## 4 Discussion
Surprisingly, the data presented in Figure 6D demonstrates that the resilience is compromised in cells frozen during spaceflight when compared to similarly treated terrestrial controls. The difference in survival was observed in samples frozen in FEP bags (Micro-14/FEP) as well as FPAs (Micro-14/FPA; ~6-fold fewer colonies in flight than terrestrial controls), demonstrating this is a feature consistent across two types of hardware. We considered the difference in survival secondary to differences in the rate of freezing. Cells are likely to freeze more slowly in the low convection environment of space, but a slower rate is typically considered to be advantageous for cell viability [46]. It was also noted that cells subjected to spaceflight experience more post-undocking temperature transitions than the terrestrial controls. Mimicking these temperature transitions in ground-based studies resulted in a loss of cell viability, but not nearly to the extent of that observed in the studies described in Figure 6D. Membrane integrity is associated with freeze stress; therefore, we are considering adaptations to the microgravity environment that might influence membrane composition. In addition to cell growth and survival, we analyzed the multicellular characteristics of _C. albicans_. A complex colony structure (HIW) had been observed in simulation experiments using the HARV bioreactor; therefore, viable cells were plated upon conclusion of the spaceflight missions to determine whether this morphological transition was also triggered in true microgravity. As demonstrated in Figure 7, HIW colonies were not observed following 24 h of growth in microgravity. Interestingly, a minimum of 5 days in the HARV was required to produce the complex colony microarchitecture observed [31], yet those cells went through a daily subculture procedure which may have delayed adaptation to the low fluid shear environment. It did not seem unreasonable to consider that a shorter term exposure to true microgravity may trigger similar adaptation responses as observed in the extended HARV studies. Longer term spaceflight studies are currently underway to address whether this colony morphology transition simply requires longer exposure to the true microgravity environment. Although the analysis of colony morphology required cells to propagate for several days in post-mission gravity conditions, the HARV-induced \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Outcome Measure** & **FPA (Micro-6)** & **FPA (Micro-14)** & **FEP (Micro-14)** \\ \hline Cell Growth & F \(>\) T * & Trend F \(>\) T & F \(>\) T \\ \hline Cell Density & Low & Intermediate & High \\ \hline Filamentation & Low, no change F vs. T & Low, no change F vs. T & Very low, no change F vs. T \\ \hline Colony Morphology & No change & No change & No change \\ \hline Colony Size & No change & F \(>\) T & F \(>\) T \\ \hline AmB Resistance & F \(>\) T & F \(>\) T & F \(>\) T \\ \hline CAS Resistance & NA & NA & F \(=\) T \\ \hline Biofilm & None & Trend T \(>\) F & NA \\ \hline Freeze Stress Resistance & NA & T \(>\) F & T \(>\) F \\ \hline \hline \end{tabular}  
* F = Flight; T = Terrestrial.  
\end{table} 
Table 3: Summary of data obtained across missions and in two types of hardware. HIW morphology had been quite stable. Re-adaptation to \(1g\) and subsequent loss of complex colony morphology does not seem a likely explanation for the observed lack of complex morphology. Biofilm communities were also undetectable in these studies, again suggesting the timeline of cultivation (24 h) was not sufficient for the development of complex macrostructures. Complex colony morphologies of _C. albicans_ are typically associated with an increase in filamented cells in the population [50]. Therefore, since complex macro-architectures were not observed in the studies herein, it is not surprising that cell filamentation was also found to be minimal.