ENH proofread the background and innovation

tex/references.bib

@@ -2647,6 +2647,19 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
   publisher = {Elsevier {BV}},
 }
 
+@Article{Rosenberg1988,
+  author    = {Steven A. Rosenberg and Beverly S. Packard and Paul M. Aebersold and Diane Solomon and Suzanne L. Topalian and Stephen T. Toy and Paul Simon and Michael T. Lotze and James C. Yang and Claudia A. Seipp and Colleen Simpson and Charles Carter and Steven Bock and Douglas Schwartzentruber and John P. Wei and Donald E. White},
+  journal   = {New England Journal of Medicine},
+  title     = {Use of Tumor-Infiltrating Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with Metastatic Melanoma},
+  year      = {1988},
+  month     = {dec},
+  number    = {25},
+  pages     = {1676--1680},
+  volume    = {319},
+  doi       = {10.1056/nejm198812223192527},
+  publisher = {Massachusetts Medical Society},
+}
+
 @Comment{jabref-meta: databaseType:bibtex;}
 
 @Comment{jabref-meta: grouping:

tex/thesis.tex

@@ -84,17 +84,31 @@
 % adding as many as possible has the added benefit of making the thesis longer
 % and making me sound more sophisticated
 
+% the many flavors of T cells
+\newcommand{\tcellacronym}[4]{
+  \newacronym[shortplural={T\textsubscript{#2}#4
+    cells}]{#1}{T\textsubscript{#2}#4}{#3 T cell}
+}
+
 \renewcommand{\glossarysection}[2][]{} % remove glossary title
 \makeglossaries
+
+\tcellacronym{tn}{n}{naive}{}
+\tcellacronym{tcm}{cm}{central memory}{}
+\tcellacronym{tscm}{scm}{stem-memory}{}
+\tcellacronym{tem}{em}{effector-memory}{}
+\tcellacronym{teff}{eff}{effector}{}
+\tcellacronym{treg}{reg}{regulatory}{}
+\tcellacronym{th}{h}{helper}{}
+\tcellacronym{tc}{c}{cytotoxic}{}
+\tcellacronym{th1}{h}{type 1 helper}{1}
+\tcellacronym{th2}{h}{type 2 helper}{2}
+% \tcellacronym{th17}{h}{\il{17} helper}{1}
+
 \newacronym{til}{TIL}{tumor infiltrating lymphocyte}
 \newacronym{tcr}{TCR}{T cell receptor}
 \newacronym{act}{ACT}{adoptive cell therapies}
 \newacronym{qc}{QC}{quality control}
-\newacronym{tn}{T\textsubscript{n}}{naive T cell}
-\newacronym{tcm}{T\textsubscript{cm}}{central memory T cell}
-\newacronym{tscm}{T\textsubscript{scm}}{stem-memory T cell}
-\newacronym{tem}{T\textsubscript{em}}{effector-memory T cell}
-\newacronym{teff}{T\textsubscript{eff}}{effector T cell}
 \newacronym{car}{CAR}{chimeric antigen receptor}
 \newacronym[longplural={monoclonal antibodies}]{mab}{mAb}{monoclonal antibody}
 \newacronym{ecm}{ECM}{extracellular matrix}
@@ -190,6 +204,7 @@
 \newacronym{cstr}{CSTR}{continuously stirred tank bioreactor}
 \newacronym{esc}{ESC}{embryonic stem cell}
 \newacronym{msc}{MSC}{mesenchymal stromal cells}
+\newacronym{scfv}{scFv}{single-chain fragment variable}
 
 %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
 % SI units for uber nerds
@@ -236,6 +251,9 @@
 \newcommand{\invivo}{\textit{in vivo}}
 \newcommand{\invitro}{\textit{in vitro}}
 \newcommand{\exvivo}{\textit{ex vivo}}
+\newcommand{\Invivo}{\textit{In vivo}}
+\newcommand{\Invitro}{\textit{In vitro}}
+\newcommand{\Exvivo}{\textit{Ex vivo}}
 
 % various CD-whatever crap
 \newcommand{\cd}[1]{CD{#1}}
@@ -577,39 +595,40 @@ conclusions as well as provide insights for how this work can be extended in the
 future.
 
 \chapter{background and significance}\label{background}
+
 \section*{background}
 
 \subsection{quality by design in cell manufacturing}
 
-The challenges facing the cell manufacturing field at large are significant.
+The challenges for the cell manufacturing field are significant. Unlike other
-Unlike other industries which manufacture inanimate products such as automobiles
+industries which manufacture inanimate products such as automobiles and
-and semiconductors, the cell manufacturing industry needs to contend with the
+semiconductors, the cell manufacturing industry needs to contend with the fact
-fact that cells are living entities which can change with every process
+that cells are living entities which can change with every process
 manipulation\cite{Kirouac2008, Little2006, Pirnay2012, Rousseau2013}. This is
 further compounded by the lack of standardization and limited regulation.
 
 In order to overcome these barriers, adopting a systemic approach to cell
-manufacturing using \gls{qbd} principles will be extremely
+manufacturing using \acrlong{qbd} principles will be extremely
 important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly
-manufacturing products which minimizes risk for downstream
+manufacturing products which minimizes risk for downstream stakeholders (in this
-stakeholders (in this case, the patient).
+case, the patient). Broadly, this entails determining \acrlongpl{cqa} and
+\acrlongpl{cpp} and incorporating them into models which can explain and predict
+the cell manufacturing process.
 
