Lecture 14 - Biological Properties PDF

Biological Properties of Biomaterials Dimitrios I. Zeugolis; BSc (Hons), MSc, PhD Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research, School of...

Biological Properties of Biomaterials Dimitrios I. Zeugolis; BSc (Hons), MSc, PhD Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research, School of Mechanical & Materials Engineering, University College Dublin (UCD), Dublin, Ireland www.ucd.ie MEEN40630 Biomaterials www.remodel.ie What types of biological response do we have in culture / upon implantation? (Hint: It takes two to tango) MEEN40630 Biomaterials  Biological response Host response: The local and systemic response, other than the intended therapeutic response of living systems to the material. Material response: The response of the material to living system. All materials will undergo tissue responses when implanted into living tissue. The specific biological response to the chosen material determines its biocompatibility. MEEN40630 Biomaterials  Terminology In vitro: in the lab In vivo: in the body Ex vivo: living cells or tissues cultured in the lab for a few hours In situ: in position In silico: modelling MEEN40630 Biomaterials  In vitro cell culture methods Cell Culture Growth of cells outside the living system Tissue Culture Growth of portion(s) or whole tissues outside the living system Organ Culture Growth of organ(s) outside the living system MEEN40630 Biomaterials  Cell culture setup MEEN40630 Biomaterials Why do we bother? MEEN40630 Biomaterials  We do we bother? To avoid the implantation toxic materials To predict the behaviour of the material or the device upon implantation To reduce the number of animals in research MEEN40630 Biomaterials  Toxicity, Necrosis, Apoptosis Cell culture systems are customarily used to evaluate the compatibility or cytotoxicity of a biomaterial or a device with living cells. The rational for the use of cultured mammalian cells for analysis of toxicity is based on the fact that whatever reaction could induce disease or death in the animal or human initiates on cellular level. Cytotoxicity: Toxic effects at a cellular level; the cell-killing property of a chemical compound Toxic material: A material that releases chemicals in sufficient quantities to kill cells either directly or indirectly through inhibition of key metabolic pathways. Potency: Number of cells affected Sensitive cells are the target cells Necrosis: Accidental cell death Apoptosis: Programmed / regulated cell death MEEN40630 Biomaterials  Two broad categories of cell types Primary cells: Obtained from fresh tissues Expensive to maintain It is subject to tissue availability Long term experiments cannot be conducted (cells lose phenotype and function in culture) Variability as a function of passaging, time in culture High relevance to in vivo setting Immortalised cells (cell lines): Derive via introduction of simian vacuolating virus 40 (SV40) and human papillomavirus sequences into cells Viral genes achieve immortalization by inactivating the tumour suppressor genes (p53, Rb, and others) that can induce a replicative senescent state in cells. Derive via expression of telomerase reverse transcriptase protein (TERT) This protein is inactive in most somatic cells, but when human TERT is exogenously expressed, the cells are able to maintain sufficient telomere lengths to avoid replicative senescence. Can be expanded indefinitely Lack functions of normal (primary) cells No variability as a function of passaging, time in culture Low relevance to in vivo setting Ethics associated with immortalised cells? The HeLa (Henrietta Lacks) cell story MEEN40630 Biomaterials Ethics associated with immortalised cells? The HeLa (Henrietta Lacks) cell story MEEN40630 Biomaterials What do we measure in culture? MEEN40630 Biomaterials  What do we measure in culture? Cell survival: toxicity, organelle & membrane integrity Cell damage: chromosomal aberration, mutagenicity and carcinogenicity Cell reproduction: growth inhibition Cell metabolic activity: energetics, synthesis & catabolism Cell effective activity: locomotion, chemotaxis, phagocytosis, alteration of cell shape and size, protein synthesis, gene expression MEEN40630 Biomaterials  Cell morphology assessment Corneal Keratocytes Corneal Fibroblasts Corneal Myofibroblasts MEEN40630 Biomaterials  Toxicity assessment Direct contact Agar diffusion Elution Eliminate extraction Eliminate extraction preparation preparation Zone of diffusion Zone of diffusion Target cell contact with the Separate extraction from Better concentration material testing gradient of toxicant Mimic physiological Dose response effect Advantages Can test one side of a conditions Extend exposure time material Standardise amount of test Choice of extract