12.3 Cell viability and function in bioprinted constructs

3D bioprinting lets us create complex tissue structures, but keeping cells alive and functioning is tricky. Factors like mechanical stress, temperature changes, and bioink properties can all affect cell survival during the printing process.

Once printed, cells need the right environment to thrive. Things like nutrient availability, growth factors, and cell organization play a big role. Researchers use various techniques to check cell health and optimize conditions for long-term tissue function.

Cell viability in 3D bioprinting

Factors affecting cell viability during bioprinting

Top images from around the web for Factors affecting cell viability during bioprinting

• 

  Frontiers | Desktop-Stereolithography 3D Printing of a Polyporous Extracellular Matrix Bioink ... View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

• 

  Frontiers | Desktop-Stereolithography 3D Printing of a Polyporous Extracellular Matrix Bioink ... View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

1 of 3

Top images from around the web for Factors affecting cell viability during bioprinting

• 

  Frontiers | Desktop-Stereolithography 3D Printing of a Polyporous Extracellular Matrix Bioink ... View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

• 

  Frontiers | Desktop-Stereolithography 3D Printing of a Polyporous Extracellular Matrix Bioink ... View original

  Is this image relevant?

• 

  Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original

  Is this image relevant?

1 of 3

• The bioprinting process exposes cells to various stresses including mechanical forces during extrusion (shear stress), changes in temperature, and alterations in the cellular microenvironment
• Bioink properties such as viscosity, shear-thinning behavior, and crosslinking mechanisms influence cell survival during printing and affect cell behavior post-printing
• Printing parameters like nozzle diameter, pressure, and printing speed can be optimized to minimize cell damage and ensure high cell viability
• Post-printing factors such as culture conditions, nutrient availability, and the presence of growth factors play a crucial role in maintaining cell viability and promoting desired cellular functions

Cell organization and interactions in bioprinted constructs

• The cell type, density, and organization within the bioprinted construct influence cell-cell interactions, signaling, and overall tissue function
• Co-culturing multiple cell types (hepatocytes and endothelial cells) can recapitulate the native tissue microenvironment and enhance cell-cell interactions, leading to improved tissue function
• Designing bioprinted constructs with appropriate porosity and interconnectivity facilitates nutrient diffusion, waste removal, and cell migration, promoting long-term cell survival and functionality
• Incorporating growth factors, cytokines, or other signaling molecules (VEGF, BMP-2) into the bioink or culture medium promotes cell proliferation, differentiation, and tissue-specific functions

Assessing cell viability in bioprinted constructs

Viability and metabolic activity assays

• Live/dead staining assays such as calcein AM/ethidium homodimer-1 can visualize and quantify the proportion of live and dead cells within a bioprinted construct
• Metabolic activity assays like MTT or alamarBlue provide a quantitative measure of cell viability and proliferation by assessing the metabolic function of cells
• Long-term cell viability and proliferation within bioprinted constructs can be assessed using live/dead staining and metabolic activity assays at various time points post-printing
• Gene expression analysis using techniques like RT-PCR or RNA sequencing provides insights into the molecular mechanisms underlying cell behavior and differentiation within the bioprinted microenvironment

Imaging and functional assessment techniques

• Imaging techniques such as confocal microscopy and two-photon microscopy enable the visualization of cell morphology, organization, and distribution within the 3D bioprinted structure
• Functional assays specific to the cell type and desired tissue function can evaluate the performance of cells within the bioprinted construct (contractility of bioprinted cardiac tissues, secretion of proteins by bioprinted liver tissues)
• Evaluating the maintenance of cell phenotype and function over extended periods is crucial for the success of bioprinted tissues, which can be achieved through cell-specific functional assays and gene expression analysis
• Assessing the remodeling and maturation of the bioprinted construct over time, including changes in the extracellular matrix composition and organization, is important for understanding the long-term behavior of cells within the tissue

Optimizing cell survival in bioprinted tissues

Bioink formulation and printing parameters

• Incorporating cell-protective additives such as antioxidants (vitamin C) or anti-apoptotic factors (caspase inhibitors) into the bioink formulation can enhance cell survival during the printing process
• Optimizing printing parameters like reducing extrusion pressure or increasing printing speed minimizes the exposure of cells to mechanical stresses and improves cell viability
• Utilizing bioinks with favorable rheological properties such as shear-thinning behavior and rapid crosslinking (alginate, gelatin methacrylate) provides a supportive microenvironment for cell survival and function
• Incorporating sacrificial materials (pluronic F-127) into the bioink can create microchannels for improved nutrient diffusion and waste removal, promoting cell survival and functionality

Co-culture systems and growth factor incorporation

• Co-culturing multiple cell types within the bioprinted construct (neurons and glial cells) recapitulates the native tissue microenvironment and enhances cell-cell interactions, leading to improved tissue function
• Incorporating growth factors, cytokines, or other signaling molecules into the bioink or the culture medium promotes cell proliferation, differentiation, and tissue-specific functions
• Designing bioprinted constructs with appropriate porosity and interconnectivity facilitates nutrient diffusion, waste removal, and cell migration, promoting long-term cell survival and functionality
• Optimizing the spatial arrangement of different cell types (endothelial cells lining vascular channels) within the bioprinted construct can enhance tissue organization and function

Long-term behavior of cells in bioprinted constructs

In vitro assessment of cell function and maturation

• Evaluating the maintenance of cell phenotype and function over extended periods is crucial for the success of bioprinted tissues, which can be achieved through cell-specific functional assays and gene expression analysis
• Assessing the remodeling and maturation of the bioprinted construct over time, including changes in the extracellular matrix composition (collagen, elastin) and organization, is important for understanding the long-term behavior of cells within the tissue
• Monitoring the formation and functionality of tissue-specific structures (bile canaliculi in bioprinted liver tissues, sarcomeres in bioprinted cardiac tissues) provides insights into the maturation and functionality of the bioprinted construct
• Studying the development of intercellular junctions (adherens junctions, gap junctions) and cell polarity within the bioprinted tissue is essential for evaluating the establishment of proper tissue architecture and function

In vivo integration and immune response

• Investigating the integration of bioprinted constructs with the surrounding host tissue in vivo is essential for evaluating their potential for tissue regeneration and functional restoration
• Studying the vascularization of bioprinted constructs, either through the incorporation of endothelial cells or the promotion of host vessel infiltration, is crucial for ensuring long-term cell survival and tissue integration
• Monitoring the immune response to bioprinted constructs, particularly when using allogeneic or xenogeneic cell sources, is important for assessing the long-term compatibility and functionality of the engineered tissue
• Evaluating the degradation kinetics of the bioprinted scaffold materials (PCL, PLA) and their replacement by cell-secreted extracellular matrix is crucial for the successful integration and remodeling of the bioprinted tissue