| 1. |
Schwann Cell Reprogramming for Functional Skeletal Muscle Innervation |
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Emmanuel Ekwueme1 and Cathryn Sundback |
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1Center for Regenerative Medicine-Laboratory of Tissue Engineering, Massachusetts General Hospital |
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Skeletal muscle regeneration is often hampered by a lack of functional innervation of the regenerated tissue. Following peripheral nervous system injury, Schwann cells (SCs) facilitate the clearing of axonal debris from injury and denervation. Later, these SCs reprogram to provide instructive signals that promote neurogenesis and reinnervation. The mechanisms that SCs utilize to facilitate neurogenesis and reinnervation during regeneration provide a strong rationale for using “reprogrammed” SCs to encourage functional innervation of tissue engineered skeletal muscle. Our preliminary results show that reprogrammed SCs provide neuroprotective and neurogenic support to motor neurons. Additionally, these reprogrammed SCs modulate acetylcholine receptor activity within differentiated myotubes. We are characterizing the impact of these cells on functional innervation in an in vitro tri-culture system containing SCs, myotubes, and motor neurons and on in vivo re-innervation in a rat sciatic nerve injury model. |
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| 2. |
Mechanical Modulation of the Mesencymal Stromal Cell Secretome for Hematopoietic Recovery |
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Frances Liu |
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Graduate Student, Van Vliet Laboratory for Material Chemomechanics, Department of Biological Engineering, Massachusetts Institute of Technology |
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Mesenchymal stromal cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum to which those cells adhere. Here, we leveraged cell-material interactions in vitro to induce human-bone-marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. Specifically, we varied the viscoelastic properties of cell culture-compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximate threefold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli < 1 kPa) prior to detectable changes in osteogenic terminal differentiation capacity. In co-culture with human CD34+ hematopoietic stem and progenitor cells (HSPCs), these mechanically modulated MSCs promoted proliferation of HSPCs without altering HSPC multi-potency. This approach enables enhanced in vitro production of both MSCs and HSPCs that potentially aid in hematopoietic recovery. |
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| 3. |
Continuous Biomanufacturing Using Micro/Nanofluidics |
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Taehong Kwon |
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Doctoral Student, Department of Electrical Engineering and Computer Science, Research Laboratory of Electronics, Micro/Nanofluidic BioMEMS Group, Massachusetts Institute of Technology |
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Continuous biomanufacturing is a growing trend in the biopharmaceutical industry. Ideas from microfluidics can be employed in all aspects of continuous biomanufacturing to significantly enhance the overall productivity as well as the efficacy and safety of the final products. First, we introduce a novel cell retention device based on inertial sorting, which can overcome challenges of existing hollow-fiber membranes, such as membrane clogging/fouling, low product recovery, and inability to remove dead cells. Second, we present a nanofluidic system for continuous-flow, multi-variate protein analysis for real-time critical quality assessments. The existing bench-type conventional analytical tools have restrictions to meet quality assurance requirements of current and future biomanufacturing systems. Finally, we introduce a recent work of integration of perfusion culture system using the microfluidic cell retention device with nanofluidic system enabling real-time product quality assessments. |
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| 4. |
Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics |
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Aniruddh Sarkar |
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Research Fellow, The Research Laboratory of Electronics, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology |
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Isolation of proteins or cells from complex mixtures is required for many diagnostic, therapeutic, research, and biomanufacturing applications. Current, affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple proteins or cell types. We present a method for rapid and multiplexed affinity-based isolation of proteins and cells using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets bind to beads of different sizes, which are then sorted by flowing through a spiral microfluidic channel. This technique performs continuous-flow, high-throughput affinity-separation of proteins or cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from whole blood or blood fractions. We use the technique to isolate antibodies specific to different HIV antigens and rare HIV-specific cells from HIV+ patients. |
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| 5. |
Application of Humanized Mice in Immunotherapy |
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Xiaocan Guo |
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Postdoctoral Associate, The Chen Lab, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology |
