Design

To create a cell swarm, we divided the task into 3 aims:

Aim 1: Making Leaders, which involved:

Designing/Cloning Chemokine Genetic Circuits with Tags
Testing HEK (Leader) Cell Viability in HL60 (Follower) Cell Culture (RPMI Media)
HEK (Leader) Cell Chemokine Secretion with Filtration + Fluorescence Reader

Aim 2: Assembling the Swarm, which involved:

Testing Chemotactic Index - Boyden Assay
HEK (Leader) Cell Chemokine Secretion with Filtration + Fluorescence Reader
Testing Chemotactic Index - Boyden Assay
Testing Motility Index - Time-Lapse Microscopy Experiments
Testing Swarming Ability - Ibidi Chamber Experiments

Aim 3: Modeling the Swarm, which involved:

Actin Protrusion Diagrams
Digital Cell Swarms with Morpheus

Aim 1: Making Leaders

To make leader cells for our swarm, we needed to design engineered cells that could secrete chemokines and could be cultured with our follower cells. These leader cells needed to be adherent cells that were easily engineered and transfected and could be capable of protein production. Based on these criteria, we decided to choose HEK-293 cells as our leader cells.

Designing/Cloning Chemokine Genetic Circuits with Tags

We had to first create plasmids for HEK cell protein secretion. Using the Type IIS Assembly Method, we designed a variety of plasmids with different types of promoters, chemokine genes, and expression indicators/tags based on all of the experiments we wanted to run (More details of our parts and their basic design can be found on our Parts Pages).

Testing HEK (Leader) Cell Viability in HL60 (Follower) Cell Culture (RPMI Media)

Next, we had to test if our leader cells were viable and transfectable in the media of our follower cells. We compared transfection rates and cell viability of our HEK cells in their preferred media, DMEM, with the preferred media of our follower cells, RPMI, for 48 hours.

Diagram by Melody Wu

HEK (Leader) Cell Chemokine Secretion with Filtration + Fluorescence Reader

Finally, we had to test the secretion levels of our transgenic proteins produced by the engineered HEK cells. We transfected HEK cells with the constitutive promoter and NeonGreen expression indicator version of our plasmid in RPMI media. Then, after 48 hours of expressing the plasmid, we collected the secreted protein supernatant of the cells by filtering the contents of their media with centrifugal filter units. We measured the amount of chemokine secreted using a Fluorescence Plate Reader.

Diagram by Maisha Prome

Aim 2: Assembling the Swarm

In order to create the swarms, we needed to perform assays that would demonstrate controlled follower cell movement. To quantify this data, experiments were separated for their contribution to a Chemotactic Index or a Motility Index. The Chemotactic Index involves measuring how the differentiated HL-60s (dHL-60s) respond to chemoattractants. The Motility Index, on the other hand, measures how the cells move in general. This includes gathering data on their speed, interactions with each other and other cell types, and other aspects of their movement.

Testing Chemotactic Index - Boyden Assay

For the chemotactic index, we used Boyden Transwell assays to measure how the cells moved towards both pure and secreted chemokines at different concentrations. Data from this test would demonstrate the viability of our chemoattractants as successful production would result in high movement to the bottom chambers.

Image from Albert Folch

Testing Motility Index - Time-Lapse Microscopy Experiments

For the motility index, we ran several of time-lapse microscopy experiments in different conditions. The most important of these was Differentiation with Optional Stimulation (see our Protocols) to see how differentiated HL-60s move in comparison to undifferentiated cells. This test would show the general movement (chemokinesis) of neutrophil-like cells.

Testing Swarming Ability - Ibidi Chamber Experiments

To assess swarming ability, an ibidi chamber experiment was created to test both the motility and chemotactic index. In this experiment, HL-60s are plated on one side of the well while HEK cells transfected with IL8-NeonGreen are plated on the opposite side with a barrier between the two cell types. The barrier is then removed and the movement of the HL-60s is observed and recorded. A significant amount of the HL-60 cells moving towards the HEK cells would demonstrate attraction to the chemokine that they are secreting.

Aim 3: Modeling the Swarm

In order to evaluate the parameters for our experiments that would produce meaningful results as well as represent real-world conditions, several computational models were developed alongside the physical experiments. The first of these models is a diagram representing actin polymerization inside of a neutrophil. This diagram conveys the mechanisms in the cell that allow for movement and determine the cell’s direction. With an accurate model, we would be able to predict cell movement from still images allowing for more complex analysis of microscopy experiments. The next set of models use the program Morpheus to study how chemokine gradients and cell swarms form using different initial conditions and assumptions. The data from these models would give us insight on how to optimize our physical experiments in order to produce strong gradients, gather the most follower cells, and plate the leader cells in efficient shapes. Information on how the models were created and their results can be found on our Modeling page. With this model, future improvements can be made in terms of design so that we might be able to engineer swarm size and movement or patterning.

Team:MIT/Design