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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster U13 - Cellular Fractionation and Characterization of Immune Subcellular Vesicles for Synthetic Biology

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Trevor Hatch

Graduate Student Researcher
Johns Hopkins University
Baltimore, Maryland, United States

Co-Author(s)

Joshua M. Devier

MSTP MD/PhD Student
University of California, San Francisco, United States
JM

Jody Mou

Student Researcher
Johns Hopkins University, United States
Sixiang Wang

Student Researcher
Johns Hopkins University, United States

Primary Investigator(s)

Joshua C. Doloff, MA, PhD (he/him/his)

Assistant Professor
Johns Hopkins University, Depts. of Biomedical Engineering and Materials Science & Engineering, Translational Tissue Engineering Center (TTEC), and the Institute for NanoBioTechnology (INBT)
Baltimore, Maryland, United States

Introduction: Researching immune cells allows us to better understand how the immune system functions and to develop new strategies for the prevention and treatment of diseases. The advent of cell-based immunotherapies demonstrates the therapeutic value of engineered immune cells and the unique functions they perform. Despite advances in immunology and cellular biology, organelle-specific contributions to immune cell behavior require further investigation. To better understand their imparted functions and how they may be leveraged therapeutically, we have developed a synthetic biology platform that allows for the separation and reconstitution of isolated organelle-containing subcellular vesicles. Through this platform, individual organelles, such as the mitochondria, nucleus, Golgi apparatus, or plasma membrane, may be isolated, characterized, and fused in desired ratios to yield synthetic cells with unique functionality. These synthetic cells may then be further characterized to evaluate how contributing organelles impact cell phenotype. This approach has potential applications in immunology, cell biology, and immunoengineering.

Materials and

Methods: Cellular fractionation and organelle isolation were accomplished via chemical destabilization of the cell cytoskeleton followed by density gradient centrifugation. A density gradient solution containing a cell cytoskeleton destabilizing agent enabled fractionation of cells into subcellular vesicles classified as karyoplasts (membrane-wrapped vesicles containing nuclei) or cytoplasts (membrane-wrapped vesicles containing cytoplasm and lacking nuclei). Organelle-specific dyes were used to label the mitochondria, nuclei, and plasma membranes of the subcellular vesicles to enable identification, characterization, and sorting. A viability dye was also utilized to assess membrane integrity. Organelle fractions were analyzed via flow cytometry and fluorescence microscopy, and sorted via fluorescence-activated cell sorting (FACS) to enrich the initial populations. FACS-enriched karyoplasts and cytoplasts were subsequently recombined using either a PEG-based fusion mixture or an enhanced fusion mixture containing additional biochemical additives. The resulting reconstituted cells were purified via FACS and characterized through phenotypical assays. These steps are outlined visually in Figure 1.

Results, Conclusions, and Discussions: The results confirm the successful generation of cytoplasts and karyoplasts from whole cells using density gradient centrifugation in combination with chemical destabilization of the cell cytoskeleton. Fluorescence microscopy images of FACS-enriched cytoplasts and karyoplasts demonstrate relatively pure populations of nuclei-deficient cytoplasmic bodies (Fig. 2A & B) and cytoplasm-deficient nuclear bodies (Fig. 2C & D), respectively. Further analysis of these populations via flow cytometry provides quantitative data regarding the frequency of cytoplasts and karyoplasts in FACS-enriched populations of subcellular vesicles, further confirming their successful generation and isolation (Fig. 3A & C). Viability dye staining and analysis via flow cytometry supports the viability of the majority of the generated subcellular vesicles (Fig. 3B & D). Further phenotypical characterization will be performed (viability, proliferation, migration, cytokine secretion, etc.) to evaluate the impact of organelle cellular origin and different stoichiometric ratios of organelles on reconstituted synthetic cells. Future work utilizing isolated organelles and the fusion techniques described here has great potential for the study and application of synthetic cells in immunology, cell biology, and immunoengineering.

Acknowledgements (Optional):