## 2 Materials and Methods
### Mission-Specific Growth Conditions and Procedures
#### 2.3.2 Micro-14/FPA, Space X CRS-16  
Similar to Micro-6/8, the hardware utilized for this payload consisted of FPAs organized eight per GAP (BioServe Space Technologies). This experiment also had two  
Figure 2: Temperature tracings comparing the experience of flight and terrestrial control samples throughout the duration of the missions. HOBO temperature sensors were incorporated into selected GAPs and retrieved during hardware de-integration at the end of the mission. **(A)** Samples in the Micro-6 payload that were activated early in the SpX CRS-1 mission, **(B)** Samples in the Micro-8 payload that were activated and then terminated late in the SpX CRS-4 mission, and **(C)** Samples in the Micro-14/FPA payload that were activated and ‘terminated’ with fresh medium late in the SpX CRS-16 mission for return of viable cells. Temperature sensors were not associated with FEP bag bioreactors in SpX CRS-17. Terrestrial control operations were performed near synchronously (2 h delay from flight) and for the purpose of this illustration the time offset was removed from the tracings to allow overlap of transitions for better comparison. Flight temperatures are traced in black, terrestrial control temperatures are traced in green, and key operations are indicated in text and highlighted with red vertical lines.  
operational windows. Some samples were cultured as described below early in flight (Early) and preserved either with the addition of paraformaldehyde or by freezing until the end of the mission. The remaining samples were maintained at 4 \({}^{\circ}\)C and cultured as late as possible in the mission (Late) and returned live and chilled (4 \({}^{\circ}\)C) for post flight analyses requiring cell growth. During each operational window, cells were cultured in microgravity for 24 h +/\(-\) 1 h at 30 \({}^{\circ}\)C. FPAs were prepared with 2 or 3 chambers as follows. (1) Samples to be frozen consisted of 2 chambers: Chamber A was loaded with 5 mL of YPD (referred to as Low due to the lower cell concentration upon inoculation) and overlaid with an air bubble to promote gas exchange. Chamber B was loaded with 0.5 mL of yeast suspended in water (4 \(\times\) 10\({}^{4}\) cells/mL). There was no chamber C. (2) Samples to be fixed consisted of 3 chambers: Chamber A was loaded with 2.7 mL YPD (referred to as High as the cell concentration was higher at the time of inoculation) and overlaid with an air bubble. Chamber B was loaded with 0.5 mL of yeast suspended in water (4 \(\times\) 10\({}^{4}\) cells/mL). Chamber C was loaded with 2.2 mL paraformaldehyde (10% \(v/v\)). (3) Samples to be returned viable consisted of 3 chambers: Chamber A was loaded with 2.7 mL YPD (referred to as High as the cell concentration was higher at the time of inoculation) and overlaid with an air bubble. Chamber B was loaded with 0.5 mL of yeast suspended in water (4 \(\times\) 10\({}^{4}\) cells/mL). Chamber C was loaded with 2.2 mL 1\(\times\) YPD. Following completion of the mission, the live samples were isolated from de-integrated hardware and processed the day they arrived in the home laboratory; fixed and frozen samples were isolated shortly thereafter. Temperature sensors were included in representative GAPs to retrospectively analyze the operational transitions as well as consistency between flight and terrestrial control samples (Figure 2).