Team:MIT/Collaborations
Collaborations
Mammalian cell culture in microfluidic devices
Purpose
Integration of microfluidic devices has advanced considerably in quantitative biological research [1][2]. The timing of the fluid flow can be regulated with high accuracy because of the short response time of in-chip membrane valves (as quick as 1ms)[1]. Moreover, with the aid of hydrogel, cells growing in microfluidics can be structured in three-dimension geometries [1]. Therefore, cell culture performed in microfluidic devices could resemble in vivo conditions due to tight control of microenvironment [2]. On the other hand, automation is another advantageous of microfluidic technology. Instead of manual intervention, programmable fluid flows can automatically change chemical conditions of the culture environment. This feature would be particularly useful to examine dynamically changing systems [1]. The ability to precisely control fluid flows and chemical species, and automation of microfluidic technology are highly beneficial to future development of our project. Thus, MIT iGEM team decided to collaborate with the Boston University Hardware iGEM team to develop a protocol for mammalian cell culture in microfluidic devices
How does microfluidics benefit our project?
- Automatically regulate estrogen and progestorone cycle in vitro cell culture
- Resemble dynamically changing estrogen and progesterone concentration
- Dynamically monitor cell reponse and output gene expression
Collaboration
We collaborated with the Boston University Hardware iGEM Team to culture mammalian cells in their microfluidic devices. We started with the simplest experiment, which is growing HEK293 (Human Embryonic Kidney) cells in a microfluidic device. We performed the experiment with two different device models.
a = b = c = 400um ; Material: PDMS. Red for controlling air exchange. Blue for fluid channel.
Model 1
Microfluidic device
The device had 2 sets of inlet-outlet channels, one for cells seeding, and the other set for controling chemical species' concentrations in media. The first model did not have any chamber or a specific region for cell cultureing. The inlet and outlet were connected by a tube with uniform dimensions. It, however, had rectangular cell traps along the channel.
Cell culturing protocol
- The cells were harvested using standard culturing protocol, and then resuspended in media to achieve 2E4cells/cm^2 seeding density.
- Sequentially sterile and wash the channel with 1ml of 70% ethanol and 1xPBS by directly pipetting into the inlet.
- Through the same inlet, pipet 1ml of cells solution (1E6 cells/ml).
- Incubate the whole device with seeded cells at 37 degree Celcius.
a = 3250um; b = 400um; volume of the chamber = ~ 4.4ul. Material: PDMS.
Model 2
Microfluidic device
Since we couldn't detect any cells under 10X light microscope (24 hrs post seeding) in the first model, we proposed several modifications to the device after doing some literature research, including:
- Create a diamond-shape chamber, a specific region for cells adhesion. The increase in cross-section area along the channel reduces the flow rate of the fluid, making it easier for the cells to stick to the surface of the chamber.
- Using a syringe pump to continuously replenish media inside the chamber after cell adhesion, preventing the media from drying out inside the chamber - which happened in model 1.
- Include a 3D-printed frame, fitting the size of a 96-well plate, so that the whold seeding and culturing process can be monitored and captured using a microscope.
Cell culturing protocol
- The cells were harvested using standard culturing protocol, and then resuspended in media to achieve 120E4 cells/ml seeding density.
- Clean and wash the chamber with 70% ethanol and 1x PBS.
- Use a 5mL synringe to load 1ml of media, o.5ml of cell solution, and another media column to a 1.59mm diameter tube.
- Use a syringe pump to load media and cell solution in the tube to the device.
Results
Model 1
After 24 hours, the channel was competely dried out. The images didn't show any cells inside the channel, even though cells in the control culture dish growed normally (image not shown), which was seeded at the same time with the microfluidic device. Since we couldn't check for cells inside the device right after seeding, We couldn't narrow down the main reasons for unsuccessful cell culturing in microfluidic device. Thus, we decided to repeat the experiment (using model 2) with dynamic monitoring right from the seeding stage.
Model 2
Right after seeding, the cells were still in suspension; therefore, they all had spherical shape. After about 20 frames (200 minutes), they started to elongate and shifted out of focal plane. At the 26th frame (260 minutes after seeding), the cells were out of focus, which could indicate adhesion of the cells to the chamber's surface. The blue arrows point at cells showing evidience of mitosis, including appearance of two nucleus within a single cell, and formation of metaphase plate. Unfortunately, at the end of the video (after 350 minutes), the media within the chamber dried out, which has been a major problem for our experiemntal set up.
Read more about our collaboration on the BU Hardware Team's website
Testing pDEST mCherry
Purpose
Collaboration
Results
Recombinases' sequences
Purpose
Collaboration
Results
REFERENCE:
- Mehling Matthias, and Tay Savas. Microfluidic Cell Culture. Science Direct 2014; 25:95-102.
- Komen Job, Wolbers Floor, Franke R. Henk, et al. Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs. Biomed Microdevices(2008); 10:727–737