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Article ; Online: Experimental and computational approach to establish fit-for-purpose cell viability assays.

Pierce, Laura / Anderson, Hidayah / Sarkar, Swarnavo / Bauer, Steven R / Sarkar, Sumona

2024 Volume 19, Issue 1, Page(s) 27–45

Abstract: Aim: ...

Abstract	Aim:
MeSH term(s)	Humans ; Cell Survival ; Apoptosis ; Cell Proliferation
Language	English
Publishing date	2024-01-22
Publishing country	England
Document type	Journal Article
ZDB-ID	2274500-2
ISSN	1746-076X ; 1746-0751
ISSN (online)	1746-076X
ISSN	1746-0751
DOI	10.2217/rme-2023-0154
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Article ; Online: Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

Leland, Pamela / Degheidy, Heba / Lea, Ashley / Bauer, Steven R / Puri, Raj K / Joshi, Bharat H

Clinical and translational medicine

2024 Volume 14, Issue 5, Page(s) e1664

Abstract: Background: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric ...

Abstract	Background: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. Methods: We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. Results: The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. Conclusion: Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.
MeSH term(s)	Humans ; Interleukin-13 Receptor alpha2 Subunit/metabolism ; Interleukin-13 Receptor alpha2 Subunit/genetics ; Mice ; Glioma/immunology ; Glioma/therapy ; Glioma/genetics ; Glioma/pathology ; Glioma/metabolism ; Animals ; Immunotherapy, Adoptive/methods ; Disease Models, Animal ; Receptors, Chimeric Antigen/metabolism ; Receptors, Chimeric Antigen/immunology
Chemical Substances	Interleukin-13 Receptor alpha2 Subunit ; IL13RA2 protein, human ; Receptors, Chimeric Antigen
Language	English
Publishing date	2024-04-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2697013-2
ISSN	2001-1326 ; 2001-1326
ISSN (online)	2001-1326
ISSN	2001-1326
DOI	10.1002/ctm2.1664
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Article ; Online: Correction: Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with

Leland, Pamela / Kumar, Dhiraj / Nimmagadda, Sridhar / Bauer, Steven R / Puri, Raj K / Joshi, Bharat H

Journal of translational medicine

2023 Volume 21, Issue 1, Page(s) 636

Language	English
Publishing date	2023-09-19
Publishing country	England
Document type	Published Erratum
ZDB-ID	2118570-0
ISSN	1479-5876 ; 1479-5876
ISSN (online)	1479-5876
ISSN	1479-5876
DOI	10.1186/s12967-023-04496-7
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Article: Characterization of Chimeric Antigen Receptor Modified T Cells Expressing scFv-IL-13Rα2 after Radiolabeling with 89Zirconium Oxine for PET Imaging.

Leland, Pamela / Kumar, Dhiraj / Nimaggada, Sridhar / Bauer, Steven R / Puri, Raj K / Joshi, Bharat H

Research square

2023

Abstract: Background Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of ... ...

Abstract	Background Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Methods To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with
Language	English
Publishing date	2023-01-13
Publishing country	United States
Document type	Preprint
DOI	10.21203/rs.3.rs-2242559/v1
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Article ; Online: Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with

Leland, Pamela / Kumar, Dhiraj / Nimmagadda, Sridhar / Bauer, Steven R / Puri, Raj K / Joshi, Bharat H

Journal of translational medicine

2023 Volume 21, Issue 1, Page(s) 367

Abstract: Background: Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism ... ...

Abstract	Background: Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products. Methods: To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with Results: We observed that radiolabeling of CAR-T cells with Conclusions: Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with
MeSH term(s)	Zirconium/pharmacokinetics ; Radioisotopes/pharmacokinetics ; Positron-Emission Tomography ; Cell Tracking/methods ; Immunotherapy, Adoptive ; Single-Chain Antibodies ; T-Lymphocytes/cytology ; Tissue Distribution ; Jurkat Cells ; Animals ; Mice ; Cell Proliferation ; Cell Survival
Chemical Substances	Zirconium (C6V6S92N3C) ; Radioisotopes ; Single-Chain Antibodies
Language	English
Publishing date	2023-06-07
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	2118570-0
ISSN	1479-5876 ; 1479-5876
ISSN (online)	1479-5876
ISSN	1479-5876
DOI	10.1186/s12967-023-04142-2
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Article ; Online: Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression.

Andrews, Seth H / Klinker, Matthew W / Bauer, Steven R / Marklein, Ross A

Biotechnology and bioengineering

2021 Volume 119, Issue 2, Page(s) 361–375

Abstract: Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs ... ...

Abstract	Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e., priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-γ) and tumor necrosis factor-⍺ (TNF-⍺) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T-cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-γ/TNF-⍺ priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors, for example, PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.
MeSH term(s)	Cell Line ; Cytokines/metabolism ; Humans ; Immune Tolerance/immunology ; Mesenchymal Stem Cells/immunology ; Molecular Imaging/methods ; Single-Cell Analysis/methods
Chemical Substances	Cytokines
Language	English
Publishing date	2021-11-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	280318-5
ISSN	1097-0290 ; 0006-3592
ISSN (online)	1097-0290
ISSN	0006-3592
DOI	10.1002/bit.27974
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Article ; Online: 3D bioprinting optimization of human mesenchymal stromal cell laden gelatin-alginate-collagen bioink.

