Team:MIT/Demonstrate

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Demonstrate

For our team to begin creating an engineered cell swarm, we had to first generate cells that produced viable chemoattractants. In order to do this, HEK cells were transfected with plasmids containing chemokines and fluorescent proteins, both in the form of fusion proteins. The data from our secretion test demonstrated that the HEK cells were able to successfully translate a fusion protein of IL-8 and Fluorescent Neon Green in significant quantities. Additionally, the supernatant of these cells also significantly fluorescenced. This suggests that the proteins were secreted in large amounts, allowing for the formation of a protein gradient. The media compatibility test also showed that production rates and efficiency were high in both RPMI as well as DMEM. As such, HL-60s would be able to swarm in their most preferred media. A favorable environment allows for both improved the viability of the follower cells (HL60s), which leads to increased production of secondary chemokines.

Our team’s biggest task in creating a cell swarm was to establish a chemokine gradient of IL-8 that could be produced by our leader cells and then detected by our follower cells. In order to do this, we needed to create an environment conducive to such a swarm. After modeling the different environmental conditions necessary to create a strong chemokine gradient, we decided that the IBIDI Chamber Well Assay would create the best environment for swarm formation. We concentrated our leader cells in the right chamber to lower the chemokine diffusivity of the system by increasing the concentration of IL8 in one centralized area. While swarming behavior in the second part of the IBIDI Chamber Well Assay was expected, the movement behavior of our follower cells in the first part of the experiment suggests that by keeping the barrier may have created better conditions for a steady gradient.

Followers and Leaders (gif)

Click the icon to watch the movie

This rightward movement of the differentiated HL-60s (dHL-60s) towards the right Chamber Well strongly suggests an IL8 chemokine gradient, causing a chemotactic response in the follower cells towards the leader cells. This chemokine-induced migration of our follower cells can be interpreted as the beginnings of a cell swarm.

While it is possible that the movement of the dHL-60s could have just been random walks, the probability is relatively low. Neutrophil migratory behavior patterns support chemokine-based movement, and for the dHL-60s to access the middle region, they had to squeeze their membranes under the left IBIDI Chamber Well Wall and then squeeze themselves between the well plate and the coverslip, similar to how neutrophils move in under-agarose assays. According to research on neutrophil chemotaxis, neutrophils will find their way past an obstacle if there is a stimulus on the other side. Additionally, cell-cell communication from other neutrophils induces this movement around or through obstacles. If one neutrophil finds a way past a barrier, other neutrophils will follow its path through a secondary chemotactic response produced by the first neutrophil that amplifies the primary chemokine gradient.

In our time lapse, there seemed to be several paths of least resistance under the wall that the dHL-60 cells found easier to squeeze themselves through to reach the middle area. In the beginning of the timelapse, near the bottom of the left Chamber Well wall, two dHL-60s almost simultaneously slide under the wall into the middle region. After they cross the barrier, several other cells seem to cross the same area of the wall, following their path. It should also be noted that no cell that crossed into the middle section returned to the left IBIDI Chamber Well. This observed movement pattern of our follower cells moving into the middle region seems homologous as to how neutrophils are observed moving past obstacles towards a chemoattractant and how neutrophils will follow each other through cell-cell communication.

Further supporting the suggestion of a chemotactic response is the migration out of the left chamber well despite the increased movement resistance between the coverslip and the well plate. The inside of the left IBIDI Chamber Well is not covered and the well plate is coated in fibronectin, making this region the best for HL-60 adherence and movement. Meanwhile, the middle portion is tightly sealed with the coverslip that connects the two IBIDI Chamber Wells. This seal is meant to be near impossible for cells to traverse under, hence why it is used as a barrier. For so many of these cells to move away from the free environment of the left IBIDI Chamber Well, there most likely was a stimulus to the right that attracted the dHL-60s away from their original region to an environment with more resistance and obstacles.

The only stimulus that was produced on the right side of the IBIDI Chamber Well Assay was the IL-8 from our leader cells. The HEK cells were transfected with the IL-8 and NeonGreen secretion plasmids for 24 hours. The NeonGreen presence in the IBIDI Chamber Well Assay indicates positive IL-8 secretion, which at a slow enough diffusion rate, would create a chemokine gradient towards the right chamber. Since a silicone barrier separated the two cell types in this segment of the experiment, a chemokine gradient could have formed under that barrier. Neutrophils are capable of receiving and responding to minute amounts of IL-8 if a gradient is present, and it is very possible that this response can be seen from our experiment. Our differentiated HL-60s, which mirrored the chemotactic and motility behavior of native neutrophils, sensed an IL-8 presence in the right IBIDI Chamber Well through an established chemokine gradient under the barrier created by the transfected HEK cells. Subsequently, some of the follower cells nearest to the barrier squeezed themselves under the wall and into the middle region. Then, more follower cells began to follow them through a secondary chemotactic response produced by other dHL-60s.

The IBIDI Chamber Well Assay may have created the conditions necessary to establish an IL-8 chemokine gradient between our HEK cells and differentiated HL-60s. Thus, there is a visual migration in our time lapse of follower cells actively moving toward leader cells, creating the beginnings of a cell swarm. If the gradient produced by our leader cells was strong enough to move our follower cells through a resistance-filled environment, then their chemokine production of our leader cells is sufficient enough to produce a full-scale cell swarm in the future.

References

  1. Ambravaneswaran, Vijayakrishnan, et al. “Directional Decisions during Neutrophil Chemotaxis inside Bifurcating Channels.” National Center for Biotechnology Information, U.S. National Library of Medicine, Nov. 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC3001269/.
  2. Heit, Bryan, and Paul Kubes. “Measuring Chemotaxis and Chemokinesis: The Under-Agarose Cell Migration Assay.” Science Signaling, American Association for the Advancement of Science, 18 Feb. 2003, stke.sciencemag.org/content/2003/170/pl5.long.

This page was written by Malik and Miles George