Microenvironment enable a highly complex and dynamic including ever changing cytokines, ligands concentration and composition. To create a similar culture environment is crucial for in vitro studies on complex life machinery such as stem cell differentiation, immune responses, and the development of organs on chip platforms. But to generate and deliver chemical signals of nanoliter volume and milliseconds accuracy is difficult using conventional biomedical approaches like pipetting.
To meet the challenges, a culture platform which is capable of alternate image acquisition generation of dynamic combinatorial and sequential signal inputs is on high demand. In this protocol, the design and fabrication procedure of a microfluidic device are presented. The proposed microfluidic chip consists of 1500 culture units, array of enhanced peristaltic pumps and on-site mixing modulus.
We demonstrate that platform is suitable for studies on single cells two dimensional cell populations and three dimensional neural spheres. The complex and dynamic environmental conditions on chip have great effects on neural stem cell differentiation and the self-renewal. The detail of design and fabrication procedures are as follows.
Chip design was performed using AutoCAD software. To fit the size of the plate holder on microscope, the microfluidic chip is approximately seven by five centimeters in size And consists of 1, 500 culture chambers. One of important features is it has peristaltic pump which consists of eight flow channels and 200 micrometer wide control channels.
The increase in the overlapping area between control and the flow channel increase 16 times as compared to the peristaltic pump, proposed by Stephen Quake in 2002, to about 15 nanoliters liquid and per pumping cycle and suffice the needs to maintain 1, 500 independent environmental conditions parallelly on chip. Another important part of the device is a two-layer culture chamber, which can maintain shear-free culture environment. The unwanted shear stress is prevented by directing it quick through the top layer.
The cells, tissues and crucial conditional medium remain untouched at the bottom layer. Numerical simulation suggests that when the liquids are directed through culture chambers, left to right. the shear forces can be effectively prevented.
Even at an input flow rate of 10 millimeter per second cell or micrometer-size tissue remain undisturbed at the bottom of the culture unit. We fabricated the replica molding or silicon wafer using UV lithography. The fluidic channels of 25 micrometers in height and 100 micrometers in weight are produced using SU-8 3025 negative photoresist.
The culture chambers of 75 micrometers and 150 micrometers in height were fabricated using SU-8 3075 photoresist. AZ50X positive photoresist is used for the round-shaped well features which overlap with the control channels to ensure a good connection. To produce the microfludic chip, the patterned and blank silicon wafers were firstly treated with TMCS.
Different quantities of PDMS 10 to 1 of monomer to catalyst ratio was thoroughly mixed and debubbled for one to two hours in the vacuum chamber at minus 0.85 megapascal. The control layer was obtained by spin coating PDMS at 2, 200 RPM. The silicon wafers were then transferred to incubator and incubated for 60 minutes at 80 centigrade.
Different layers were aligned and bonded together using customized optical device and plasma etching machine. The inlet holes were then punched after another two hours thermal bonding at 80 centigrade. The chip was bonded to a PDMS-coated cover slip, which is the same size of 96-well plate and cured for at least 12 hours at 80 centrigrade before use.
For operation, control channels were connected to miniature pneumatic solenoid valves through plastic tubing. The turning all of penumatic wells were controlled by a custom MATLAB program through the graphical user interface. Optimal closing pressures of push-up PDMS membrane valves were determined individually for each chip, which typically ranges from 25 to 30 PSI.
By turning all of series of wells dynamically combinatorial and sequential inputs can be generated on chip. Cells were harvested at 80%confluency and resuspended with culture medium DMEM at a density about 10 to the power six per milliliter, which were then loaded into the chip by pressurizing the cell containing solution. For both adherent and the suspension culture of neuro stem cells, cells were collected and the spheres were directly loaded into the chip.
For image acquisition, an inverted microscope with an automatic translational stage and the digital complementary metal-oxide semiconductor camera were used. The stage and image acquisition were controlled via the software shows the upper left corner and the lower right corner chambers as selecting points and move the translational stage to the points orderly to get the preliminary imaging with 4x objective lens. Change the 4x objective lens with the 20x one and adjust imaging parameters including light intensity, expose time, et cetera.
Then locate the selecting points so the position of each chamber leaving 13 by 15 chamber matrix or to be defined by the program automatically. With determining the coordinates of the chambers, translational stage moves to each chamber where X, Y, Z focal plane can be fine tuned. Set the interval and the duration of imaging cycle.
Save the path and then start imaging. This system is available for various target dynamic tracking, such as neural stem cells differentiation in brightfield and the fluorescence field of the process of neural stem cells sphere formation. Data analysis.
Change the format in nd2 to Tif using the custom MATLAB program. And divide the data according to points. Select object to be analyzed is cell or tissue.
Enter the threshold, object size, noise size to analysis. Or you can choose step-by-step analysis to optimal parameters. And then analyzing all the data.
After save the result in MAT format, using the plot to draw the results or traces. In this paper, we described the protocol of the and culture of high-throughput microfluidic chip system based on the photolithography technology for the live cell microenvironment dynamic control. We also described some parameters that one should carefully control such as the closing pressure of PDMS membrane valves and the spin control of fluid in parallel of the chamber.
In traditional cell in vitro culture the treatment of cell culture environment is performed manual, which is time consuming and laborious and fluid filled is not easy to standard control. Particularly minutes of solution. There are some problems such as generating inputs, immune drug concentration and large consumption.
However, using our microfluidic system, it is possible to repeat it as stimulator at a fixed dose single fluid equal amount of uniform concentration, compare different cultures in different culture chambers at the same time. Different with some miniature temporal resolution back stimulus and with subsetting the resolution by regulating the fluid. In addition, our system can obtain a large number of comparative experimental results by simple operations.
Our system is based on the principle of diffusion, creates minimal mechanical disturbance to the cellular microenvironment. For more stable cellular microenvironment chips you can increase the list of the buffer layer as the depth of the culture layer or reduce the input fluid of medium exchange, that is reduce the pressure, which will be here for the research on cell behavior. It is to be noticed that this study has examined only the culture of 3T3 and NSC cells.
For other cells or cell lines, the culture conditions comparable with our parameters to adjust it.