-This requires identification of \glspl{cqa}, which are measurable properties of
+\Glspl{cqa} are measurable properties of the product that can be used to define
-the product that can be used to define its functionality and hence quality.
+its functionality and hence quality. \glspl{cqa} are important for defining the
-\glspl{cqa} are important for defining the characteristics of a `good' product
+characteristics of a `good' product (release criteria) but also for ensuring
-(release criteria) but also for ensuring that a process is on track to making
+that a process is on track to making such a product (process control). In the
-such a product (process control). In the space of cell manufacturing,
+space of cell manufacturing, examples of \glspl{cqa} include markers on the
-examples of \glspl{cqa} include markers on the surface of cells and readouts
+surface of cells and readouts from functional assays such as killing assays. In
-from functional assays such as killing assays. In general, these are poorly
+general, these are poorly understood if they exist at all.
-understood if they exist at all.
 
-In addition to \glspl{cqa}, the \glspl{cpp} pertinent to the manufacturing
+\glspl{cpp} are parameters which may be tuned and varied to control the outcome
-process are poorly understood. \glspl{cpp} are parameters which may be tuned and
+of process and the quality of the final product. In cell manufacturing, these
-varied to control the outcome of process and the quality of the final product.
+are poorly understood. Examples in the cell manufacturing space include the type
-Examples in the cell manufacturing space include the type of media used and the
+of media used and the amount of \il{2} added. Once \glspl{cpp} are known, they
-amount of \il{2} added. Once \glspl{cpp} are known, they can be optimized to
+can be optimized to ensure that costs are minimized and potency of the cellular
-ensure that costs are minimized and potency of the cellular product is
+product is maximized.
-maximized.
 
 The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of
 this work are discussed further in \cref{sec:background_doe} and
@@ -617,30 +636,39 @@ this work are discussed further in \cref{sec:background_doe} and
 
 \subsection{T cells for immunotherapies}
 
+A variety of T cell therapies have been utilized with varying degrees of
+success, and we describe a few of the most prominent below. We should note that
+while this work focuses on the application of \gls{car} T cell therapies, in
+theory the technology developed in this dissertation could theoretically apply
+to any T cell-based therapy with little to no modification.
+
 One of the first successful T cell-based immunotherapies against cancer is
 \glspl{til}\cite{Rosenberg2015}. This method works by taking tumor specimens
 from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{}, and
-then administered back to the patient along with a high dose of \il{2} [44]. In
+then administered back to the patient along with a high dose of
-particular, \gls{til} therapy has shown robust results in treating melanoma [1],
+\il{2}\cite{Rosenberg1988}. In particular, \gls{til} therapy has shown robust
-although \gls{til} have been found in other solid tumors such as
+results in treating melanoma\cite{Rosenberg2011}, although \glspl{til} have been
-gastointestinal, cervical, lung, and ovarian\cite{Rosenberg2015, Wang2014,
+found in other solid tumors such as gastointestinal, cervical, lung, and
-  Foppen2015, Solinas2017, June2007, Santoiemma2015}, and their presence is
+ovarian\cite{Rosenberg2015, Wang2014, Foppen2015, Solinas2017, June2007,
-generally associate with favorable outcomes\cite{Clark1989}. \glspl{til} are
+  Santoiemma2015}, and their presence is generally associate with favorable
-heterogenous cell mixtures and generally are comprised of CD3 T cells and
+outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and
-$\upgamma\updelta$ T cells\cite{Nishimura1999, Cordova2012}. To date, there are
+generally are comprised of CD3 T cells and $\upgamma\updelta$ T
-over 250 open clinical trials using \glspl{til}.
+cells\cite{Nishimura1999, Cordova2012}. To date, there are over 250 open
+clinical trials using \glspl{til}.
 
-Besides \gls{til}, the other broad class of T cell immunotherapies that has
+Besides \glspl{til}, the other broad class of T cell immunotherapies that has
 achieved great success in treating cancer in recent decades are gene-modified T
 cells. Rather than expand T cells that are present natively (as is the case with
 \gls{til} therapy), gene-modified T cell therapies entail extracting T cells
 from either the cancer patient (autologous) or a healthy donor (allogeneic) and
 reprogramming them genetically to target a tumor antigen (see
-\cref{sec:background_source}). In theory this offers much more
+\cref{sec:background_source} for an overview of how T cells can be sourced).
-flexibility\cite{Rosenberg2015}.
+This approach offers much more flexibility, as the degree of reprogramming is
+only limited by the scale and possibilities of gene-editing technology, which
+has rapidly accelerated in recent decades\cite{Rosenberg2015}.
 
 T cells with transduced \glspl{tcr} were first designed to overcome the
-limitations of \gls{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
+limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
 are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr}
 targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
 results in melanoma patients\cite{Robbins2011}, synovial
@@ -649,147 +677,35 @@ sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over
 
 While transduced \glspl{tcr} offer some flexibility in retargeting T cells
 toward relevant tumor antigens, they are still limited in that they can only
-target antigens that are presented via \gls{mhc} complexes. \gls{car} T cells
+target antigens that are presented via \gls{mhc} complexes. \acrlong{car} T
-overcome this limitation by using a the heavy and light chains (scFv) from a
+cells overcome this limitation by using linking a \gls{tcr}-independent antigen
-\gls{mab} which can target any antigen recognizable by antibodies. \gls{car} T
+recognition domain with the stimulatory and costimulatory machinery of a T cell
-cells were first demonstrated in 1989, where the author swapped the
+\gls{car} T cells were first demonstrated in 1989, where the authors swapped the
 antigen-recognition domains of a native \gls{tcr} with a that of a foreign
 \gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
-scFv with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or 4-1BB
+\gls{scfv} with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or
-domains for costimulation. Since these can all be expressed with one protein
+4-1BB domains for costimulation. Since these can all be expressed with one
-sequence, \gls{car} T cells are relatively simple to produce and require only a
+protein sequence, \gls{car} T cells are relatively simple to produce and require
-single genetic transduction step (usually a lentiviral vector) to reprogram a
+only a single genetic transduction step (usually a lentiviral vector) to
-batch T cells \exvivo{} toward the desired antigen. \gls{car} T cells have
+reprogram a batch T cells \exvivo{} toward the desired antigen. \gls{car} T
-primarily found success in against CD19- and CD20-expressing tumors such as
+cells have primarily found success in against CD19- and CD20-expressing tumors
-\gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011, Brentjens2011,
+such as \gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011,
-  Kochenderfer2010, Maude2014, Till2012, Till2008}.
+  Brentjens2011, Kochenderfer2010, Maude2014, Till2012, Till2008}.
 
