ENH integrate the kinetics figure
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tex/thesis.tex
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tex/thesis.tex
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@ -1255,6 +1255,32 @@ appeared that the \gls{mab} binding was quadratically related to biotin binding
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critical to controlling the amount and \glspl{mab} and thus the amount of signal
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critical to controlling the amount and \glspl{mab} and thus the amount of signal
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the T cells receive downstream.
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the T cells receive downstream.
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\begin{figure*}[ht!]
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\begingroup
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\includegraphics{../figures/dms_qc.png}
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\phantomsubcaption\label{fig:dms_qc_doe}
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\phantomsubcaption\label{fig:dms_qc_ph}
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\phantomsubcaption\label{fig:dms_qc_washes}
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\phantomsubcaption\label{fig:dms_snb_decay_curves}
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\endgroup
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\caption[\gls{dms} Quality Control]
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{\gls{dms} quality control investigation and development
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\subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass
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of microcarriers, reaction temperature, and biotin concentration on
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biotin attachment.}
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\subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.}
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\subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the
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microcarriers on biotin attachment.}
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\subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in
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\gls{pbs} or \gls{di} water.}
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All statistical tests where p-values are noted are given by two-tailed t
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tests.
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}
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\label{fig:dms_flowchart}
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\end{figure*}
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To answer this question, we first performed a \gls{doe} to understand the effect
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To answer this question, we first performed a \gls{doe} to understand the effect
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of reaction parameters on biotin binding. The parameters included in this
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of reaction parameters on biotin binding. The parameters included in this
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\gls{doe} were temperature, microcarrier mass, and \gls{snb} input mass. These
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\gls{doe} were temperature, microcarrier mass, and \gls{snb} input mass. These
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@ -1305,64 +1331,6 @@ to the microcarrier suspension (which itself is in \gls{pbs}) this result
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indicated that hydrolysis is not of concern when adding \gls{snb} within
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indicated that hydrolysis is not of concern when adding \gls{snb} within
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minutes.
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minutes.
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\begin{figure*}[ht!]
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\begingroup
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\includegraphics{../figures/dms_qc.png}
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\phantomsubcaption\label{fig:dms_qc_doe}
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\phantomsubcaption\label{fig:dms_qc_ph}
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\phantomsubcaption\label{fig:dms_qc_washes}
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\phantomsubcaption\label{fig:dms_snb_decay_curves}
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\endgroup
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\caption[\gls{dms} Quality Control]
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{\gls{dms} quality control investigation and development
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\subcap{fig:dms_qc_doe}{\gls{doe} investigating the effect of initial mass
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of microcarriers, reaction temperature, and biotin concentration on
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biotin attachment.}
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\subcap{fig:dms_qc_ph}{Effect of reaction ph on biotin attachment.}
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\subcap{fig:dms_qc_washes}{effect of post-autoclave washing of the
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microcarriers on biotin attachment.}
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\subcap{fig:dms_snb_decay_curves}{Hydrolysis curves of \gls{snb} in
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\gls{pbs} or \gls{di} water.}
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All statistical tests where p-values are noted are given by two-tailed t
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tests.
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}
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\label{fig:dms_flowchart}
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\end{figure*}
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We also investigated the reaction kinetics of all three coating steps.
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To quantify the reaction kinetics of the biotin binding step, we reacted
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multiple batches of \SI{20}{\mg\per\ml} microcarriers in \gls{pbs} at \gls{rt}
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with \gls{snb} in parallel and sacrificially analyzed each at varying timepoints
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using the \gls{haba} assay. This was performed at two different concentrations.
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We observed that for either concentration, the reaction was over in
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\SIrange{20}{30}{\minute} (\cref{fig:dms_biotin_rxn_mass}). Furthermore, when
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put in terms of fraction of input \gls{snb}, we observed that the curves are
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almost identical (\cref{fig:dms_biotin_rxn_frac}). Given this, the reaction step
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for biotin attached was set to \SI{30}{\minute}.
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% TODO these numbers might be totally incorrect
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Next, we quantified the amount of \gls{stp} reacted with the surface of the
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biotin-coated microcarriers. Different batches of biotin-coated \glspl{dms} were
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coated with \SI{40}{\ug\per\ml} \gls{stp} and sampled at various timepoints
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using the \gls{bca} assay to indirectly quantify the amount of attached
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\gls{stp} mass. We found this reaction took \SI{45}{\minute}
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(\cref{fig:dms_stp_per_time}).
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% TODO find real numbers for this
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Finally, we used the reaction data from the \gls{stp} binding curve to estimate
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the \gls{mab} binding curve. Assuming a quasi-steady-state paradigm, we
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estimated that the diffusion rate coefficient for the microcarriers was
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{\#}{diffusion rate}. Using this diffusion rate and the maximum mass of
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\glspl{mab} bound the microcarriers (\cref{fig:mab_coating}), we estimated that
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the \gls{mab} reaction should proceed in {\#}{mab curve}.