conditions Independent of the material material or test Choice of solvents density indeterminate shapes Use filter paper disk to test Can extend exposure time liquids or extracts by adding fresh media Cellular trauma if material Requires flat surface moves Solubility of toxicant in agar Cellular trauma with high Risk of thermal shock when Disadvantages density materials preparing agar overlay Additional time and steps Decrease cell population Limited exposure time with highly soluble Risk of absorbing water toxicants from agar MEEN40630 Biomaterials  Cell confluency assessment Confluency: No restriction to the growth of the cells in/on a medium; it is assumed that cells are in a growth phase Experiments are usually run when cells at ~90 % confluency MEEN40630 Biomaterials  Cell viability assessment Trypan blue: Live cells have intact membranes that do not allow absorbance of trypan blue Dead cells have damaged cell membranes that allow the passage of trypan blue Live/Dead®: Simultaneous staining of live (green) and dead (red) cells Live cells (green) are distinguished by the presence of ubiquitous intracellular esterase activity as determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, which is well-retained within live cells. The red component of the Live/Dead® is cell-impermeant and therefore only enters cells with damaged membranes. In dying and dead cells a bright red fluorescence is generated upon binding to DNA. The live cell component produces an intense, uniform green fluorescence in live cells (excitation / emission: 488 nm / 515 nm). The dead cell component produces a predominantly nuclear red fluorescence (excitation / emission: 570 nm / 602 nm) in cells with compromised cell membranes, a strong indicator of cell death and cytotoxicity. MEEN40630 Biomaterials  Cell proliferation assessment PicoGreen®: Cell proliferation assay that uses a dye (PicoGreen®) that fluoresces upon interacting with double-stranded DNA (dsDNA). The fluorescence generated from a sample can be compared with that of a dsDNA standard and used to measure the DNA in that sample. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1,000-fold enhancement in its fluorescence (green cells). Nuclei counting: An alternative method is nuclei counting 4′,6-diamidino-2-phenylindole (DAPI), is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA Cell nuclei are stained blue MEEN40630 Biomaterials  Cell metabolic activity assessment MTT: It is a colorimetric assay that is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting coloured solution is quantified by measuring absorbance at 500-600 nm using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells. alamarBlue®: The alamarBlue® assay incorporates a fluorometric / colorimetric growth indicator. alamarBlue® is a cell metabolic activity assay, which uses the cell permeable, non-toxic and weakly fluorescent blue indicator dye called resazurin. Resazurin is used as an oxidation-reduction (REDOX) indicator that undergoes colorimetric change in response to cellular metabolic reduction. As cells being tested grow, innate metabolic activity results in a chemical reduction of alamarBlue®. Continued growth maintains a reduced environment while inhibition of growth maintains an oxidized environment. Reduction related to growth causes the REDOX indicator to change from oxidized (non-fluorescent, blue) form to reduced (fluorescent, red) form. Fluorescence is monitored at 530-560 nm excitation wavelength and 590 nm emission wavelength. Absorbance is monitored at 570 nm and 600 nm. MEEN40630 Biomaterials  Cell proliferation, metabolic activity and viability; All are needed Product Description & Name Collagen / Oxidised regenerated cellulose, Promogran (CORC-PG), Acelity , USA Ovine forestomach, Endoform (OF-EF), Hollister Wound Care, USA Porcine urinary bladder, MatriStem® (PUB-MS), ACell®, USA Porcine mesothelium, Meso Biomatrix® (PM-MB), DSM Biomedical, Netherlands Porcine mesothelium, Puracol® Ultra ECM (PM- PC), Medline Industries, USA MEEN40630 Biomaterials  Protein synthesis: Gel electrophoresis Vertical setup Horizontal setup Vertical polyacrylamide gels are also used for some nucleic acid separations, for example, Horizontal submarine agarose gels are used for the majority of nucleic acid separations. purification of synthetic oligonucleotides. An agarose electrophoresis gel can be used to separate a much wider range of DNA and RNA Polyacrylamide electrophoresis gels yield sharper bands than agarose gels. sizes than a polyacrylamide gel. Another advantage of horizontal submarine gels is that multiple rows of wells can be formed, enabling the screening of large numbers of samples on a single gel. MEEN40630 Biomaterials  Protein synthesis: Gel electrophoresis Principle: Electrophoresis is