-% BACKGROUND where else have they been approved?
 Out of all the T cell therapies discussed thus far, \gls{car} T cells have
 experienced the most commercial success and excitement. In 2017, Novartis and
 Kite Pharma acquired FDA approval for \textit{Kymriah} and \textit{Yescarta}
 respectively, both of which are \gls{car} T cell therapies against B-cell
-malignancies.
+malignancies. \gls{car} T cells are under further exploration for use in many
-% BACKGROUND beef this up, this is a big deal
+other tumors, including multiple myeloma, mesothelioma, pancreatic cancer,
-\gls{car} T cells are under further exploration for use in many other tumors,
+glioblastoma, neuroblastoma, and prostate cancer, breast cancer, non-small-cell
-including multiple myeloma, mesothelioma, pancreatic cancer, glioblastoma,
+lung cancer, and others\cite{Rosenberg2015, Wang2014, Fesnak2016, Guo2016}. To
-neuroblastoma, and prostate cancer, breast cancer, non-small-cell lung cancer,
+date, there are almost 1000 clinical trials using \gls{car} T cells.
-and others\cite{Rosenberg2015, Wang2014, Fesnak2016, Guo2016}. To date, there
-are almost 1000 clinical trials using \gls{car} T cells.
 
 % TODO there are other T cells like virus-specific T cells and gd T cells, not
 % that they matter...
 
-\subsection{cell sources in T cell manufacturing}\label{sec:background_source}
+\subsection{scaling T cell expansion}
-
-T cells for cell manufacturing can be obtained broadly via two paradigms:
-autologous and allogeneic. The former involves obtaining T cells from a patient
-and giving them back to the same patient after \exvivo{} expansion and genetic
-modification. The latter involves taking T cells from a (usually) healthy donor,
-expanding them and manipulating them as desired, storing them long term, and
-then giving them to multiple patients. There are advantages and disadvantages to
-both, and in some cases such as \gls{til} therapy, the only option is to use
-autologous therapy.
-
-Autologous T cells by default are much safer. By definition, they will have no
-cross-reactivity with the patient and thus \gls{gvhd} is not a
-concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
-therapies are over 20X more costly as the process needs to be repeated for every
-patient\cite{Harrison2019}. To highlight how resource-intensive this can be,
-many cell products are manufactured at a centralized location, so patient T
-cells need to be shipped twice on dry ice from the hospital and back. In
-additional to being expensive, this can add days to the process, which is
-critical for patients with fast moving diseases. Manufacturing could be done
-on-site in a decentralized manner, but this requires more equipment and
-personnel overall. Using cells from a diseased patient has many drawbacks in
-itself. Cancer patients (especially those with chronic illnesses) often have
-exhausted T cells which expand far less readily and are consequently less
-potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high
-frequencies of T\textsubscript{reg} cells which inhibitory\cite{Decker2012}.
-Removing these cells as well as purifying Th1 cells may enhance the potency of
-the final product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013,
-  Grotz2015}; however, this would make the overall process more expensive as an
-additional step would be required.
-
-Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
-\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
-they can be stored easily under liquid nitrogen, and donors could be screened to
-find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
-Obviously this could be done the same way as done for transplants where patients
-find a `match' for their \gls{hla} type, but this severally limits options. This
-can be overcome by using gene-editing (eg \glspl{zfn}, \glspl{talen}, or
-\gls{crispr} to remove the native \gls{tcr} which would prevent the donor T
-cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012,
-  Berdien2014, Themeli2015}. To date there are about 10 open clinical trials
-utilizing allogeneic T cell therapies edited with \gls{crispr} to reduce the
-likelihood of \gls{gvhd}.
-
-\subsection{microcarriers in bioprocessing}
-
-Microcarriers have historically been used to grow a number of adherent cell
-types for a variety of applications. They were introduced in 1967 as a means to
-grow adherent cells `in suspension', effectively turning a 2D flask system into
-a 3D culture system\cite{WEZEL1967}. Microcarriers are generally spherical and
-are several hundred \si{\um} in diameter, which means they collectively have a
-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
-\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
-choice of material and macroporous structure. Key concerns have been cell
-attachment at the beginning of culture and cell detachment at the harvesting
-step; these have largely driven the nature of the material and structures
-employed\cite{Derakhti2019}. Many microcarriers simply use polystyrene (the
-material used for tissue culture flasks and dishes in general) with no
-modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
-(SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
-Fact III). Other base materials have been used such as dextran (GE Healthcare
-Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767),
-all with none or similar surface modifications. Additionally, some microcarriers
-such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
-cases, porcine collagen) which also allows the microcarriers to be enzymatically
-degraded. In the case of non-protein materials, cells may still be detached
-using enzymes but these require similar methods to those currently used in
-flasks such as trypsin which target the cellular \gls{ecm} directly. Since
-trypsin and related enzymes tends to be harsh on cells, an advantage of using
-entirely protein-based microcarriers is that they can be degraded using a much
-safer enzyme such as collagenase, at the cost of being more expensive and also
-being harder to make \gls{gmp}-compliant\cite{Derakhti2019}. Going one step
-further, some microcarriers are composed of a naturally degrading scaffold such
-as alginate, which do not need an enzyme for degradation but are limited in that
-the degradation process is less controllable. Finally, microcarriers can differ
-in their overall structure. \gls{cug} and \gls{cus} microcarriers as well as the
-Cytopore microcarriers are macroporous, meaning they have a porous network in
-which cells can attach throughout their interior. This drastically increases the
-effective surface area and consequently the number of cells which may be grown
-per unit volume. Other microcarriers are microporous (eg only to small
-molecules) or not porous at all (eg polystyrene) in which case the cells can
-only grow on the surface.
-
-Microcarriers in general have seen the most use in growing \gls{cho} cells and
-hybridomas in the case of protein manufacturing (eg \gls{igg}
-production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more
-recently in the case of cell manufacturing\cite{Heathman2015, Sart2011,
-  Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored
-using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell
-therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016,
-  Park2013, Malda2006}. However, the characteristic shared by all the cell types
-in this application is the fact that they are adherent. In this work, we explore
-the use of microcarrier for T cells, which are naturally non-adherent.
-
-The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the
-former) which are both composed of cross-linked gelatin and have a macroporous
-morphology. These specific carriers have been used in the past for pancreatic
-islet cells\cite{Guerra2001}, \glspl{esc}\cite{Fernandes2007, Storm_2010}, and
-\glspl{msc}\cite{Eibes2010}. Furthermore, they are readily available in over 30
-countries and are used in an FDA fast-track-approved combination retinal pigment
-epithelial cell product (Spheramine, Titan Pharmaceuticals)\cite{purcellmain}.
-This regulatory history will aid in clinical translation.
-
-\subsection{methods to scale T cells}
 