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% TODO add additional paragraph about how this diffusion coefficient was used to
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% estimate the wash step times.
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% RESULT talk about the kinetic stuff in this figure more
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\begin{figure*}[ht!]
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\begin{figure*}[ht!]
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\begingroup
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\begingroup
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@ -1387,6 +1355,64 @@ the \gls{mab} reaction should proceed in {\#}{mab curve}.
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\label{fig:dms_kinetics}
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\label{fig:dms_kinetics}
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\end{figure*}
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\end{figure*}
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We also investigated the reaction kinetics of all three coating steps.
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To quantify the reaction kinetics of the biotin binding step, we reacted
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multiple batches of \SI{20}{\mg\per\ml} microcarriers in \gls{pbs} at \gls{rt}
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with \gls{snb} in parallel and sacrificially analyzed each at varying timepoints
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using the \gls{haba} assay. This was performed at two different concentrations.
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We observed that for either concentration, the reaction was over in
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\SIrange{20}{30}{\minute} (\cref{fig:dms_biotin_rxn_mass}). Furthermore, when
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put in terms of fraction of input \gls{snb}, we observed that the curves are
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almost identical (\cref{fig:dms_biotin_rxn_frac}). Given this, the reaction step
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for biotin attached was set to \SI{30}{\minute}.
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% TODO these numbers might be totally incorrect
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% TODO state what the effective diffusivity is
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Next, we quantified the amount of \gls{stp} reacted with the surface of the
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biotin-coated microcarriers. Different batches of biotin-coated \glspl{dms} were
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coated with \SI{40}{\ug\per\ml} \gls{stp} and sampled at intermediate timepoints
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using the \gls{bca} assay to indirectly quantify the amount of attached
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\gls{stp} mass. We found this reaction took approximately \SI{30}{\minute}
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(\cref{fig:dms_stp_per_time}). Assuming a quasi-steady-state paradigm, we used
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this experimental binding data to fit a continuous model for the \gls{stp}
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binding reaction. Using the diffusion rate of the \gls{stp}, we then calculated
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the effective diffusivity of the microcarriers to be {\#}.
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Using this effective diffusivity and the known diffusion coefficient of a
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\gls{mab} protein in water, we calculated predict the binding of \glspl{mab} per
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time onto the microcarriers (this obviously assumes that the effectively
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diffusivity is independent of the protein used, which should be reasonable given
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that the pores of the microcarriers are huge compared to the proteins, and we
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don't expect any significant reaction between the protein and the microcarrier
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surface save for the \gls{stp}-biotin binding reaction). According to this
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model, the \gls{mab} binding reaction should be complete within \SI{15}{\minute}
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under the conditions used for our protocol (\cref{fig:dms_mab_per_time}). Note
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that our unoptimized coated steps were done in \SI{45}{\minute}, which seemed
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reasonable given the slightly larger hydrodynamic radius of \glspl{mab} compared
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to \gls{stp} which was shown to react in \SI{30}{\minutes} experimentally. The
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results of this model should be experimentally verified.
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% TODO find the actual numbers for this
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Finally, we used the effective diffusivity of the microcarriers to predict the
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time needed for wash steps. This is important, as failing to wash out residual
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free \gls{snb} (for example) could occupy binding sites on the \gls{stp}
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molecules, lowering the effective binding capacity of the \gls{mab} downstream.
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Once again, we assumed the microcarriers to be porous spheres, this time with an
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initial concentration of \gls{snb}, \gls{stp}, or \glspl{mab} equal to the final
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concentration of the bulk concentration of the previous binding step, and
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calculated the amount of time it would take for the concentration profile inside
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the microcarriers to equilibrate to the bulk in the wash step. Using this model,
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we found that the wash times for \gls{snb}, \gls{stp}, and \glspl{mab} was
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\SI{10}{\minute}, {\#} minutes, and {\#} minutes respectively. Note that
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\gls{snb}, \gls{stp}, and \glspl{mab} each required 3, 2, and 2 washes to reduce
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the concentration down to a level that was 1/1000 of the starting concentration
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(which was deemed to be acceptable for preventing downstream inhibition). Using
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this in our protocol, we verified that the \gls{snb} was totally undetectable
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after washing (\cref{fig:dms_biotin_washed}). The other two species need to be
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verified, but note that the consequences of residual \gls{stp} or \gls{mab} are
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far less severe than that of \gls{snb}.
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\subsection{DMSs can efficiently expand T cells compared to beads}
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\subsection{DMSs can efficiently expand T cells compared to beads}
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% RESULT add other subfigures here
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% RESULT add other subfigures here
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