Sawyer, Stephen W / Takeda, Kazuyo / Alayoubi, Alaadin / Mirdamadi, Eman / Zidan, Ahmed / Bauer, Steven R / Degheidy, Heba

Biomedical materials (Bristol, England)

2022 Volume 18, Issue 1

Abstract: 3D bioprinting technology has gained increased attention in the regenerative medicine and tissue engineering communities over the past decade with their attempts to create functional living tissues and ... ...

Abstract	3D bioprinting technology has gained increased attention in the regenerative medicine and tissue engineering communities over the past decade with their attempts to create functional living tissues and organs
MeSH term(s)	Humans ; Gelatin ; Alginates ; Reproducibility of Results ; Collagen ; Mesenchymal Stem Cells
Chemical Substances	Gelatin (9000-70-8) ; Alginates ; Collagen (9007-34-5)
Language	English
Publishing date	2022-12-08
Publishing country	England
Document type	Journal Article
ZDB-ID	2265222-X
ISSN	1748-605X ; 1748-6041
ISSN (online)	1748-605X
ISSN	1748-6041
DOI	10.1088/1748-605X/aca3e7
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Article ; Online: GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line.

Lewis, Andrew M / Foseh, Gideon / Tu, Wei / Peden, Keith / Akue, Adovi / KuKuruga, Mark / Rotroff, Daniel / Lewis, Gladys / Mazo, Ilya / Bauer, Steven R

PloS one

2023 Volume 18, Issue 12, Page(s) e0293406

Abstract: The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less ... ...

Abstract	The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells.
MeSH term(s)	Animals ; Mice ; Zinc Finger Protein GLI1/metabolism ; Cell Differentiation ; Mesenchymal Stem Cells ; Cell Line ; Stem Cells ; Neoplasms/metabolism ; Kidney ; Cells, Cultured
Chemical Substances	Zinc Finger Protein GLI1
Language	English
Publishing date	2023-12-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0293406
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Article ; Online: Cytocentric measurement for regenerative medicine.

Henn, Alicia D / Pereira, Taci / Hunsberger, Joshua / Mitra, Kunal / Izadifar, Zohreh / Somara, Sita / Lindström, Lisa / Forest Farb-Horch, Thomas / Boy, Jake / Muschler, George F / Bauer, Steven R / Yerden, Randy

Frontiers in medical technology

2023 Volume 5, Page(s) 1154653

Abstract: Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions ...

Abstract	Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions can be controlled to meet the needs of cells and tissues in culture for RM, we have to know what those needs are and be able to quantify them. Therefore, identification and measurement of critical cell quality attributes at a cellular or pericellular level is essential to generating reproducible cell and tissue products. Here, we identify some of the critical cell and process parameters for cell and tissue products as well as technologies available for sensing them. We also discuss available and needed technologies for monitoring both 2D and 3D cultures to manufacture reliable cell and tissue products for clinical and non-clinical use. As any industry matures, it improves and standardizes the quality of its products. Cytocentric measurement of cell and tissue quality attributes are needed for RM.
Language	English
Publishing date	2023-04-27
Publishing country	Switzerland
Document type	Journal Article
ISSN	2673-3129
ISSN (online)	2673-3129
DOI	10.3389/fmedt.2023.1154653
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Article ; Online: Functionally-Relevant Morphological Profiling: A Tool to Assess Cellular Heterogeneity.

Marklein, Ross A / Lam, Johnny / Guvendiren, Murat / Sung, Kyung E / Bauer, Steven R

Trends in biotechnology

2018 Volume 36, Issue 1, Page(s) 105–118

Abstract: Heterogeneity in cell function has presented a significant hurdle to the successful clinical translation of many cellular therapies. Current techniques for assessing cell quality and the effects of microenvironmental cues and manufacturing processes on ... ...

Abstract	Heterogeneity in cell function has presented a significant hurdle to the successful clinical translation of many cellular therapies. Current techniques for assessing cell quality and the effects of microenvironmental cues and manufacturing processes on cell behavior often inadequately address heterogeneity due to issues such as population versus single-cell measurements and the therapeutic relevance and throughput/robustness of the assay. Due to the well-established relationship between morphology and cellular function, morphological profiling has become increasingly utilized to better understand functional heterogeneity and its impact on therapeutic development. In this review, we introduce an emerging field we term functionally-relevant morphological profiling with great potential to improve our understanding of cellular heterogeneity through discovering novel quality attributes, optimizing manufacturing, and screening drugs/biomaterials.
MeSH term(s)	Cell Culture Techniques/methods ; Cell Shape ; Cell- and Tissue-Based Therapy/methods ; Cytological Techniques/methods ; Humans ; Image Processing, Computer-Assisted/methods ; Microscopy/methods ; Optical Imaging/methods ; Technology, Pharmaceutical/methods
Language	English
Publishing date	2018
Publishing country	England
Document type	Journal Article ; Review ; Research Support, U.S. Gov't, P.H.S. ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	47474-5
ISSN	1879-3096 ; 0167-7799
ISSN (online)	1879-3096
ISSN	0167-7799
DOI	10.1016/j.tibtech.2017.10.007
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