ADD a bunch of future work fluff

tex/references.bib

@@ -2583,6 +2583,19 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
   publisher = {The American Association of Immunologists},
 }
 
+@Article{Stephan2014,
+  author    = {Sirkka B Stephan and Alexandria M Taber and Ilona Jileaeva and Ericka P Pegues and Charles L Sentman and Matthias T Stephan},
+  journal   = {Nature Biotechnology},
+  title     = {Biopolymer implants enhance the efficacy of adoptive T-cell therapy},
+  year      = {2014},
+  month     = {dec},
+  number    = {1},
+  pages     = {97--101},
+  volume    = {33},
+  doi       = {10.1038/nbt.3104},
+  publisher = {Springer Science and Business Media {LLC}},
+}
+
 @Comment{jabref-meta: databaseType:bibtex;}
 
 @Comment{jabref-meta: grouping:

tex/thesis.tex

@@ -163,6 +163,7 @@
 \newacronym{qbd}{QbD}{quality-by-design}
 \newacronym{aws}{AWS}{amazon web services}
 \newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction}
+\newacronym{cstr}{CSTR}{continuously stirred tank bioreactor}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 % SI units for uber nerds
@@ -733,7 +734,7 @@ much higher surface area than a traditional flask when matched for volume.
 Consequently, this means that microcarrier-based cultures can operate with much
 lower footprints than flask-like systems. Microcarriers also allow cell culture
 to operate more like a traditional chemical engineering process, wherein a
-stirred tank bioreactor may be employed to enhance oxygen transfer, maintain pH,
+\gls{cstr} may be employed to enhance oxygen transfer, maintain pH,
 and continuously supply nutrients\cite{Derakhti2019}.
 
 A variety of microcarriers have been designed, primarily differing in their
@@ -4315,7 +4316,8 @@ group.
 
 \section{future directions}
 
-There are several important next steps to perform with this work:
+There are several important next steps to perform with this work, many of which
+will be relevent to using this technology in a clinical trial:
 
 \subsection{Translation to GMP process}
 
@@ -4341,12 +4343,33 @@ as dynabeads and thus the research-grade proteins used here could be easily
 replaced. The \gls{snb} is a synthetic small molecule and thus does not have any
 animal-origin concerns.
 
-\subsection{Mechanistic investigation}
+\subsection{mechanistic investigation}
 
-% why do the dms work?
-% can we put anything on the dms to enhance their potency?
+Despite the improved outcomes in terms of expansion and phenotype relative to
+beads, we don't have a good understanding of why they \gls{dms} platform works
+as well as it does. Several broad areas remain to be investigated, including the
+role of the increased cytokine output (including \il{15} which was explored to
+some extent in this work), the role of cells on the interior of the \gls{dms}
+relative to those outside the \gls{dms}, and the role of the physical surface
+properties of the \gls{dms} (including the morphology and the stiffness).
 
-\subsection{Assessing performance using unhealthy donors}
+\subsection{additional ligands and signals on the DMSs}
+
+In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated
+on the surface of the \gls{dms}. The chemistry used for the \gls{dms} is very
+general, and any molecule or protein that could be engineered with a biotin
+ligand could be attached without any further modification. There are many other
+ligands that could have profound effects on the expansion and quality of T cells
+which may be utilized. The simplest next step is to simply vary the ratio of
+\acd{3} and \acd{28} signal. Another obvious example is to attach
+\il{15}/\il{15R$\upalpha$} complexes to the surface to mimic \textit{trans}
+presentation from other cell types\cite{Stonier2010}. Other adhesion ligands or
+peptides such as GFOGER could be used to stimulate T cells and provide more
+motility on the \glspl{dms}\cite{Stephan2014}. Finally, viral delivery systems
+could theoretically be attached to the \gls{dms}, greatly simplifying the
+transduction step.
+
+\subsection{assessing performance using unhealthy donors}
 
 All the work presented in this dissertation was performed using healthy donors.
 This was mostly due to the fact that it was much easier to obtain healthy donor
@@ -4360,8 +4383,23 @@ expansion technology given that even in healthy donors, we observed the
 
 \subsection{translation to bioreactors}
 
-% use dms in non-static bioreactors such as wave by first activating in a static
-% environment
+In this work we performed some preliminary experiments demonstrating that the
+\gls{dms} platform can work in a Grex bioreactor. While an important first step,
+more work needs to be done to optimize how this system will or can work in a
+scalable environment using bioreactors. There are several paths to explore.
+Firstly, the Grex itself has additional automation accessories which could be
+tested, which would allow continuous media exchange and cytokine
+administration. While this is an improvement from the work done here, it is
+still a Grex and has all the disadvantages of an open system. Secondly, other
+static bioreactors such as the Quantum hollow fiber bioreactor (Terumo) could be
+explored. Essentially the \gls{dms} would be an additional matrix that could be
+supplied to this system which would enhance its compatibility with T cells.
+Finally, suspension bioreactors such as the classic \gls{cstr} or WAVE
+bioreactors could be tried. The caveat with these is that the T cells only seem
+to be loosely attached to the \gls{dms} throughout culture, so an initial
+activation/transduction step in static culture might be necessary before moving
+to a suspension system (alternatively the \gls{dms} could be coated with
+additional adhesion ligands to make the T cells attach more strongly).
 
 \onecolumn
 \clearpage