 In order to scale T cell therapies to meet clinical demands, automation and
 bioreactors will be necessary. To this end, there are several choices that have
@@ -799,8 +715,8 @@ The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical
 applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a
 broader class of bioreactors that consist of rocking platforms that agitate a
 bag filled with media and cells. Importantly, it has built-in sensors for
-measuring media flow rate, carbon dioxide, oxygen, pH, and nutrient consumption
+measuring media flow rate, \ce{CO2}, \ce{O2}, pH, and nutrient consumption which
-which enables automation. Unfortunately, in some settings this is not considered
+enables automation. Unfortunately, in some settings this is not considered
 scalable as only one bag per bioreactor is allowed at once\cite{Roddie2019}. The
 other disadvantage with the WAVE system is that it keeps cells far apart by
 design, which could have negative impact on cross-talk, activation, and
@@ -830,6 +746,56 @@ operator to load, feed, and harvest the cell product. Grex bioreactors have been
 using to grow \glspl{til}\cite{Jin2012} and virus-specific T
 cells\cite{Gerdemann2011}.
 
+Much work is still required in the space of bioreactor design for T cell
+manufacturing, but novel T cell expansion technologies such as that described in
+this work need to consider how it may be used at scale in such a system.
+
+\subsection{cell sources in T cell manufacturing}\label{sec:background_source}
+
+T cells for cell manufacturing can be obtained broadly via two paradigms:
+autologous and allogeneic. The former involves obtaining T cells from a patient
+and giving them back to the same patient after \exvivo{} expansion and genetic
+modification. The latter involves taking T cells from a healthy donor, expanding
+them and manipulating them as desired, storing them long term, and then giving
+them to multiple patients. There are advantages and disadvantages to both, and
+in some cases such as \gls{til} therapy, the only option is to use autologous
+therapy.
+
+Autologous T cells by default are much safer. By definition, they will have no
+cross-reactivity with the patient and thus \gls{gvhd} is not a
+concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
+therapies are over 20 times more costly as the process needs to be repeated for
+every patient\cite{Harrison2019}. Compounding this, many cell products are
+manufactured at a centralized location, so patient T cells need to be shipped
+twice on dry ice from the hospital and back. This adds days to the process,
+which is critical for patients with fast moving diseases. Manufacturing could be
+done on-site in a decentralized manner, but this requires more equipment and
+personnel overall. Using cells from a diseased patient has many drawbacks in
+itself. Cancer patients (especially those with chronic illnesses) often have
+exhausted T cells which expand far less readily and are consequently less
+potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high
+frequencies of \glspl{treg} which inhibitory\cite{Decker2012}. Removing these
+cells as well as purifying \glspl{th1} may enhance the potency of the final
+product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013, Grotz2015};
+however, this would make the overall process more expensive as an additional
+separation step would be required.
+
+Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
+\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
+they can be stored easily under liquid nitrogen, and donors could be screened to
+find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
+Obviously this could be done the same way as done for transplants where patients
+find a `match' for their \gls{hla} type, but this severally limits options. This
+can be overcome by using advanced gene-editing tools which can both add and
+delete genetic material (eg \glspl{zfn}, \glspl{talen}, or \gls{crispr}) to
+remove the native \gls{tcr} which would prevent the donor T cells from attacking
+host tissue\cite{Liu2019, Wiebking2020, Provasi2012, Berdien2014, Themeli2015}.
+This obviously complicates the process, as additional edits besides the
+insertion of the \gls{car} would be required, and these technologies are not yet
+very efficient. To date there are about 10 open clinical trials utilizing
+allogeneic T cell therapies edited with \gls{crispr} to reduce the likelihood of
+\gls{gvhd}.
+
 \subsection{overview of T cell quality}\label{sec:background_quality}
 
 T cells are highly heterogeneous and can exist in a variety of states and
@@ -840,39 +806,39 @@ criteria, and initial cell source screening.
 
