diff --git a/tex/thesis.tex b/tex/thesis.tex index fe90a4e..eb9c75c 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -61,6 +61,7 @@ \newacronym{bca}{BCA}{bicinchoninic acid assay} \newacronym{bsa}{BSA}{bovine serum albumin} \newacronym{stp}{STP}{streptavidin} +\newacronym{stppe}{STP-PE}{streptavidin-phycoerythrin} \newacronym{snb}{SNB}{sulfo-nhs-biotin} \newacronym{cug}{CuG}{Cultispher G} \newacronym{cus}{CuS}{Cultispher S} @@ -132,6 +133,8 @@ \newcommand{\inlinecode}{\texttt} +\newcommand{\subcap}[2]{\subref{#1}) #2} + %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % ditto for environments @@ -662,6 +665,16 @@ better retention of memory phenotype compared to current bead-based methods. \section{methods} \subsection{dms functionalization} +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/dms_flowchart.png} + + \endgroup + \caption[\gls{dms} Flowchart]{Overview of \gls{dms} manufacturing process.} + \label{fig:dms_flowchart} +\end{figure*} + Gelatin microcarriers (\gls{cus} or \gls{cug}, GE Healthcare, DG-2001-OO and DG-0001-OO) were suspended at \SI{20}{\mg\per\ml} in 1X \gls{pbs} and autoclaved. All subsequent steps were done aseptically, and all reactions were @@ -781,7 +794,7 @@ threshold. Media glucose was measured using a \product{GlucCell glucose % added to a final concentration of \SI{50}{\ug\per\ml} during media addition. Cells on the \glspl{dms} were visualized by adding \SI{0.5}{\ul} -\product{\gls{stp}-\gls{pe}}{\bl}{405204} and \SI{2}{ul} +\product{\gls{stppe}}{\bl}{405204} and \SI{2}{ul} \product{\acd{45}-\gls{af647}}{\bl}{368538}, incubating for \SI{1}{\hour}, and imaging on a spinning disk confocal microscope. @@ -868,21 +881,113 @@ context of pure error). Statistical significance was evaluated at $\upalpha$ = \subsection{DMSs can be fabricated in a controlled manner} +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/dms_coating.png} + \phantomsubcaption\label{fig:stp_carrier_fitc} + \phantomsubcaption\label{fig:mab_carrier_fitc} + \phantomsubcaption\label{fig:cug_vs_cus} + \phantomsubcaption\label{fig:biotin_coating} + \phantomsubcaption\label{fig:stp_coating} + \phantomsubcaption\label{fig:mab_coating} + + \endgroup + \caption[\gls{dms} Coating] + {\gls{dms} functionalization results. + \subcap{fig:stp_carrier_fitc}{\gls{stp}-coated or uncoated \glspl{dms} + treated with biotin-FITC and imaged using a lightsheet microscope.} + \subcap{fig:mab_carrier_fitc}{\gls{mab}-coated or \gls{stp}-coated + \glspl{dms} treated with \anti{\gls{igg}} \glspl{mab} and imaged using a + lightsheet microscope.} \subcap{fig:cug_vs_cus}{Bound \gls{stp} surface + density on either \gls{cus} or \gls{cug} microcarriers. Surface density + was estimated using the properties in~\cref{tab:carrier_props}} Total + binding curve of \subcap{fig:biotin_coating}{biotin}, + \subcap{fig:stp_coating}{\gls{stp}}, and + \subcap{fig:mab_coating}{\glspl{mab}} as a function of biotin added. } + \label{fig:dms_flowchart} +\end{figure*} + +% TODO these caption titles suck +% TODO combine this DOE figure into one interaction plot \begin{table}[!h] \centering \caption{Properties of the microcarriers used} - \label{tab:ci_treat} + \label{tab:carrier_props} \input{../tables/carrier_properties.tex} \end{table} -% fabrication flow chart -% microcarrier properties -% green carrier figure -% quality control figure -% DMS manufacturing timing figure +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/dms_qc.png} + \phantomsubcaption\label{fig:dms_qc_doe} + \phantomsubcaption\label{fig:dms_qc_ph} + \phantomsubcaption\label{fig:dms_qc_washes} + \phantomsubcaption\label{fig:dms_snb_decay_curves} + + \endgroup + \caption[\gls{dms} Quality Control] + {\gls{dms} quality control investigation and development + \subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass + of microcarriers, reaction temperature, and biotin concentration on + biotin attachment.} + \subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.} + \subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the + microcarriers on biotin attachment.} + \subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in + \gls{pbs} of differing pH.} + All statistical tests where p-values are noted are given by two-tailed t + tests. + } + \label{fig:dms_flowchart} +\end{figure*} + +\begin{table}[!h] \centering + \caption{Properties of the microcarriers used} + \label{tab:carrier_props} + \input{../tables/carrier_properties.tex} +\end{table} + +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/dms_timing.png} + \phantomsubcaption\label{fig:dms_biotin_rxn_mass} + \phantomsubcaption\label{fig:dms_biotin_rxn_frac} + \phantomsubcaption\label{fig:dms_stp_per_time} + + \endgroup + \caption[\gls{dms} Reaction timing] + {Reaction kinetics for microcarrier functionalization. + \subcap{fig:dms_biotin_rxn_mass}{Biotin mass bound per time} + \subcap{fig:dms_biotin_rxn_frac}{Fraction of input biotin bound per time} + \subcap{fig:dms_stp_per_time}{\Gls{stp} bound per time.} + } + \label{fig:dms_flowchart} +\end{figure*} \subsection{DMSs can efficiently expand T cells compared to beads} -% cells growing on DMSs +% TODO make sure the day on these is correct +\begin{figure*}[ht!] + \begingroup + + \includegraphics{../figures/cells_on_dms.png} + \phantomsubcaption\label{fig:dms_cells_phase} + \phantomsubcaption\label{fig:dms_cells_fluor} + + \endgroup + \caption[\gls{dms} Reaction timing] + {Cells grow in tight clusters in and around functionalized \gls{dms}. + \subcap{fig:dms_cells_phase}{Phase-contrast image of T cells growing on + \glspl{dms} on day 7} + \subcap{fig:dms_cells_fluor}{Confocal images of T cells in varying z-planes + growing on \glspl{dms} on day 9. \Glspl{dms} were stained using + \gls{stppe} (red) and T cells were stained using \acd{45}-\gls{af647}.} + } + \label{fig:dms_flowchart} +\end{figure*} + % 3-donor expansion figure \subsection{DMSs lead to greater expansion and memory and CD4+ phenotypes}