Experimental Test Sequence

As part of this project, we traveled to ESA headquarters in the Netherlands to conduct a trial run in April 2013.  The trial run, formally called the Experimental Sequence Test (EST), was a full-dress rehearsal for flight, and provided us with the opportunity to mimic the flight experiment from start to finish. We moved our entire lab operation, supplies, and small equipment from San Francisco to the Netherlands, set up and took down the lab all within the course of a few weeks.


For the test run, we isolated T-cells from five donors and loaded them into flight cassettes provided by Kaiser Italia (KI). For the flight experiment, we will be using ten donors.  Each cassette has four compartments and each compartment has three separate chambers.  The bottom chamber contained isolated T-cells from an individual donor.  The middle chamber contained magnetic beads coated with antibodies against the TCR/CD3 and costimulatory CD28 receptors. Interactions between T-cells and the beads mimic a normal physiological infection and activate the T-cells.  The upper chamber in the cassettes contains a fixative that immediately preserves and stabilizes RNA transcripts.  When mixed with the cells, the fixative essentially takes a ‘snapshot’ of the cell at that moment in time and allows us to later identify and quantify what genes are being expressed at different stages of T-cell activation.  At the start of the experiment when we load the cassettes, all of the chambers are separated (Figure 1). 


Once we finish assembling the cassettes, we hand them over to the KI team for leak testing.  After, they were placed into a stowage bag and loaded onto the spacecraft.  At EST, the loading and launch were simulated, but for our first flight, the cells will be travelling to the ISS on SpaceX’s Falcon 9 rocket. Once the cassettes are unloaded on the ISS, a crew member will place them in ESA’s KUBIK centrifuge at 37°C, where cassettes will be in either a fixed position (microgravity environment) or placed on the centrifuge (1g environment).  All cassettes will be activated after a two-hour pre-incubation period.  A crew member will use a special tool that is designed to push the activation chamber into the cell chamber, so that the T-cells and beads can mix (Figure 2).  The fixative chamber remains sealed.  At different time points – 1.5 hours (both flights), 4 hours (first flight) and 24 hours (second flight), the crew member will use a different tool to add the fixative to the cells and ‘freeze’ them at that moment in time (Figure 3).  Once the cells are fixed, they are stored in a freezer until they returned to us back on earth for analysis.

 

 

 

 

 

Figure 1: Incubation before activator injection

 

Figure 2: Activator injection

 

Figure 3: Fixative injection

 

Because we work with living cells, time is of the essence – cells must be fixed within 120 hours from our isolation or else they start to die.  Consequently, EST was very important to ensure that the teams could coordinate all these steps in a timely fashion. 


You can read more about our test run in the Netherlands here.
Additional information about our project can also be found on the NASA Mission website.

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