Name: generation of dynamical environmental conditions using a high-throughput microfluidic device
Uploaded: 2021-04-17T13:00:00
Description: Watch this Scientific Journal Video about Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device at JoVE.com

Authors acknowledge the technical support from Zhifeng Cheng of Chansn Instrument (China) LTD. This work was supported by grants (National Natural Science Foundation of China,51927804).

1. Microfluidic chips design
<ol>
	<li>Design the microfluidic multiplexer consisting of 18 inlets, each of which is controlled by an individual valve and a peristaltic pump. To increase the liquid volume driven by per pumping cycle, have the peristaltic pump be composed of 3 control channels, which was purposely widened to 200 &#181;m, and 10 connected flow lines.</li>
	<li>Design the shear-free culture chamber. Replication of the 2-level culture unit is composed by a lower cell culture chamber (400 &#181;m x 400 &#18...

<ol>
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Various microfluidic devices have been developed to perform multiplexed and complex experiments17,18,19,20. For example, microwells made of an array of topological recesses can trap individual cells without the use of external force, showing advantageous characters including small sample size, parallelization, lower material cost, faster response, high sensitivity21...

High throughput techniques are crucial for biomedical and clinical studies. By parallelly conducting millions of chemical, genetic, or live cell and organoid tests, researchers can rapidly identify genes that modulate a bio-molecular pathway, and customize sequential drug input to one's specific needs. Robotics1 and microfluidic chips in combination with a device control program allow complex experimental procedures to be automated, covering cell/tissue manipulation, liquid handling, imaging, and data processing/control2,3. Therefore, hundreds and thousands of experimental conditions can be maintained on a single chip, according to the desired throughput4,5. 
In this protocol, we described the design and fabrication procedure of a microfluidic device, which consists of 1500 culture units, an array of enhanced peristaltic pumps and on-site mixing modulus. The 2-level cell culture chamber prevents unnecessary shear during medium exchange, which ensures an undisturbed culture environment for long-term live cell imaging. The studies demonstrate that the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery. Moreover, the advanced features of the microfluidic chip allow automated reconstitution of highly complex and dynamic microenvironmental conditions in vivo, like the everchanging cytokines and ligands compositions6,7, the completion of which takes months for conventional platforms like 96-well plate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>2713 Loker Avenue West</td>
			<td>Torrey pines scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AZ-50X</td>
			<td>AZ Electronic Materials, Luxembourg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane(TMCS) 92360-25mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator HP151</td>
			<td>Heal Force</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Hole Puncher for PDMS chips WH-CF-14</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM(L-glutamine, High Glucose, henol Red)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Balance UTP-313 Max:600g, e:0.1g, d:0.01g</td>
			<td>Shanghai Hochoice Apparatus Manufacturer Co.,LTD.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronection 0.25 mg/mL</td>
			<td>Millipore, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax 100x</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Incubator BGG-9240A</td>
			<td>Shanghai bluepard instruments Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Model Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep 10 Units/mL Penicillin 10 ug/mL Streptomycin</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner PDC-002</td>
			<td>Harrick Plasma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polydimethylsiloxane(PDMS)</td>
			<td>Momentive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polylysine 0.01%</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater ARE-310</td>
			<td>Awatori Rentaro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater TDZ5-WS</td>
			<td>Cence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater WH-SC-01</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3075</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of dynamical environmental conditions using a high-throughput microfluidic device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

The conventional on-chip peristaltic pump was firstly described by Stephen Quake in 2000, using which the peristalsis was actuated by the pattern 101, 100, 110, 010, 011, 001 8,10. The number 0 and 1 indicate &#34;open&#34; and &#34;close&#34; of the 3 horizontal control lines. Studies using more than 3 valves (e.g., five) have also been reported11. Even though the peristaltic pump composed by 3 control lines and 3 flow lines provides nano...

Watch this Scientific Journal Video about Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device at JoVE.com

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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