 One of the most important dimensions of T cell quality is that of
 differentiation. T cells begin their life in circulation (eg after they exit the
-thymus) as naive T cells. When they become activated in the secondary lymph node
+thymus) as \glspl{tn}. When they become activated in the secondary lymph node
-organs, they differentiate from \gls{tn} to \gls{tscm}, \gls{tcm}, \gls{tem},
+organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm},
-and finally \gls{teff}\cite{Gattinoni2012}. Subtypes earlier in this process are
+\glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in
-generally called `memory' or `memory-like' cells (eg \gls{tscm} and \gls{tcm}),
+this process are generally called `memory' or `memory-like' cells (eg \gls{tscm}
-and have been shown to have increased potency toward a variety of tumors,
+and \gls{tcm}), and have been shown to have increased potency toward a variety
-presumably due to their higher capacity for self-renewal and replication,
+of tumors, presumably due to their higher capacity for self-renewal and
-enhanced migratory capacity, and/or increased engraftment potential\cite{Xu2014,
+replication, enhanced migratory capacity, and/or increased engraftment
-  Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}. The capacity for
+potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}.
-self-renewal is especially important for T cells therapies, as evidenced by the
+The capacity for self-renewal is especially important for T cells therapies, as
-fact that \gls{til} therapies with longer telomeres tend to work
+evidenced by the fact that \gls{til} therapies with longer telomeres tend to
-better\cite{Donia2012}. Additionally, clonal diversity decreases following the
+create more durable responses\cite{Donia2012}. Additionally, clonal diversity
-infusion of \gls{car} T cell therapies, which demonstrates that only a few
+decreases following the infusion of \gls{car} T cell therapies, which
-clones are self-renewing and therefore responsible for the overall
+demonstrates that only a few clones are self-renewing and therefore responsible
-response\cite{Sheih2020}. Memory T cells can be quantified easily using surface
+for the overall response\cite{Sheih2020}. Memory T cells can be quantified
-markers such as CD62L, CCR7, CD27, CD45RA, and CD45RO. Furthermore, memory
+easily using surface markers such as CD62L, CCR7, CD27, CD45RA, and CD45RO.
-markers are inversely related to exhaustion markers which are negatively
+Furthermore, memory markers are inversely related to exhaustion markers which
-associated with clinical outcomes\cite{Lee2013}. These cells in particular are
+are negatively associated with clinical outcomes\cite{Lee2013}. These cells in
-seen in patients with chronic immune activation such as patients with chronic
+particular are seen in patients with chronic immune activation such as patients
-cancers.
+with chronic cancers.
 
 In addition to memory, the other major axis by which T cells may be classified
-is the CD4/CD8 ratio. CD4 (`helper') T cells are responsible for secreting
+is the CD4/CD8 ratio. \Glspl{th} are CD4+ are responsible for secreting
-cytokines which coordinate the immune response while CD8 (`killer') T cell
+cytokines which coordinate the immune response while CD8+ \glspl{tc} are
-responsible for killing tumor or infected cells using specialized lytic enzymes.
+responsible for killing tumors or infected cells using specialized lytic
-Since CD8 T cells actually perform the killing function, it seems intuitive that
+enzymes. Since \glspl{tc} actually perform the killing function, it seems
-CD8 T cells would be most important for anti-tumor immunotherapies. However, in
+intuitive that \glspl{tc} would be most important for anti-tumor
-mouse models with glioblastoma, survival was negatively impacted when CD4 T
+immunotherapies. However, in mouse models with glioblastoma, survival was
-cells were removed\cite{Wang2018}. Furthermore, CD4 T cells have been shown to
+negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore,
-have cytotoxic properties on their own and also show resistance to T cell
+\glspl{th} have been shown to have cytotoxic properties on their own and also
-exhaustion compared to CD8 T cells\cite{Yang2017}. While T cell products with a
+show resistance to exhaustion compared to \glspl{tc}\cite{Yang2017}. While T
-defined ratio of CD4 and CD8 T cells have been utilized, they are more expensive
+cell products with a defined ratio of CD4 and CD8 T cells have been utilized,
-than products with undefined ratios as the T cells need to be sorted and
+they are more expensive than products with undefined ratios as the T cells need
-recombined, adding additional complexity\cite{Turtle2016}.
+to be sorted and recombined, adding additional complexity\cite{Turtle2016}.
 
 While less of a focus in this dissertation, other quality markers exists to
 assess the overall killing potential and safety of the T cell product. Numerous
@@ -887,14 +853,10 @@ using retro- or lentiviral vectors as their means of gene-editing must be tested
 for replication competent vectors\cite{Wang2013} and for contamination via
 bacteria or other pathogens.
 
-\subsection*{T cell activation}
+\subsection*{T cell activation methods}\label{sec:background_activation}
-
-% Despite these success of T cell therapies (especially \gls{car} T cell
-% therapies) they are constrained by an expensive and difficult-to-scale
-% manufacturing process\cite{Roddie2019, Dwarshuis2017}.
 
