Biomaterials and Cell Culture Quiz

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Questions and Answers

What is the primary benefit of using hTERT-immortalized cells in research?

They avoid the need for ethical considerations.

They enhance the effectiveness of chemotherapy.

They are less expensive to maintain.

They can replicate indefinitely. (correct)

What is a common artifact encountered in electrophoresis?

Chemical precipitation on the gel. (correct)

Noise from background signals.

Overexposure of gel images.

Misinterpretation of migration patterns.

What technique is utilized to visualize proteins in polyacrylamide gels?

Electrochemical detection.

Radioactive labeling.

Fluorescent dye staining.

Silver staining. (correct)

Which of the following is a focus of immunocytochemistry?

Analyzing specific proteins or antigens in cells. Signup and view all the answers

Which publication discusses the mechanism of immortalization?

Mechanism of Immortalization in Aging. Signup and view all the answers

What defines the host response in the context of biomaterials?

The local and systemic response of living systems to the material Signup and view all the answers

What does the term 'biocompatibility' refer to?

The specific biological response to a material when implanted Signup and view all the answers

Which of the following best describes the process of in vitro?

Culturing cells outside a living organism Signup and view all the answers

What type of culture involves the growth of whole organs outside the living system?

Organ culture Signup and view all the answers

What is the significance of the material response in biomaterials?

It describes how the material reacts to living tissue Signup and view all the answers

Which term accurately describes the process of living cells or tissues cultured in a lab for a brief period?

Ex vivo Signup and view all the answers

The phrase 'it takes two to tango' in the context of biological responses refers to which interactions?

The interaction between the host and the implanted material Signup and view all the answers

What is one advantage of in vitro testing compared to in vivo testing?

It provides quantifiable results more easily. Signup and view all the answers

What aspect of biomaterials does the term 'in situ' describe?

Studying materials in their natural position within the body Signup and view all the answers

Which limitation is associated with in vitro characterisation?

It lacks the ability to maintain a three-dimensional environment. Signup and view all the answers

What effect does prolonged immobilization have on bone culture in vitro?

Leads to increased bone resorption. Signup and view all the answers

How can in vitro studies potentially misrepresent material toxicity?

They may overestimate toxicity levels due to short cell lifespan. Signup and view all the answers

What is often missing in in vitro models that limits their effectiveness in simulating in vivo conditions?

Systemic factors including vascularization. Signup and view all the answers

Which challenge arises from phenotypic drift in in vitro cultures?

Altered responses compared to natural conditions. Signup and view all the answers

What is one consequence of using a two-dimensional surface for cell growth in vitro?

It may lead to altered cellular behavior. Signup and view all the answers

What is a common misconception about in vitro testing regarding material quality screening?

It can effectively predict long-term health outcomes. Signup and view all the answers

What needs to be considered when selecting a primary antibody for a sample?

It should be raised from a different species than the sample. Signup and view all the answers

What is an important requirement for secondary antibodies in immunocytochemistry?

They should be against the host species of the primary antibody. Signup and view all the answers

Why is it important to predict cross-reactivity when selecting antibodies?

To confirm the antibody's affinity for the primary target protein across species. Signup and view all the answers

For dual immunostaining, what is necessary regarding the primary antibodies?

They should be raised in different species. Signup and view all the answers

What must be verified when choosing a secondary antibody?

It must be listed as suitable for the intended application. Signup and view all the answers

Which of the following statements is true regarding the choice of species for antibodies?

The primary antibody should not be from the species of the sample. Signup and view all the answers

What is the benefit of using directly conjugated primary antibodies?

They do not need any secondary antibody. Signup and view all the answers

Which type of collagen is mentioned in relation to physiological protein deposition?

Collagen I Signup and view all the answers

What is the significance of the REDOX indicator changing from blue to red?

It reflects continued cell proliferation and metabolic activity. Signup and view all the answers

Which absorbance wavelengths are used to monitor cell viability and growth?

570 nm and 600 nm Signup and view all the answers

What advantage do horizontal submarine agarose gels have for nucleic acid separation?

They allow for the screening of multiple samples simultaneously. Signup and view all the answers

How do polyacrylamide gels compare to agarose gels in terms of band sharpness?

Polyacrylamide gels yield sharper bands. Signup and view all the answers

What does the term electrophoresis refer to?

The migration and separation of charged particles according to size in an electric field. Signup and view all the answers

Why is the growth inhibition of cells significant in the context of metabolic activity?

It maintains an oxidized environment. Signup and view all the answers

What type of biomaterials is Endoform (OF-EF) made from?

Ovine forestomach Signup and view all the answers

In what application are vertical polyacrylamide gels primarily used?

Purification of synthetic oligonucleotides Signup and view all the answers

What is the primary goal of metabolomics?

Determining a sample’s profile of compounds under specific conditions Signup and view all the answers

How do glioblastoma stem cells (GSC) and normal cells cluster in the study?

By patient, while normal cells cluster together Signup and view all the answers

What do the heatmaps of gene expression represent in relation to GSCs?

Relative gene expression sorted by principal component analysis Signup and view all the answers

Which genes are primarily focused on when analyzing GSCs based on PC1?

Genes with the lowest and highest PC1 loading values Signup and view all the answers

What does the mutually exclusive expression of CD24 and CD44 imply about GSCs?

Expression of these markers indicates different subtypes of GSCs Signup and view all the answers

In the flow cytometry analysis of GSCs, what was contrasted?

CD24 and CD44 expression levels Signup and view all the answers

In the context of gene expression analysis, what do the cNMF signatures indicate?

Distinct groupings corresponding to various cell types Signup and view all the answers

What does a high expression indicated by the red color in the gene expression heatmaps signify?

High gene activity within the sample Signup and view all the answers

Study Notes

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.

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Description

Test your knowledge on biomaterials, hTERT-immortalized cells, and techniques for culturing cells. This quiz covers various aspects of immunocytochemistry, biocompatibility, and in vitro methodologies. Perfect for students and researchers in the biomedical field!