diff --git a/tex/references.bib b/tex/references.bib index cbc2c9b..196a489 100644 --- a/tex/references.bib +++ b/tex/references.bib @@ -2200,7 +2200,7 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec country = {United States}, issn-linking = {1538-4047}, issue = {6}, - keywords = {Acute Disease; Animals; Cell Line, Tumor; Cell Transplantation; Clone Cells; Collagen, metabolism; Culture Media, Conditioned, analysis; Cytokines, immunology; Drug Combinations; Infections, blood, immunology; Interferon-gamma, analysis, immunology; Interleukin-10, deficiency; Laminin, metabolism; Listeria monocytogenes, pathogenicity; Lymphocytic choriomeningitis virus, pathogenicity; Melanoma, Experimental, blood, immunology, pathology; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Neovascularization, Pathologic; Proteoglycans, metabolism; Th1 Cells, immunology; Time Factors; Toxoplasma, pathogenicity; Toxoplasmosis; Vascular Endothelial Growth Factor A, analysis, genetics, metabolism}, + keywords = {Acute Disease; Animals; Cell Line, Tumor; Cell Transplantation; Clone Cells; Collagen, metabolism; Culture Media, Conditioned, analysis; Cytokines, immunology; Drug Combinations; Infections, blood, immunology; Interferon-gamma, analysis, immunology; Interleukin-10, deficiency; Laminin, metabolism; Listeria monocytogenes, pathogenicity; Lymphocytic choriomeningitis virus, pathogenicity; Melanoma, Experimental, immunology, pathology; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Neovascularization, Pathologic; Proteoglycans, metabolism; Th1 Cells, immunology; Time Factors; Toxoplasma, pathogenicity; Toxoplasmosis; Vascular Endothelial Growth Factor A, genetics, metabolism}, nlm-id = {101137842}, owner = {NLM}, pii = {557}, @@ -2463,6 +2463,18 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec url = {https://www.ebook.de/de/product/8324352/wu\_hamada\_experiments\_2e.html}, } +@Article{Lam2020, + author = {Norris Lam and Nathan D. Trinklein and Benjamin Buelow and George H. Patterson and Namrata Ojha and James N. Kochenderfer}, + journal = {Nature Communications}, + title = {Anti-{BCMA} chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains}, + year = {2020}, + month = {jan}, + number = {1}, + volume = {11}, + doi = {10.1038/s41467-019-14119-9}, + publisher = {Springer Science and Business Media {LLC}}, +} + @Comment{jabref-meta: databaseType:bibtex;} @Comment{jabref-meta: grouping: diff --git a/tex/thesis.tex b/tex/thesis.tex index d70e7ee..134cc49 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -162,6 +162,7 @@ \newacronym{talen}{TALEN}{transcription activator-like effector nuclease} \newacronym{qbd}{QbD}{quality-by-design} \newacronym{aws}{AWS}{amazon web services} +\newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds @@ -243,6 +244,7 @@ \newcommand{\catnum}[2]{(#1, #2)} \newcommand{\product}[3]{#1 \catnum{#2}{#3}} \newcommand{\thermo}{Thermo Fisher} +\newcommand{\sigald}{Sigma Aldrich} \newcommand{\miltenyi}{Miltenyi Biotech} \newcommand{\bl}{Biolegend} \newcommand{\bd}{Becton Dickenson} @@ -743,7 +745,7 @@ 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), +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 @@ -1281,7 +1283,7 @@ two washes were done and the agitation time was \SI{30}{\minute}. Biotinylated \glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904} were combined in a 1:1 mass ratio and added to the carriers at \SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile -\product{\gls{bsa}}{Sigma}{A9576} was added to a final concentration of +\product{\gls{bsa}}{\sigald}{A9576} was added to a final concentration of \SI{2}{\percent} in order to prevent non-specific binding of the antibodies to the reaction tubes. \glspl{mab} were allowed to bind to the carriers for \SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were @@ -1299,12 +1301,12 @@ was then manually counted to obtain a concentration. Surface area for \subsection{dms quality control assays} -Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In -the case of quantifying \gls{snb} prior to adding it to the microcarriers, the -sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH -and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester -linkages from binding to the avidin proteins in the \gls{haba}/avidin premix. -All quantifications of \gls{haba} were performed on an Eppendorf D30 +Biotin was quantified using the \product{\gls{haba} assay}{\sigald}{H2153-1VL}. +In the case of quantifying \gls{snb} prior to adding it to the microcarriers, +the sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} +NaOH and allowed to react for \SI{1}{\minute} in order to prevent the reactive +ester linkages from binding to the avidin proteins in the \gls{haba}/avidin +premix. All quantifications of \gls{haba} were performed on an Eppendorf D30 Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be \SI{34000}{\per\cm\per\molar}. @@ -1457,25 +1459,52 @@ Briefly, cells were washed once and stained with \product{biotinylated BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls (\gls{pe}-\gls{stp} with no \gls{ptnl}). -% TODO add BCMA-CAR stuff \subsection{car plasmid and lentiviral