 In order for T cells to be expanded \exvivo{} they must be activated with a
-stimulatory signal (Signal 1) and a costimulatory signal (Signal 2). \invivo{}
+stimulatory signal (Signal 1) and a costimulatory signal (Signal 2). \Invivo{},
 Signal 1 is administered via the \gls{tcr} and the CD3 receptor when \glspl{apc}
 present a peptide via \gls{mhc} that the T cell in question is able to
 recognize. Signal 2 is administered via CD80 and CD86 which are also present on
@@ -906,12 +868,13 @@ and \glspl{apc} have corresponding ligands for these depending on the nature of
 the pathogen they have detected\cite{Azuma2019}. Furthermore, T cells exist in
 high cell density within the secondary lymphoid organs, which allows efficient
 cytokine cross-talk in an autocrine and paracrine manner. These cytokines are
-responsible for expansion (in the case of \il{2}) and subset differentiation (in
+responsible for triggering proliferation (in the case of \il{2}) and subset
-the case of many others)\cite{Luckheeram2012}. By tuning the signal strength and
+differentiation (in the case of many others)\cite{Luckheeram2012}. By tuning the
-duration of Signal 1, Signal 2, the various costimulatory signals, and the
+signal strength and duration of Signal 1, Signal 2, the various costimulatory
-cytokine milieu, a variety of T cell phenotypes can be actualized.
+signals, and the cytokine milieu, a variety of T cell phenotypes can be
+actualized.
 
-\invitro{}, T cells can be activated in a number of ways but the simplest and
+\Invitro{}, T cells can be activated in a number of ways but the simplest and
 most common is to use \glspl{mab} that cross-link the CD3 and CD28 receptors,
 which supply Signal 1 and Signal 2 without the need for antigen (which also
 means all T cells are activated and not just a few specific clones). Additional
@@ -921,21 +884,21 @@ or feeder cells\cite{Forget2014}.
 As this is a critical unit operation in the manufacturing of T cell therapies, a
 number of commercial technologies exist to activate T cells\cite{Wang2016,
   Piscopo2017, Roddie2019, Bashour2015}. The simplest is to use \acd{3} and
-\acd{28} \gls{mab} bound to a 2D surface such as a plate, and this can be
+\acd{28} \glspl{mab} bound to a 2D surface such as a plate, and this can be
-ackomplished in a \gls{gmp} manner as soluble \gls{gmp}-grade \glspl{mab} are
+accomplished in a \gls{gmp} manner (at least from a reagents perspective) as
-commericially available. A similar but distinct method along these lines is to
+soluble \gls{gmp}-grade \glspl{mab} are commericially available. A similar but
-use multivalent activators such as ImmunoCult (StemCell Technologies) or Expamer
+distinct method along these lines is to use multivalent activators such as
-(Juno Therapeutics) which may have increased cross-linking capacity compared to
+ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which may have
-traditional \glspl{mab}. Beyond soluble protein, \glspl{mab} against CD3 and
+increased cross-linking capacity compared to traditional \glspl{mab}. Beyond
-CD28 can be mounted on magnetic microbeads (\SIrange{3}{5}{\um} in diameter)
+soluble protein, \glspl{mab} against CD3 and CD28 can be mounted on magnetic
-such as DynaBeads (Invitrogen) and MACSbeads (\miltenyi{}), which are easier to
+microbeads (\SIrange{3}{5}{\um} in diameter) such as DynaBeads (Invitrogen) and
-separate using magnetic washing plates. Magnetic nanobeads such as TransAct
+MACSbeads (\miltenyi{}), which are easier to separate using magnetic washing
-(\miltenyi{}) work by a similar principle except they can be removed via
+plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar
-centrifugation rather than a magnetic washing plate. Cloudz (RnD Systems) are
+principle except they can be removed via centrifugation rather than a magnetic
-another bead-based T cell expansion which mounts \acd{3} and \acd{28}
+washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion
-\glspl{mab} on alginate microspheres, which are not only easily degradable but
+which mounts \acd{3} and \acd{28} \glspl{mab} on alginate microspheres, which
-are also softer, which can have a positive impact on T cell activation and
+are not only easily degradable but are also softer, which can have a positive
-phenotype\cite{Lambert2017, OConnor2012}.
+impact on T cell activation and phenotype\cite{Lambert2017, OConnor2012}.
 
