diff --git a/tex/thesis.tex b/tex/thesis.tex index 5ecf4f5..c30bb19 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -569,6 +569,68 @@ present our final conclusions in Chapter~\ref{conclusions}. % TODO consider adding a separate section on microcarriers and their use in % bioprocess % TODO add stuff about T cell licensing + +\subsection{microcarriers in bioprocessing} + +% https://www.sciencedirect.com/science/article/pii/S0928493118338001#bb0010 + +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 (https://www.nature.com/articles/216064a0.pdf). +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 stirred tank bioreactor may +be employed to enhance oxygen transfer, maintain pH, and continuously supply +nutrients (). + +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. 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 (Sigma Aldrich 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. 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. + +% https://www.sciencedirect.com/science/article/pii/S0734975013000657 +% https://link.springer.com/article/10.1023/A:1008038609967 +% https://onlinelibrary.wiley.com/doi/full/10.1002/bit.23289 +Microcarriers have seen the most use in growing \gls{cho} cells and hybridomas +in the case of protein manufacturing (eg \gls{igg} production) as well as +pluripotent stem cells and mesenchymal stromal cells more recently in the case +of cell manufacturing\cite{Heathman2015, Sart2011}. Interestingly, some groups +have even explored using biodegradable microcarriers \invivo{} as a delivery +vehicle for stem cell therapies in the context of regenerative medicine. +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. + \subsection*{current T cell manufacturing technologies} \Gls{car} T cell therapy has received great interest from both academia and @@ -621,20 +683,6 @@ cytokine release properties and ability to resist exhaustion\cite{Wang2018, Yang2017}, and no method exists to preferentially expand the CD4 population compared to state-of-the-art systems. -Here we propose a method using microcarriers functionalized with \acd{3} and -\acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have -historically been used throughout the bioprocess industry for adherent cultures -such as stem cells and \gls{cho} cells, but not with suspension cells such as T -cells\cite{Heathman2015, Sart2011}. The carriers have a macroporous structure -that allows T cells to grow inside and along the surface, providing ample -cell-cell contact for enhanced autocrine and paracrine signaling. Furthermore, -the carriers are composed of gelatin, which is a collagen derivative and -therefore has adhesion domains that are also present within the lymph nodes. -Finally, the 3D surface of the carriers provides a larger contact area for T -cells to interact with the \glspl{mab} relative to beads; this may better -emulate the large contact surface area that occurs between T cells and -\glspl{dc}. - \subsection*{integrins and T cell signaling} Because the microcarriers used in this work are derived from collagen, one key