12.3 Cell viability and function in bioprinted constructs
5 min read•july 30, 2024
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 , 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
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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
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Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original
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Frontiers | Hydrogel-Based Bioinks for 3D Bioprinting in Tissue Regeneration View original
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Frontiers | Desktop-Stereolithography 3D Printing of a Polyporous Extracellular Matrix Bioink ... View original
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- 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 , differentiation, and tissue-specific functions
Assessing cell viability in bioprinted constructs
Viability and metabolic activity assays
- assays such as calcein AM/ethidium homodimer-1 can visualize and quantify the proportion of live and dead cells within a bioprinted construct
- 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
- 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 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
Key Terms to Review (15)
Apoptosis: Apoptosis is a programmed process of cell death that occurs in a regulated manner, allowing for the removal of unwanted or damaged cells without causing an inflammatory response. This essential biological mechanism plays a crucial role in maintaining tissue homeostasis, regulating development, and ensuring the proper functioning of multicellular organisms. The process is characterized by distinct morphological changes and biochemical events, such as DNA fragmentation and membrane blebbing, which serve to safely eliminate cells.
Cell Adhesion: Cell adhesion refers to the process by which cells interact and attach to neighboring cells or the extracellular matrix (ECM) through specific proteins known as cell adhesion molecules (CAMs). This process is crucial for tissue formation, maintenance, and repair, as well as for cell signaling and communication.
Cell Proliferation: Cell proliferation is the process by which cells grow and divide to produce new cells, playing a critical role in tissue growth, repair, and regeneration. This process is tightly regulated by various internal and external factors, ensuring that cells proliferate in a controlled manner, which is essential for maintaining healthy tissues and organ systems.
Extracellular matrix: The extracellular matrix (ECM) is a complex network of proteins and carbohydrates that provides structural and biochemical support to surrounding cells. It plays a critical role in regulating various cellular functions, including cell adhesion, migration, proliferation, and differentiation, significantly influencing tissue architecture and homeostasis.
Extrusion-based bioprinting: Extrusion-based bioprinting is a technique used to create three-dimensional biological structures by depositing bioink, which consists of living cells and biomaterials, layer by layer. This method enables the precise placement of cells and materials to form complex tissue constructs, which is crucial for applications in regenerative medicine and tissue engineering.
Flow Cytometry: Flow cytometry is a powerful analytical technique used to measure and analyze the physical and chemical characteristics of cells or particles as they flow in a fluid stream through a laser or other light source. It allows for rapid quantification of multiple parameters per cell, enabling researchers to assess cell size, granularity, and the presence of specific surface markers, which is crucial in various biological applications.
Gene expression: Gene expression is the process by which information from a gene is used to synthesize functional gene products, typically proteins. This process involves two main steps: transcription, where the DNA sequence of a gene is transcribed to produce messenger RNA (mRNA), and translation, where the mRNA is decoded to build a protein. The regulation of gene expression is crucial for cell differentiation, development, and response to environmental stimuli, making it a key aspect in fields like regenerative medicine and tissue engineering.
Immunohistochemistry: Immunohistochemistry (IHC) is a laboratory technique used to visualize the presence and location of specific proteins in tissue sections using antibodies. This method is crucial for understanding the spatial distribution of proteins within the extracellular matrix (ECM) and can provide insights into cellular behaviors, such as cell viability and function in bioprinted constructs. By employing specific antibodies, researchers can obtain detailed images that reveal the interactions between cells and their surrounding environment, shedding light on important biological processes.
Inkjet bioprinting: Inkjet bioprinting is a 3D printing technique that utilizes inkjet technology to deposit viable cells and biomaterials layer by layer to create complex biological structures. This method allows for precise placement of cells in a controlled manner, which is essential for constructing tissue-like structures that mimic natural biological environments.
Live/dead staining: Live/dead staining is a technique used to differentiate between live and dead cells in a biological sample using specific fluorescent dyes. This method allows researchers to evaluate cell viability and health, which is especially crucial in assessing the quality of bioprinted constructs where cell function and survival are essential for successful tissue engineering applications.
Mechanotransduction: Mechanotransduction is the process by which cells convert mechanical stimuli from their environment into biochemical signals that can influence cellular behavior. This key mechanism is vital for understanding how cells interact with their extracellular matrix (ECM), migrate, and adapt to various physical forces, playing a crucial role in tissue engineering and regenerative medicine.
Metabolic activity: Metabolic activity refers to the biochemical processes that occur within cells to maintain life, including energy production, synthesis of biomolecules, and waste elimination. In the context of bioprinted constructs, understanding metabolic activity is crucial as it influences cell viability and function, impacting how well the cells can survive and perform their roles in engineered tissues.
MTT Assay: The MTT assay is a colorimetric assay used to assess cell viability by measuring the reduction of a yellow tetrazolium salt (MTT) to purple formazan crystals by metabolically active cells. This method is widely utilized in various fields, including regenerative medicine, to evaluate the health and proliferation of cells within bioprinted constructs, offering insights into the effectiveness of biomaterials and cellular responses.
Nutrient Availability: Nutrient availability refers to the accessibility and supply of essential nutrients that cells require for optimal growth, survival, and function. In the context of bioprinted constructs, this availability is crucial as it directly influences cell viability, proliferation, and the overall performance of tissue-engineered constructs. Adequate nutrient availability ensures that cells can carry out metabolic processes effectively, contributing to the functionality of the engineered tissues.
Organoids: Organoids are miniaturized and simplified versions of organs produced in vitro from stem cells that mimic some functions and structures of real organs. They serve as powerful tools for studying organ development, disease modeling, and drug testing due to their ability to replicate the architecture and functionality of actual tissues. Their relevance expands into regenerative medicine, where they offer insights into potential therapeutic applications and challenges.