Revision as of 07:02, 18 October 2016

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Collaborations

Mammalian cell culture in microfluidic devices

Purpose

Microfluidic device fabricated by BU Hardware iGEM Team.

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?

1. Automatically regulate estrogen and progestorone cycle in vitro cell culture
2. Resemble dynamically changing estrogen and progesterone concentration
3. 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.

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

1. The cells were harvested using standard culturing protocol, and then resuspended in media to achieve 2E4cells/cm^2 seeding density.
2. Sequentially sterile and wash the channel with 1ml of 70% ethanol and 1xPBS by directly pipetting into the inlet.
3. Through the same inlet, pipet xxxxxxul of cells solution.
4. Incubate the whole device with seeded cells at 37 degree Celcius.

Model 2

Microfluidic device

Since we couldn't find any cells under 10X microscope in the first-model device, we proposed several modifications for the device to the BU hardware team after doing some literature research, which included:

1. 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 the cells to stick to the surface of the chamber.
2. 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.
3. 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

Figure. A) General setup for automatic cells seeding using a syringe pump. B) Input and output wires are connected to inlet and outlet of the microfluidic device. A 3D-printed frame is used to put the device on the microscope's placeholder, allowing dynamic monitoring. C) A seringe pump is set up to comtinuously pump the solution containing cells into the channel during seeding (seeding flow rate was: 20ul/min, cell density: 120E4 cells/ml).

Results

Model 1

Figure. Images of microfluidic channel, 24hrs after cells seeding.

Model 2

Video. Images taken during 350 minutes after seeding. Frame rate: 3fps. Each image frame was taken at 10 minute interval.

Testing pDEST mCherry

Purpose

Collaboration

Results

Recombinases' sequences

Purpose

Collaboration

Results