Biomaterials and Cell Culture Quiz

Biological Properties of Biomaterials

• Biomaterials are substances intended to interact with biological systems for medical, therapeutic, or diagnostic purposes.
• Host response refers to the local and systemic reaction of living systems to a material, aside from the intended therapeutic response.
• Material response involves how the material interacts with the living system, often eliciting a tissue response upon implantation.
• Biocompatibility is determined by the specific biological response the material provokes in the living tissue.

Terminology

• In vitro: experiments conducted in a laboratory, outside a living organism
• In vivo: experiments conducted within a living organism
• Ex vivo: experiments conducted on cells or tissues removed from a living organism but still kept alive
• In situ: experiments conducted in the original location or position of the cells/tissue in the organism
• In silico: experiments or modeling uses computational methods

In Vitro Cell Culture Methods

• Cell culture: growing cells outside the living organism
• Tissue culture: growing portions or whole tissues outside the living organism
• Organ culture: growing organs outside the living organism

Cell Culture Setup

• Images depict various equipment used in cell culture laboratories.
• Equipment includes air handling units (biological safety cabinets, class 14/11), automated liquid handling systems, incubators, cell culture plates, and containers.

Why Study Biomaterials?

• To avoid using implantation toxic materials
• To predict how a material or device functions upon implantation
• To minimise the use of animals in research

Toxicity, Necrosis, Apoptosis

• Cell cultures are used to assess biomaterial compatibility.
• A toxic material releases substances to kill cells directly or by inhibiting metabolic pathways.
• Necrosis is accidental cell death; apoptosis is programmed cell death.

Two Broad Categories of Cell Types

• Primary cells: derived from fresh tissues, expensive to maintain, availability is a concern, their phenotype and function may degrade in culture; highly relevant for in vivo studies
• Immortalized cells (cell lines): Derived through viral transformation or introduction of telomerase reverse transcriptase (TERT); potentially infinite lifespan but may lose normal functions; less relevant for in vivo.
• Ethical considerations regarding the use of immortalized cells include the HeLa cells story (Henrietta Lacks).

What Do We Measure In Culture?

• Cell survival (toxicity); the integrity of organelles and membranes, damage to chromosomes
• Cell production, growth inhibition
• Cell metabolic activity (energetics, synthesis and catabolism)
• Cell effective activity (locomotion, chemotaxis, phagocytosis; alteration of cell shape and size, proteins and gene expression)

Cell Morphology Assessment

• Visual analysis of cells (e.g., microscopy) helps to understand morphology and identify cell types.
• Images in the slides show examples of cell morphology analysis.

Toxicity Assessment

• Direct contact, agar diffusion, and elution are crucial methods for determining toxicity using biological models.
• Each method varies in the specific conditions and preparation required.

Cell Confluency Assessment

• Confluency reflects the density of cells in a culture and usually experiments are run when the confluency is ~90%.

Cell Viability Assessment

• Trypan blue exclusion tests; live cells have intact membranes that do not allow trypan blue to enter.
• Live/Dead® assay measures live and dead cells simultaneously through calcein AM fluorescence and propidium iodide fluorescence.

Cell Proliferation Assessment

• PicoGreen® dye fluoresces upon interacting with double-stranded DNA, allowing for the measurement of DNA quantities.
• Nuclei counting, using 4',6-diamidino-2-phenylindole (DAPI), also provides a way to determine the number of cells or nuclei present.

Cell Metabolic Activity Assessment

• MTT assay: measures cell metabolic activity using a colorimetric assay based on the reduction of MTT to formazan.
• AlamarBlue assay: measures cell metabolic activity using a fluorometric/colorimetric assay; the dye resazurin is reduced to a fluorescent dye.

Cell Proliferation, Metabolic Activity, and Viability

• Graphs presented in presentation show the measured data from different material and cell combinations across multiple days of culture.

Protein synthesis: Gel Electrophoresis

• Electrophoresis is used to separate molecules based on size, charge, and shape, providing insights into the composition of proteins.
• Polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gel electrophoresis are described.

Immunocytochemistry / Immunohistochemistry

• Immunocytochemistry (ICC) and immunohistochemistry (IHC) methods detect protein/antigen presence in tissue or cells via antibodies.
• The method involves using antibodies to target specific antigens within the sample.

Immunocytochemistry/Immunohistochemistry; Flow chart and optimisation

• The presented flowcharts guide the steps in performing and optimising the immunocytochemical/immunohistochemical method for various sample types.

Immunocytochemistry / Immunohistochemistry; Considerations.

• Choosing the appropriate species for the antibodies used against the sample
• Antibody testing for cross-reactivity
• Validation of the secondary antibody for the application is essential

Immunocytochemistry in physiological protein deposition

• The images are demonstrating successful use of immunocytochemistry techniques to detect certain proteins in tissues.

Immunocytochemistry in physiological cell function

• Immunocytochemistry and various fluorescent stains (e.g., Hoechst 33342, BODIPY TR-X phallaoidin, DiOC6) allow for the visualisation of structures like the nucleus, actin filaments, and organelles.

Immunocytochemistry in non-physiological protein deposition

• The presented images are illustrating the use of immunocytochemistry for examining collagen deposition in response to different concentrations of carrageenan (CR) in cells.

Immunohistochemistry in skin pathophysiologies

• Visualising skin tissue for pathology using stained samples using immunohistochemistry.

Immunohistochemistry for tissue graft / biomaterial composition verification

• Images show immunohistochemistry to detect different proteins in tissue and biomaterial samples, which validates the composition and structure.

Gene expression analysis: Polymerase chain reaction (PCR)

• PCR is a technique that amplifies DNA portions, useful for detecting or quantifying nucleic acid contents.

Hall of Fame - Kary B. Mullis

• Kary B. Mullis invented the Polymerase Chain Reaction (PCR) method, allowing for the amplification of small DNA fragments into large copies.

Real-time PCR in action

• Real-time PCR is used to measure gene expression levels across different groups of cells and/or types of cartilage at different time points in culture.

Gene array

• A gene array is used to assess the expression levels of various genes in response to environmental or experimental factors.
• Gene expression data is displayed as heatmaps showing which genes are up- or down-regulated.

Omics Era

• Genomic, transcriptomic, proteomic, and metabolomic are related and used for studying complete systems of life processes.

Omics Data

• The slide presents the use of single-cell RNA sequencing to analyze the diversity, variety, and transcriptomics heterogeneity in cancer cells.

Advantages and disadvantages of in vitro culture

• In vitro assays provide information on cellular factors relevant to the material.
• Limitations include the lack of the physiological in vivo context.