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Mouse models are most commonly used in vivo system for developing gene and cell therapy. However, there are many differences between mice and humans. A human immune system-based in vivo model is required for advancing cancer immunotherapy. We have established B cell lymphoma as well as acute myeloid leukemia humanized mouse models by introducing oncogenes into hemopoietic stem cells and transplanting hemopoietic stem cells into immunodeficient mice. Those mice have human hematological malignancies and autologous immune system and resemble human patients both in clinical presentation and pathology. We provide a powerful pre-clinical platform for evaluating the therapeutic efficacy of antibodies. Our group also aims to develop chimeric antigen receptor T-cell therapy targeting neo-antigen of hematological malignancies that will potentially benefit human patients. Humanized mice bridge the gap between mice and human, thus advancing immune-based drug development. |
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| 6. |
Comprehensive Characterization of a Series of Therapeutic Proteins Produced from a Platform Developed for Distributed Manufacturing (InSCyT) System by Tandem Mass Spectrometry |
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Di Wu |
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Graduate Student, Hancock Lab, Departments of Chemistry and Biological Chemistry, Northeastern University |
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Biosimilar therapeutics are marketed worldwide nowadays, and stability assessment is necessary to validate the long-term safety and purity of the products. We have used Pichia as the host system instead of the traditional E. coli system to improve platform stability and achieve less host cell protein contamination. The InSCyT system is designed as a portable microbial manufacturing platform for producing a wide range of biotherapeutics. This poster describes the characterization of recombinant hGH, G-CSF and interferon produced from the system. The analysis ranges from primary structure identification to possible post-translational modifications (PTMs) by the use of sensitive LC-MS. We demonstrated the analysis of any degradation product, and the manufactured biopharmaceuticals are at a high-quality level compared with the corresponding reference drugs. Thus, the methodology developed is suitable for the identification of product variants and can be used in the future to ensure that a manufacturing platform is efficient, convenient, and consistent. |
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| 7. |
Elucidating Mechanisms of Heterogeneity Onset During Mesenchymal Stem Cell Culture Expansion |
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Deena Rennerfeldt |
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Graduate Student, Van Vliet Laboratory for Material Chemomechanics, Department of Biological Engineering, Massachusetts Institute of Technology |
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Mesenchymal stem cells (MSCs) have been cited as a promising therapeutic for indications ranging from autism to liver cirrhosis to muscular dystrophy, yet there remain no FDA-approved treatments using this malleable class of stem cells. Despite numerous clinical trials pursuing MSC therapy, the in vitro expansion of functionally homogeneous populations to therapeutically relevant quantities remains an elusive goal. Our work has recently shown that the emergence of functional heterogeneity occurs even within single-cell-derived MSC colonies, and administration of heterogeneous cell populations can lead to diminished clinical outcomes. Here, we demonstrate our findings on the extent, mechanisms, and possible causes of heterogeneity onset through long-term time lapse image analysis and single-cell RNA sequencing. |
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| 8. |
Effects of Culture Media and Suspension Expansion Technologies In Mesenchymal Stem Cell Manufacturing – A Computational Bioprocess and Bioeconomics Study |
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Catia Bandeiras |
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Graduate Student, Institute for Data, Systems, and Society, Massachusetts Institute of Technology |
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Mesenchymal stem cell (MSC) based therapies are promising for unmet medical needs. Despite this promise, the scaling-up of production of clinical grade MSCs is hindered by the use of planar technologies that require intensive labor and are not enough to meet market demands, as well as high product and process variability introduced by the use of xenogeneic materials. This work presents a new bioprocess and bioeconomics model of stem cell expansion to support informed decisions for stem cells process scaling-up at reduced annual costs. The intrinsic equations and parameters that capture the cell biological features, according to their source and media used, are embedded in the model. Three variables were evaluated: the type of culture medium, the source of MSCs, and 2D vs. 3D expansion technologies. The findings of this study may be used to improve the design of expansion methods with fully xeno-free materials. |
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| 9. |
Industrialization Of Adenoviral Vector Production and Purification in Packed-Bed Icellis® Bioreactor: From Bench to Clinical Scale |
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Rachel Legmann1 and Heather Mallory |
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1Pall Life Sciences |
