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1. Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

   https://doi.org/10.1038/s41598-020-77288-4  

   Bijonowski, Brent M. ; Yuan, Xuegang ; Jeske, Richard ; Li, Yan ; Grant, Samuel C. ( December 2020 , Scientific Reports)

   Abstract Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinicalmore »relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.« less
2. Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction

   https://doi.org/10.1038/s41419-020-03173-7  

   Sullivan, Matthew Ryan ; Ugolini, Giovanni Stefano ; Sarkar, Saheli ; Kang, Wenjing ; Smith, Evan Carlton ; Mckenney, Seamus ; Konry, Tania ( November 2020 , Cell Death & Disease)

   The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8⁺T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8⁺T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8⁺T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations ofmore »CD8⁺T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8⁺T cells.

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3. Development and Optimization of a Novel Centrifugal Bioreactor with a Real-Time Monitoring Sensor for T Cell Exhaustion with Applications in Cancer Immunotherapy

   Brenden Fraser-Hevlin, Kitana M. ( November 2021 , Annual meeting American Institute of Chemical Engineers)

   Cancer has been one of the most significant and critical challenges in the field of medicine. It is a leading cause of death both in the United States and worldwide. Common cancer treatments such as radiation and chemotherapy can be effective in destroying cancerous tissue but cause many detrimental side effects. Thus, recent years have seen new treatment methods that do not harm healthy tissue, including immunotherapy. Adoptive cell therapy (ACT) is one form of immunotherapy in which patients’ immune cells are modified to target cancer cells and then reintroduced into the body. ACT is promising, but most current treatments are inefficient and costly. Widespread implementation of ACT has been a difficult task due to the high treatment cost and inefficient methods currently used to expand the cells. Additionally, if the manufacturing process is not carefully controlled, it can result in the cells losing their cancer-killing ability after expansion. To address the need for an economically feasible culture process to expand immune cells for immunotherapy, our laboratory has designed a centrifugal bioreactor (CBR) expansion system. The CBR uses a balance of centrifugal forces and fluid forces, as shown in Figure 1, to quickly expand infected CD8+ T-cells from a bovinemore »model up to high population densities. With other applications, the CBR has achieved cell densities as high as 1.8 x 108 cells/mL over 7 days in an 11.4-mL chamber. For this study, our goal is to begin validating the CBR by optimizing the growth of CEM (human lymphoblastic leukemia) cells, which are similar cell to cytotoxic T lymphocytes (CTLs). This can be accomplished by measuring kinetic growth parameters based on the concentrations of glucose and inhibitory metabolites in the culture. We hypothesize that by designing a kinetic model from static culture experiments, we can predict the parameters necessary to achieve peak CEM and eventually CTL growth in the CBR. We will report on kinetic growth studies in which different glucose concentrations are tested, and a maximum specific growth rate and Monod constant determined, as well as studies where varying levels of the inhibitory growth byproducts, lactate and ammonium, are added to the culture and critical inhibitor concentrations are determined. Another recent conceptual development for the design of the CBR is a real-time monitoring and feedback control system to regulate the cellular environment, based on levels of surface co-receptors and mRNA signaling within the culture. Prior studies have pinpointed T cell exhaustion as a significant issue in achieving successful immunotherapy, particularly in treatments for solid tumors; T cell exhaustion occurs during a period of chronic antigen stimulation when the cells lose their ability to target and kill cancer cells, currently theorized to be associated with particular inhibitory receptors and cytokines in the immune system. Designing a system with a fiber optic sensor that can monitor the cell state and use feedback control to regulate the pathways involved in producing these receptors will ensure the cells maintain cytotoxic properties during the expansion process within a Centrifugal Fluidized Expansion we call the CentriFLEX. In this presentation, we will also report on early results from development of this exhaustion monitoring system. In brief, achieving optimal kinetic models for the CBR system and methods to prevent T cell exhaustion has the potential to significantly enhance culture efficiency and availability of immunotherapy treatments.« less
4. Autologous Protein Solution processing alters lymphoid and myeloid cell populations and modulates gene expression dependent on cell type

   https://doi.org/10.1186/s13075-022-02875-x  

   Peña, Alexis N. ; Sommerfeld, Sven D. ; Anderson, Amy E. ; Han, Jin ; Maestas, Jr, David R. ; Mejias, Joscelyn C. ; Woodell-May, Jennifer ; King, William ; Ganguly, Sudipto ; Elisseeff, Jennifer H. ( September 2022 , Arthritis Research & Therapy)

   Osteoarthritis (OA) is a degenerative disease associated with cartilage degradation, osteophyte formation, and fibrillation. Autologous Protein Solution (APS), a type of autologous anti-inflammatory orthobiologic, is used for pain management and treatment of OA. Various compositions of autologous PRP formulations are in clinical use for musculoskeletal pathologies, by nature of their minimal processing and source of bioactive molecules. Currently, there is no consensus on the optimal composition of the complex mixture. In this study, we focused on elucidating the immune cell subtypes and phenotypes in APS. We identified the immune cell types in APS from healthy donors and investigated phenotypic changes in the immune cells after APS processing. Based on flow cytometric analysis, we found that neutrophils and T cells are the most abundant immune cell types in APS, while monocytes experience the largest fold change in concentration compared to WBCs. Gene expression profiling revealed that APS processing results in differential gene expression changes dependent on immune cell type, with the most significantly differentially regulated genes occurring in the monocytes. Our results demonstrate that the mechanical processing of blood, whose main purpose is enrichment and separation, can alter its protein and cellular composition, as well as cellular phenotypes in themore »final product.

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5. Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State

   https://doi.org/10.1007/s12195-019-00602-2  

   Deng, Wentao ; Fernandez, Audry ; McLaughlin, Sarah L. ; Klinke, David J. ( April 2020 , Cellular and Molecular Bioengineering)

   Introduction—Cellular communication network factor 4 (CCN4/WISP1) is a secreted matricellular protein that stimulates metastasis in multiple malignancies but has an unclear impact on phenotypic changes in melanoma. Recent data using cells edited via a double-nickase CRISPR/Cas9 approach suggest that CCN4/WISP1 stimulates invasion and metastasis of melanoma cells. While these data also suggest that loss of CCN4/WISP1 increases cell proliferative, the CRISPR approach used may be an alternative explanation rather than the loss of gene function. Methods—To test whether CCN4/WISP1 also influences the proliferative phenotype of melanoma cells, we used mouse melanoma models and knocked out Ccn4 using a homologydirected repair CRISPR/Cas9 system to generate pools of Ccn4-knockout cells. The resulting edited cell pools were compared to parental cell lines using an ensemble of in vitro and in vivo assays. Results—In vitro assays using knockout pools supported previous findings that CCN4/WISP1 promoted an epithelial– mesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. While Ccn4 knockout also enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions in vitro, quantitative analysis of tumor growth assays in vivo, and transcriptomicsmore »analysis of human melanoma cell lines were also used to quantify changes in phenotype and generalize the findings. Conclusions—In addition to stimulating invasion and metastasis of melanoma cells, the results suggested that CCN4/WISP1 repressed cell growth and simultaneously enhanced cell survival.« less