 A problem with all of these commercial solutions is that they only focus on
 Signal 1 and Signal 2 and ignore the many other physiological cues present in
@@ -952,9 +915,78 @@ microrods\cite{Cheung2018}, 3D-scaffolds via either Matrigel\cite{Rio2018} or
 \gls{mab}-conjugated \gls{pdms} beads\cite{Lambert2017} that respectively
 recapitulate the cellular membrane, large interfacial contact area,
 3D-structure, or soft surfaces T cells normally experience \textit{in vivo}.
-While these are in theory much easier to produce and \gls{qc} compared to feeder
+None have been demonstrated to demonstrably expand high quality T cells as
-cells, none have been demonstrated to demonstrably expand high quality T cells
+outlined in \cref{sec:background_quality}.
-as outlined in \cref{sec:background_quality}.
+
+\subsection{microcarriers in bioprocessing}
+
+In this work, we explored microcarriers as the basis for an alternative to the
+methods described in \cref{sec:background_activation}.
+
+Microcarriers have historically been used to grow a number of adherent cell
+types for a variety of applications. They were introduced in 1967 as a means to
+grow adherent cells `in suspension', effectively turning a 2D flask system into
+a 3D culture system\cite{WEZEL1967}. Microcarriers are generally spherical and
+are several hundred \si{\um} in diameter, which means they collectively have a
+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
+\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
+choice of material and macroporous structure. Key concerns driving these
+choiceshave been cell attachment at the beginning of culture and cell detachment
+at the harvesting step\cite{Derakhti2019}. Many microcarriers simply use
+polystyrene (the material used for tissue culture flasks and dishes in general)
+with no modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface
+charge (SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen
+(SoloHill Fact III). Other base materials have been used such as dextran (GE
+Healthcare Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{}
+G2767), all with none or similar surface modifications. Additionally, some
+microcarriers such as \gls{cus} and \gls{cug} are composed entirely out of
+protein (in these cases, porcine collagen) which also allows the microcarriers
+to be enzymatically degraded. In the case of non-protein materials, cells may
+still be detached using enzymes but these require similar methods to those
+currently used in flasks such as trypsin which target the cellular \gls{ecm}
+directly. Since trypsin and related enzymes tends to be harsh on cells, an
+advantage of using entirely protein-based microcarriers is that they can be
+degraded using a much safer enzyme such as collagenase, at the cost of being
+more expensive and also being harder to make
+\gls{gmp}-compliant\cite{Derakhti2019}. Going one step further, some
+microcarriers are composed of a naturally degrading scaffold such as alginate,
+which do not need an enzyme for degradation. Finally, microcarriers can differ
+in their overall structure. \gls{cug} and \gls{cus} microcarriers as well as the
+Cytopore microcarriers are macroporous, meaning they have a porous network in
+which cells can attach throughout their interior. This drastically increases the
+effective surface area and consequently the number of cells which may be grown
+per unit volume. Other microcarriers are microporous (eg only to small
+molecules) or not porous at all (eg polystyrene); in either case the cells can
+only grow on the surface.
+
+Microcarriers in general have seen the most use in growing \gls{cho} cells and
+hybridomas in the case of protein manufacturing (eg \gls{igg}
+production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more
+recently in the case of cell manufacturing\cite{Heathman2015, Sart2011,
+  Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored
+using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell
+therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016,
+  Park2013, Malda2006}. However, the characteristic shared by all the cell types
+in this application is the fact that they are adherent. In this work, we explore
+the use of microcarrier for T cells, which are naturally non-adherent.
+
+The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the
+former) which are both composed of cross-linked gelatin and have a macroporous
+morphology. Their protein-based composition makes functionalization easy; the
+surface is rich in lysine residues which can be easily bonded with a
+base-reactive linker such as \gls{snb}. These specific carriers have been used
+in the past for pancreatic islet cells\cite{Guerra2001},
+\glspl{esc}\cite{Fernandes2007, Storm_2010}, and \glspl{msc}\cite{Eibes2010}.
+Furthermore, they are readily available in over 30 countries and are used in an
+FDA fast-track-approved combination retinal pigment epithelial cell product
+(Spheramine, Titan Pharmaceuticals)\cite{purcellmain}. This regulatory history
+will aid in clinical translation.
 
 \subsection*{integrins and T cell signaling}
 
@@ -1007,22 +1039,23 @@ components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or
 \il{15R$\upalpha$} \invitro{} has been shown to inhibit homeostatic
 proliferation of resting human T cells\cite{MirandaCarus2005}.
 
+% ACRO fix the il2R and IL15R stuff
 \il{15} has been puzzling historically because it shares almost the same pathway
 as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994,
-  Giri1995}. In particular, both cytokines share the common gamma subchain
+  Giri1995}. In particular, both cytokines share the common $\upgamma$ subchain
 (CD132) as well as the \il{2} $\upbeta$ receptor (CD122). The main difference in
 the heterodimeric receptors for \il{2} and \il{15} is the \il{2} $\upalpha$
-chain (CD25) and the \il{15} $\upalpha$ chain respectively, both of which have
+receptor (CD25) and the \il{15} $\upalpha$ chain respectively, both of which
-high affinity for their respective ligands. The \il{2R$\upalpha$} chain itself
+have high affinity for their respective ligands. The \il{2R$\upalpha$} chain
-does not have any signaling capacity, and therefore all the signaling resulting
+itself does not have any signaling capacity, and therefore all the signaling
-from \il{2} is mediated thought the $\upbeta$ and $\upgamma$ chains, namely via
+resulting from \il{2} is mediated thought the $\upbeta$ and $\upgamma$ chains,
-JAK1 and JAK3 leading to STAT5 activation consequently T cell activation.
+namely via JAK1 and JAK3 leading to STAT5 activation consequently T cell
-\il{15R$\upalpha$} itself has some innate signaling capacity, but this is poorly
+activation. \il{15R$\upalpha$} itself has some innate signaling capacity, but
-characterized in lymphocytes. Thus there is a significant overlap between the
+this is poorly characterized in lymphocytes\cite{Stonier2010}. Thus there is a
-functions of \il{2} and \il{15} due to their receptors sharing the $\upbeta$ and
+significant overlap between the functions of \il{2} and \il{15} due to their
-$\upgamma$ chains in their heterodimeric receptors, and perhaps the main driver
+receptors sharing the $\upbeta$ and $\upgamma$ chains in their heterodimeric
-of their differential functions it the half life of each respective
+receptors, and perhaps the main driver of their differential functions it the
-receptor\cite{Osinalde2014}.
+half life of each respective receptor\cite{Osinalde2014}.
 
 Where \il{15} is unique is that many (or possibly most) of its functions derive
 from being membrane-bound to its receptor\cite{Stonier2010}. Particularly,
@@ -1045,16 +1078,19 @@ described\cite{Budagian2004}.
 
 \subsection*{overview of design of experiments}\label{sec:background_doe}
 
-The \gls{dms} system has a number of parameters that can be optimized, and a
+The \gls{dms} system described in this dissertation has a number of parameters
-\gls{doe} is an ideal framework to test multiple parameters simultaneously. The
+that can be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal
-goal of \gls{doe} is to answer a data-driven question with the least number of
+framework to test multiple parameters simultaneously and determine which are
-resources\cite{Wu2009}. It was developed in many non-biological industries
+relevant \glspl{cpp}.
+
+The goal of \gls{doe} is to answer a data-driven question with the least number
+of resources\cite{Wu2009}. It was developed in many non-biological industries
 throughout the \nth{20} century such as the automotive and semiconductor
 industries where engineers needed to minimize downtime and resource consumption
 on full-scale production lines.
 