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Bioprocessing of therapeutic viruses for Cell and Gene Therapy still faces many challenges during scale-up. Conventional viral vector production processes are not adaptable to manufacturing scales. To overcome this challenge, Pall has developed an upstream and downstream industrialized process that has successfully generated the required number of adenoviruses for pre-clinical study. By optimizing culture and infection parameters such as cell seeding density and process parameters, yield was increased to 1.4E10 infectious virus particles per cm2 fixed bed. Under optimized conditions of pH and conductivity, adenoviral vector was bound to the membrane. The Adenovirus 5 bind/elute strategy managed to reduce significantly impurities such as Host Cell Protein (HCP) and residual host cell DNA. We have demonstrated that the eluted virus peak from Mustang Q membrane contains more than 90% ratio of full to empty capsid. Final purified product was fully functional and comparable to the virus produced by the conventional, non-industrial process. |
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| 10. |
Downstream Purification Solutions for Viral Vectors: Enabling Platform Approaches to Advance Gene Therapies |
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Scott Zobbi |
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Thermo Fisher Scientific |
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Over the past decade, gene therapy applications and their importance in the biopharmaceutical industry have been increasing. Vectors centered on the nonpathogenic adeno-associated virus (AAV) have emerged as the vector of choice for many therapies. Although current purification strategies can accommodate AAV doses on a scale suitable for orphan indications, generating industrialized platform technologies that will maximize productivity and enable efficient, simple, and inexpensive purification of biologically active viral vectors for large-scale commercial manufacturing has been a challenge. Thermo Fisher Scientific has developed downstream purification solutions to enable the scale-up of these viral vectors. |
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| 11. |
Rapid, Real Time Quantification of Adeno-Associated Virus Particles using Antibody-Based Detection on the Virus Counter 3100 |
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Katherine D. Shives, PhD1 and Antje Schickert, PhD |
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1Virus Analyticsm, Sartorius-Stedim |
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Due to their persistent infection and lack of pathogenicity, adeno-associated virus (AAV) particles have become valuable vectors for modern gene therapies. Biologically relevant quantitation methods are required to support production and therapeutic use of these vectors. Quantification of AAV particles is difficult, often relying on costly and time-demanding electron microscopy methods. Rapid analysis methods are needed to monitor vector production and quantify particle counts in final formulations. The flow-based Virus Counter 3100 and antibody-based ViroTag AAV reagents allow for the rapid quantitation of AAV particles by utilizing serotype-specific fluorescently labeled antibodies to detect intact, properly assembled AAV vector particles. Utilizing a patented, no-wash assay, AAV samples are stained in 30 minutes and samples are quantified in 3 minutes per sample. This speed allows for in-process monitoring and production optimization of AAV vector products, making the Virus Counter 3100 and ViroTag reagents a valuable addition to bioprocessing applications utilizing AAV particles. |
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| 12. |
Scale Up of Pluripotent Stem Cell Cultivation and Differentiation in a Novel Single-Use Bioreactor System |
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Joe Petrosky |
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PBS Biotech |
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Growing and differentiating pluripotent stem cells as aggregates suspended in bioreactors is a viable option for scaling up. Cells grown as aggregates can be very sensitive to hydrodynamic shear. The cells respond to the microenvironment during cell growth as well as differentiation process in ways that may affect final product yield, quality, and potency. A single-use bioreactor using Vertical-Wheel technology offers homogenous dissipation energy distribution and uniform cell aggregate suspension with low power input. The low shear environment remains constant across the range of vessel sizes from 0.5 to 80 liters. Homogeneous distribution of turbulent energy dissipation rates inside the Vertical-Wheel bioreactor generates a uniform size and shape of cell aggregates. Controlling the size of cell aggregates can be beneficial for cell growth and the differentiation process of pluripotent stem cells. The results of physical measurements of Kolmogorov scale and the growth and the differentiation kinetics of PSCs will be discussed. |
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| 13. |
Evaluation of Hollow Fiber Based Bioreactor System for Efficient Cell Expansion and Virus Production |
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Jonathan Lee, Shelley Alvarado, Martina Kopp, Jennifer Rattan, Houman Dehghani |
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Process Development, Amgen |
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With the recent approvals of cell therapy products, solutions for efficient manufacture of cell therapies are gaining momentum. Current cell therapy manufacturing can be a manual and open process and would benefit from a closed and scalable process. We have evaluated a single-use, closed hollow fiber perfusion bioreactor to scale various cancer cell lines as well as producing retroviruses. This closed cell culture system allows for efficient cell expansion and transduction in a small enclosed unit. Utilizing a closed perfusion based hollow fiber bioreactor for T-Cell expansion and transduction offers the possibility of manufacturing a personalized cell therapy drug product in a small footprint modular manufacturing environment. Data will be presented on virus production and cell expansion using this system. |
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