(228ab) Applying Standardized Three-Dimensional Mechanoculture to Multi-Well Plates

Introduction: Although multi-well plates mark the pinnacle standard for in vitro cell culture studies, they are essentially limited in emulating 3D and dynamic extracellular milieu in the body that are critical in restoring cellular functions. Various scaffolds and bioreactor designs have been introduced to reduce this gap, and their biological significance has been evidently demonstrated. Yet, these platforms have been sparingly accepted in biological experiments and pharmaceutical screenings partly because the benefits of using multi-well plates (e.g. reproducible, quantitative and observable experiments with affordable cost) outweigh the advantages of 3D bioreactor cultures. Here, we introduce an integrative approach to realize a standardized 3D mechanoculture in a multi-well plate using a hydrogel scaffold and a magnetically responsive floating disc.

Materials and Methods: 3D polyacrylamide hydrogel scaffolds with controlled microporous structure (D=250-300µm) and defined thickness (H=1mm) were prepared following listed method [1]. A magnetically responsive floating disc was fabricated by attaching a medical grade stainless steel ball (D=4.76mm) in a cylindrical (D=12mm, H=3mm) PDMS slap containing air sac (V=0.1mL). An array of neodymium magnets (D=H=9.5mm) were placed on an acrylic stage. A pneumatic piston was used to adjust the distance between the floating disc and magnet-acrylic plate. As the pump pushed the magnet-acrylic plate up, the discs move to the bottom of the well in response to the magnetic field, and generate shear and mechanical stimulus. An acrylic O-ring (D=15.4mm) was placed on the bottom of some wells to prevent the floating disc from striking the bottom. The frequency of pneumatic actuation was controlled by Arduino Uno (Adafruit). Human Bone Marrow Stromal Cells (hBMSCs) and human prostate cancer cells (PC-3) were used for proof of concept studies. The confluent areas in each well were progressively analyzed using the open source program ImageJ.

Results and Discussion: hBMSCs response to three distinct culture conditions were analyzed: disc only, disc with O-Ring, and control. Optical clarity of the disc allowed us to image the cells without the risk of airborne contaminants from open-lead operation. BMSC in the disc and O-Ring well have much more densely aligned cellular morphology in the direction of the applied shear force compared to that of the control well. Physical interaction between the disc and the well plate may have caused some cellular damage. A Similar phenomenon was observed for PC-3 cells, thus conclusive that scaffolds further increased the complexity of in-vitro culture systems by offering 3D matrices. Current efforts are in directing stimuli to the 3D scaffolds.

Conclusion: We demonstrated a simple and versatile approach to apply 3D and mechanically active culture in a multi-well plate. Our modular approach allows simulating specific structure and geometry of extracellular matrices as well as shear and mechanical stimuli of a diverse range of tissues/organs. We envision our multi-well plate based 3D bioreactor design will facilitate practical implementation of 3D mechanoculture to broader research communities.