transduction} -The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$ +The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} with the EF1$\upalpha$ promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a \product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core. Lentiviral vectors were synthesized by the Emory Viral Vector Core or the -Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce -primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto -non-TC treated 96 well plates and used to immobilize lentiviral vector particles -according to the manufacturer's instructions. Briefly, retronectin solution was -adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using -\gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at -\SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were -activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and -then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated -plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms} -were removed from the retronectin plates using vigorous pipetting and -transferred to another 96 well plate wherein expansion continued. +Cincinnati Children's Hospital Medical Center Viral Vector Core. RNA titer was +calculated using a \product{Lenti-X \gls{qpcr} titer kit}{Takara}{631235}. To +transduce primary human T cells, \product{retronectin}{Takara}{T100A} was coated +onto non-TC treated 96 well plates and used to immobilize lentiviral vector +particles according to the manufacturer's instructions. Briefly, retronectin +solution was adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next +day using \gls{bsa}. Prior to transduction, lentiviral supernatant was +spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. +T cells were activated in 96 well plates using beads or \glspl{dms} for +\SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred onto +lentiviral vector coated plates and incubated for another \SI{24}{\hour}. Cells +and beads/\glspl{dms} were removed from the retronectin plates using vigorous +pipetting and transferred to another 96 well plate wherein expansion continued. + +% METHOD fill in missing product numbers +\gls{bcma} \gls{car} lentiviral vector was synthesized in house as +follows\footnote{lentiviral synthesis was performed by Ritika Jain in our + laboratory and included here for reference}. \SI{10}{\ng} of +\anti{\gls{bcma}}-CD8-CD137-CD3$\upzeta$ plasmid (generously provided by Jim +Kochenderfer at the NIH)\cite{Lam2020} was added to \SI{50}{\ul} +\product{DH5$\upalpha$ cells}{\thermo}{18265017} and incubated for +\SI{30}{\minute} on ice. The cell mixture was then heat-shocked at +\SI{42}{\degreeCelsius} for \SI{20}{\minute} before being placed on ice for +another \SI{2}{\minute}. \SI{950}{\ul} \product{LB Broth}{TODO}{TODO} was added +to the cells which were then centrifuged for \SI{1}{\hour} at \SI{225}{\rpm}. +\SI{20}{\ul} of the cell mixture was then spread over selection plates and +incubated overnight at \SI{37}{\degreeCelsius}. Colonies were selected the +following day and incubated in \product{LB Broth}{TODO}{TODO} with +\product{ampicillin}{\sigald{}}{A9518-5G} at \SI{37}{\degreeCelsius} for +\SIrange{12}{16}{\hour} prior to using the \product{miniprep kit}{Qiagen}{27104} +as per the manufacturer's instructions to isolate the plasmid DNA. Transfer +plasmid along with \product{pMDLg/pRRE}{Addgene}{12251}, +\product{pRSV-Rev}{Addgene}{12253}, and \product{pMD2.G}{Addgene}{12259} +(generously provided by the Sloan lab at Emory University) in +\product{Opti-Mem}{\thermo}{31-985-070} with \product{lipfectamine + 2000}{\thermo}{11668019} were added dropwise to HEK 293T cells and incubated +for \SI{6}{\hour}, after which all media was replaced with fresh fresh media. +After \SI{24}{\hour} and \SI{48}{\hour}, supernatent was collected, pooled, and +concentrated using a \product{Lenti-X concentrator}{Takara}{631231} prior to +storing at \SI{-80}{\degreeCelsius}. \subsection{sulfo-NHS-biotin hydrolysis quantification} @@ -3375,8 +3404,8 @@ interest using \glspl{mab}. % METHOD The concentration for the surface marker cleavage experiment was much % higher, if that matters \glspl{dms} were digested in active T cell cultures via addition of sterile -\product{\gls{colb}}{Sigma}{11088807001} or -\product{\gls{cold}}{Sigma}{11088858001}. Collagenase was dissolved in +\product{\gls{colb}}{\sigald}{11088807001} or +\product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in \product{\gls{hbss}}{Gibco}{14025-076} or \product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}. This solution was added to T cell cultures at a 1:1 ratio in place of plain @@ -3405,8 +3434,8 @@ calculation neighborhood size of 5 and local density approximation factor of \subsection{integrin blocking experiments} To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were -supplemented with \product{\anti{\gls{a2b1}}}{Sigma}{MAB1973Z} and -\product{\anti{\gls{a2b2}}}{Sigma}{MAB1950Z} (both \gls{leaf}) at indicated +supplemented with \product{\anti{\gls{a2b1}}}{\sigald}{MAB1973Z} and +\product{\anti{\gls{a2b2}}}{\sigald}{MAB1950Z} (both \gls{leaf}) at indicated concentrations and timepoints. T cells were grown as described in \cref{sec:tcellculture}.