Poster N15 - Development of Biomimetic Extracellular Matrix for Cell Spheroid Encapsulation

Introduction: Cell spheroid encapsulation has emerged as a promising technique in tissue engineering and regenerative medicine due to its potential to enhance cell viability, functionality, and therapeutic efficacy. Current advances in cell spheroid encapsulation methods have enabled the development of various coating materials and techniques to provide structural support, selective permeability, and protection from physical stress. However, limitations such as insufficient biocompatibility, instability of coatings, and limited control over permeability remain challenges that need to be addressed. Our study aims to generate a biomimetic extracellular matrix (BECM) for cell spheroid encapsulation using a combination of oligonucleotides and polymers, providing a novel approach to improving the stability, biocompatibility, and functionality of encapsulated cell spheroids.

Materials and

Methods: Bone marrow derived mesenchymal stem cell (MSC) spheroids were formed using ultralow attachment well plates. Oligonucleotides used in this study were obtained from Integrated DNA Technologies (Coralville, IA). The cholesterol conjugated DNA initiator (DI) was inserted into the cell membranes through hydrophobic insertion. To form a DNA template through hybridization chain reaction (HCR), DNA monomer 1 (DNA1) and DNA monomer 2 (DNA2) were added. Further crosslinking of polymers through polyelectrolyte complexation led to biomimetic extracellular matrix (BECM) synthesis. The formation of BECM on the cell membrane was examined using confocal microscopy and flow cytometry. FITC-conjugated dextran was used for the permeability assay to examine the selective permeability of BECM. Cell viability was examined using MTS assay and live/dead staining. The protection ability of BECM was analyzed using fluorescence images after applying the physical stress. The maintained cell behavior of MSC spheroids after encapsulation was assessed using cytokine array and ELISA.

Results, Conclusions, and Discussions: Confocal microscopy and flow cytometry show that BECM was synthesized on the cell spheroid surface from DNA sequences and polyelectrolytes. Fluorescence imaging showed the BECM was stably generated on the cell membrane. Permeability assay results showed that penetration of dextran with large molecular weights into BECM coated cell spheroids were partially inhibited. Dextran with small molecular weight could freely penetrate the BECM. Diffusion profiles from Transwell assays further show that BECM coating can allow the penetration of small molecules but prevent large molecules. BECM coated MSC spheroids remained their spheroid morphology without attaching to the well plate surface. In addition, BECM coating can protect the host spheroids from physical stress. Cell viability assays revealed that BECM coating was biocompatible, with showing negligible cytotoxicity. Human cytokine array and VEGF ELISA results demonstrated that BECM coating did not compromise the cell behaviors of the host cell spheroid. The results of this study indicate that the BECM synthesized from oligonucleotides and polymers provides a robust encapsulation for MSC spheroids. The successful formation of BECM, as confirmed by confocal microscopy and flow cytometry, demonstrate the feasibility of our approach. The permeability assays highlight the selective nature of the BECM, allowing small molecules to penetrate while restricting larger molecules, which is crucial for maintaining cell function and nutrient exchange. The BECM's protective properties against physical stress and its biocompatibility suggests its potential for various biomedical applications. The maintenance of cell behaviors post-encapsulation, as shown by cytokine array and ELISA results, underscores the efficacy of our BECM coating in preserving the functional integrity of the spheroids. The development of BECM has demonstrated significant advancements in enhancing the stability, selective permeability, and protection of MSC spheroids. The BECM coating not only preserves cell viability and function but also provides a controlled environment that supports the maintenance of spheroid morphology and resistance to physical stress. These findings suggest that BECM encapsulation holds great promise for improving the efficacy and applicability of cell spheroids in tissue engineering and regenerative medicine. Future studies will focus on optimizing the BECM formulation and exploring its potential in various biomedical applications.

Acknowledgements (Optional): This work was supported by the Pennsylvania State University Huck Chair Professorship.