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

Summary

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.

Abstract

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.

Introduction

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/control^2,3. Therefore, hundreds and thousands of experimental conditions can be maintained on a single chip, according to the desired throughput^4,5.

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 compositions^6,7, the completion of which takes months for conventional platforms like 96-well plate.

Protocol

1. Microfluidic chips design

1. 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 µm, and 10 connected flow lines.
2. Design the shear-free culture chamber. Replication of the 2-level culture unit is composed by a lower cell culture chamber (400 µm x 400 µm x 150 µm) and a higher buffer layer (400 µm x 400 µm x 75 µm), which prevents unwanted shear stress on cells during medium exchange (Figure 1).
3. Design high-throughput features. Duplicate the culture unit to form a 30 x 50 matrix layout, occupying an area of approximately 7 cm by 5 cm in size.

2. Chip fabrication and operation

1. Fabrication of the replica molding using UV lithography
   NOTE: The replica molding was fabricated on silicon wafer according to the standard photolithography protocol⁸.
   1. Fabrication the channel structures
      1. Spinning photoresist: Spin coat 5 mL of the SU-8 3025 negative photoresist on a silicon wafer at 500 rpm for 10 s and 3000 rpm for 30 s.
      2. Soft bake: Put the wafer on a hotplate at 65 °C for 2 min and then 95 °C for 10 min, Cool it down to room temperature.
      3. Alignment and curing: Fix the wafer and mask on the holder of the aligner and turn on the light source for 18 s to cure the exposed photoresist.
      4. Pre post exposure bake: Ramp up the wafer to 95°C at 110°C/h from room temperature and keep it at least 40 min till removing.
      5. Develop: Dip the wafer in the developing solution (SU-8 developer) and agitate it for 2.5 min to wash off redundant photoresist and get the 25 µm-high channel structure.
      6. Hard bake: Cover the wafer with glass Petri dish and bake at 65 °C for 2 min, then ramp up to 160°C at 120 °C/h and keep it for 3 hours.
   2. Fabrication of the cell culture chamber with the buffer layer
      1. Use the parameters of step 2.1.1.1 to spin coat 7 mL of the SU-8 3075 negative photoresist on the above wafer.
      2. Soft bake (described in step 2.1.1.2) the wafer and change the mask to align well with markers on the wafer. Then turn on the light source for 24 s to cure the exposed photoresist.
      3. Dip the wafer to the developing solution (SU-8 developer) and agitate it for 4 minutes and 40 seconds to wash off redundant photoresist and get a 75 µm-high layer structure that around the 25 µm-high channel structure fabricated before. Hard bake (described in step 2.1.1.6) the wafer to make the complex structure stronger.
      4. Repeat steps 2.1.2.1-2.1.2.3 to fabricate a 75 µm-high chamber structure that stack on the layer structure.
   3. Fabrication of the valve structures
      1. Using the parameter of step 2.1.1.1 to spin coat 5 mL of the AZ 50x positive photoresist on the above wafer.
      2. Soft bake (described in step 2.1.1.2) the wafer and make the mask aligned well with markers on the wafer, then keep the light source on for 20 s and off immediately for 30 s. Repeat the light on/ off procedure in 5 circles to cure the exposed photoresist.
      3. Dip the wafer to matched developer and agitate it for about 8 min to wash off redundant photoresist and get the round-shaped valves that overlap with the control channels to ensure good connection. Hard bake (described in step 2.1.1.6) the patterned wafer to make the whole model stronger.
2. Microfluidic chip production using soft lithography
   1. Treat the patterned and blank silicon wafers with rimethylchlorosilane for 15 min.
   2. Prepare 3 portions of PDMS gel (10:1 of monomer/catalyst ratio) corresponding to 50 g of flow layer, 20 g of control layer 2, and 20 g of membrane, respectively.
   3. Cast 50 g of PDMS gel on the patterned silicon wafer, and de-gas them for 1-2 hours in the vacuum chamber at -0.85 MPa to copy the flow layer.
   4. Degas the 2 portions of 20 g of PDMS and spin it on the patterned wafer and a blank silicon wafer at 2000-2800 rpm for 30 s as to prepare a control layer and membrane layer.
   5. Put the PDMS-covered wafers into ventilating oven for 60 min at 80 °C for incubation.
   6. Align and bond the different layers together through customized optical device (zoom in 100x) and plasma etching machine. Then keep it in a ventilating oven for 2 hours at 80 °C to enhance the bonding of the chip.
   7. Punch the inlet holes on the chip, and then bond it onto a PDMS-coated coverslip and cured for at least 12 hours at 80 °C before use.
3. Chip operation
   1. Connect miniature pneumatic solenoid valves to the control layer of the chip, and open the customized MATLAB graphical user interface⁸ to link and control the switch.
   2. Set the closing pressures of push-up PDMS membrane valves to 25 psi.
   3. Deliver dynamically changing combinatorial/sequential inputs to designated chambers (Figure 2d) by timely on-off of the valves.

3. Generation of dynamic inputs in cellular microenvironments

1. Chip treatment and cell loading
   1. Maintain the standard culture conditions (37 °C, 5% CO₂) on the microscope for at least 5 hours.
   2. Fill the chip with coating medium (i.e., mentioned in the NOTE) and incubate it at the standard culture conditions (described in step 3.1.1) for at least one hour.
   3. Flush the chip by phosphate-buffered saline (PBS) or cell culture medium (Dulbecco's Modified Eagle's medium, DMEM) to build a healthy culture environment.
   4. Harvest cells at 80% confluency, and resuspend the cells using culture media (DMEM) at a density of ~ 10⁶/mL. Then load cells into the chip by pressurizing the cell-containing solution.
      NOTE: Culturing different cell lines on chip requires corresponding coating medium to treat the cell culture chamber. Typically, for experiments on 3T3 fibroblast and adherent culture of hen all valves controlling the culture chambers are open, cells flow into all culture chambers within the same column.
2. Setup for high throughput live-cell imaging
   NOTE: For image acquisition, an inverted microscope with an automated translational stage and a digital complementary metal-oxide semiconductor (CMOS) camera were used. The stage and image acquisition were controlled via the customized software.
   1. Visualize the matrix of culture chambers using 10x objective lens in bright field to affirm and define the location coordinates of each chamber of the 30 by 50 chamber matrix.
   2. Transform the objective lens to the 20x or 40x, then select the location coordinates of the desired chamber and the translational stage moves to the assigned position after confirmation. Fine tune the x, y, z focal plane to get an optimal image.
   3. Optimal the light intensity, expose time, and other imaged parameters were determined individually for each channel (i.e., bright-field and fluorescence imaging).
   4. Set the interval and duration of imaging cycle, save the path and then start imaging.

Results

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 "open" and "close" 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 nanoliter accuracy, ...

Discussion

Various microfluidic devices have been developed to perform multiplexed and complex experiments^17,18,19,20. 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 sensitivity^21...

Materials

List of materials used in this article

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