplet ejection, set pulse generator to "NORM" mode</li>
</ol></li>
<li>Change signal settings<ol>
<li>Set high level and low level output voltage: HIL to 5 V and LOL to 0 V and make sure the"LIM"LED is on</li>
<li>Set signal as a square pulse</li>
<li>Change the amount of time the solenoid valve is open for droplet ejection by changing the value for "WID" or changing duty cycle ("DUTY")</li>
<li>Change the frequency of ejection by changing the value for "PER"</li>
<li>Change the number of droplets ejected in a burst by changing the value for "BUR"</li>
</ol></li>
<li>Eject cell solution onto prepared substrate for imaging with microscope</li>
</ol>
C. Staining
<ol>
<li>Make up dye solution with 0.5 &micro;L calcein-AM and 2 &micro;L ethidium homodimer per mL of DPBS</li>
<li>Immerse prepared substrate in dye solution</li>
<li>Allow sample to incubate for 10 minutes at 37&deg;C before imaging</li>
</ol>
Experiment Validation
<ol>
<li>On a Nikon Eclipse TE-2000 U Fluorescent Microscope<ol>
<li>Spot advanced software (Diagnostics, Inc.)</li>
<li>Live/Dead Assay</li>
</ol></li>
</ol>

The authors have nothing to disclose.

<table><tbody><tr><td>Fluorescent Microscope</td><td></td><td>Nikon Instruments</td><td>Eclipse TE-2000 U</td><td></td></tr><tr><td>Solenoid valve cell ejector</td><td></td><td></td><td></td><td>Operation frequency: 1 KHz, 30psi, 50nl~0.5ul. 12V Valve Driver: 2.5 Amp drive current</td></tr><tr><td>5-Gallon Portable air tank</td><td></td><td>Coleman</td><td>Powermate CT5</td><td>Pressurized air: 30 PSI </td></tr><tr><td>Pressure regulators</td><td></td><td>Marsh Bellofram</td><td></td><td></td></tr><tr><td>Pulse Generator</td><td></td><td></td><td>HP8112A</td><td>Actuation frequency generation: 50 MHz</td></tr><tr><td>XYZ-stage</td><td></td><td>Newmark Systems</td><td></td><td>With Stepping motor: 6inch travel xy-stage(NLS4-6-25), 2.5inch travel z-stage(NLS4-2.5-25), 3axis controller(NSC-G3), 0.1um resolution</td></tr><tr><td>NIH 3T3</td><td>Cell-line</td><td></td><td></td><td>fibroblasts</td></tr><tr><td>Trypsin</td><td></td><td></td><td></td><td>0.05% solution</td></tr><tr><td>NIH 3T3 cell medium</td><td></td><td></td><td></td><td></td></tr><tr><td>DPBS</td><td>Buffer</td><td></td><td></td><td></td></tr><tr><td>T75</td><td></td><td></td><td></td><td>Tissue culture flasks</td></tr><tr><td>Plastic conical tubes</td><td></td><td></td><td></td><td>15 ml, for tissue culture</td></tr></tbody></table>

title cell encapsulation by droplets

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

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Learn cell encapsulation by droplets for 3D bioprinting using a solenoid valve system. This method enables precise cell ejection and supports tissue engineering applications.

Title Cell Encapsulation by Droplets

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