 At its core, a \gls{doe} is simply a matrix of conditions to test where each row
-is usually called a `run' and corresponds to one experimental unit to which the
+(usually called a `run') corresponds to one experimental unit for which the
 conditions are applied, and each column represents a parameter of concern to be
 tested. The values in each cell represent the level at which each parameter is
 to be tested. When the experiment is performed using this matrix of conditions,
@@ -1073,11 +1109,11 @@ principles:
 \begin{description}
 \item [randomization --] The order in which the runs are performed should
   ideally be as random as possible. This is to mitigate against any confounding
-  factors that may be present which depend on the order or position of the
+  factors that may be present which depend on the order or position of the runs.
-  experimental runs. For an example in context, the evaporation rate of media in
+  For an example in context, the evaporation rate of media in a tissue culture
-  a tissue culture plate will be much faster at the perimeter of the plate vs
+  plate will be much faster at the perimeter of the plate vs the center. While
-  the center. While randomization does not eliminate this bias, it will ensure
+  randomization does not eliminate this error, it will ensure the error is
-  the bias is `spread' evenly across all runs in an unbiased manner.
+  `spread' evenly across all runs in an unbiased manner.
 \item [replication --] Since the analysis of a \gls{doe} is inherently
   statistical, replicates should be used to ensure that the underlying
   distribution of errors can be estimated. While this is not strictly necessary
@@ -1108,14 +1144,14 @@ principles:
 \end{description}
 
 \Glspl{doe} served three purposes in this dissertation. First, we used them as
-screening tools, which allowed us to test many input parameters and filter out
+screening tools for potential \glspl{cpp}, which allowed us to test many input
-the few that likely have the greatest effect on the response. Second, they were
+parameters and filter out the few that likely have the greatest effect on the
-used to make a robust response surface model to predict optimums using
+response. Second, they were used to make a robust response surface model to
-relatively few resources, especially compared to full factorial or
+predict optimums using relatively few resources, especially compared to full
-one-factor-at-a-time approaches. Third, we used \glspl{doe} to discover novel
+factorial or one-factor-at-a-time approaches. Third, we used \glspl{doe} to
-effects and interactions that generated hypotheses that could influence the
+discover novel effects and interactions that generated hypotheses that could
-directions for future work. To this end, the types of \glspl{doe} we generally
+influence the directions for future work. To this end, the types of \glspl{doe}
-used in this work were fractional factorial designs with three levels, which
+we generally used were fractional factorial designs with three levels, which
 enable the estimation of both main effects and second order quadratic effects.
 
 \subsection*{identification and standardization of CPPs and
@@ -1175,7 +1211,7 @@ interesting cell types and the markers that define them.
 
 Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein
 putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be
-set and optimized to maximize products with these \glspl{cpp} and then
+set and optimized to maximize products with these \glspl{cpp}, and then
 additional data will be collected in the clinic as the product is tested on
 various patients with different indications. Additional \glspl{cqa} may be
 identified which better predict specific clinical outcomes, which can be fed
@@ -1183,10 +1219,8 @@ back into the process model and optimized again.
 
 \section{Innovation}
 
-\subsection{Innovation}
+Several aspects of the \gls{dms} platform described in this dissertation are
-
+novel considering the state-of-the-art technology for T cell manufacturing:
-Several aspects of this work are novel considering the state-of-the-art
-technology for T cell manufacturing:
 
 \begin{itemize}
 \item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic
@@ -1194,26 +1228,22 @@ technology for T cell manufacturing:
   the licenses for these techniques is controlled by only a few companies
   (Invitrogen and Miltenyi respectively). Because of this, bead-based expansion
   is more expensive to implement and therefore hinders companies from entering
-  the rapidly growing T cell manufacturing arena. Providing an alternative as we
+  the rapidly growing T cell manufacturing arena. Providing an alternative will
-  are doing will add more options, increase competition among both raw material
+  provide more options for manufacturers, leading to increased competition,
-  and T cell manufacturers, and consequently drive down cell therapy market
+  lower costs, and higher innovation in the T cell manufacturing space.
-  prices and increase innovation throughout the industry.
 \item This is the first technology for T cell immunotherapies that selectively
   expands memory T cell populations with greater efficiency relative to
   bead-based expansion Others have demonstrated methods that can achieve greater
   expansion of T cells, but not necessarily specific populations that are known
   to be potent.
-\item We propose to optimize our systems using \gls{doe} methodology, which is a
+\item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a
-  strategy commonly used in other industries and disciplines but has yet to gain
+  strategy commonly used in non-biological industries but has yet to gain wide
-  wide usage in the development of cell therapies. \Glspl{doe} are advantageous
+  usage in the development of cell therapies where research often employs a
-  as they allow the inspection of multiple parameters simultaneously, allowing
+  one-factor-at-a-time approach. We believe this method is highly relevant to
-  efficient and comprehensive analysis of the system vs a one-factor-at-a-time
+  the development of cell therapies, not only for process optimization but also
-  approach. We believe this method is highly relevant to the development of cell
+  hypotheses generation. Furthermore, it is a perfectly natural strategy to use
-  therapies, not only for process optimization but also hypotheses generation.
+  even at small scale, where the cost of reagents, cells, and materials often
-  Of further note, most \textit{in vivo} experiments are not done using a
+  precludes large sample sizes.
-  \gls{doe}-based approach; however, a \gls{doe} is perfectly natural for a
-  large mouse study where one naturally desires to use as few animals as
-  possible.
 \item The \gls{dms} system is be compatible with static bioreactors such as the
   G-Rex which has been adopted throughout the cell therapy industry. Thus this
   technology can be easily incorporated into existing cell therapy process that