a chromatography technique and in general the term describes the migration and separation of charged particles (ions) according to their size under the influence of an electric field. An electrophoretic system consists of two electrodes of opposite charge (anode, cathode), connected by a conducting medium called an electrolyte. The separation effect on the ionic particles results from differences in their velocity (V), which is the product of the particle's mobility (m) and the field strength (E): V = m x E The mobility (m) of an ionic particle is determined by particle size, shape, and charge, and the temperature during the separation, and is constant under defined electrophoretic conditions. Electrophoretic conditions are characterized by the electrical parameters (current, voltage, power) and factors (ionic strength, pH value, viscosity, pore size, etc.), which describe the medium in which the particles are moving. Coomassie blue staining Coomassie Brilliant Blue binds non-specifically to virtually all proteins. Coomassie Blue staining is approximately 50-fold less sensitive than silver staining. Silver staining Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups. Silver staining allows for the detection of nanogram quantities of proteins. Drawbacks of silver staining: Many silver staining methods use glutaraldehyde as a sensitizer, which acts as a protein crosslinker making subsequent protein elution more difficult and modifies lysine residues preventing complete digestion and recovery of peptides. Silver ions inhibit trypsin digestion by binding at the active site of trypsin and chymotrypsin, thus preventing peptide fragmentation for mass spectroscopy analysis. Silver staining methods are unable to detect glycoproteins and proteins with large modified groups attached to their side-chains. MEEN40630 Biomaterials  Protein synthesis: Reduced and delayed gel electrophoresis Principle: Most biological molecules carry a net charge at any pH other than their isoelectric point and will migrate at a rate proportional to their charge density. The mobility of a molecule through an electric γ field will depend on the following factors: field strength, net charge on the molecule, size and shape of the molecule, ionic strength, and properties of the matrix through which the molecule migrates (e.g., viscosity, pore size). Polyacrylamide and agarose are two support matrices commonly used in electrophoresis. These matrices serve as porous media and behave like a molecular sieve. Agarose has a large pore size β and is suitable for separating nucleic acids and large protein complexes. Polyacrylamide has a smaller pore size and is ideal for separating majority of proteins and smaller nucleic acids. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by α1(III) mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Thus, when a current is applied, SDS-bound proteins in a sample will migrate α1(I) through the gel toward the positively charged electrode. Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel. α2(I) Delayed: Because it is stopped. 2 3 Reduced: Because a reducing agent (e.g. β-mercaptoethanol and dithiothreitol) is added to break disulfide bonds. 1 2 3 Samples on the right hand side image 1. Pepsin extracted rat tail tendon 2. Pepsin extracted bovine Achilles tendon 3. Pepsin extracted porcine Achilles tendon MEEN40630 Biomaterials  SDS-PAGE 1 2 3 MEEN40630 Biomaterials  Immunocytochemistry / Immunohistochemistry Immunocytochemistry (ICC) and immunohistochemistry (IHC) are techniques for the detection and visualization of proteins or other antigens, in cells and tissues, respectively, using antibodies specifically recognizing the target of interest. The antibody is directly or indirectly linked to a reporter, such as a fluorophore or enzyme. Antibody specificity can either be viewed as a measure of the goodness of fit between the antibody-combining site (paratope) and the corresponding antigenic determinant (epitope), or the ability of the antibody to discriminate between similar or even dissimilar antigens. MEEN40630 Biomaterials Immunocytochemistry / Immunohistochemistry; Flow chart and optimisation MEEN40630 Biomaterials Immunocytochemistry / Immunohistochemistry MEEN40630 Biomaterials  Immunocytochemistry / Immunohistochemistry; Considerations Sample species: If possible, choose an antibody that has been raised against the same species your sample is from. The antibody may react with the same target protein from other species sharing sufficient amino acid sequence homology. If your sample is not from one of the species listed in the datasheet, this means that the species has not been tested and we suitability has not been demonstrated. A prediction of cross-reactivity is made based on sequence similarity. Choosing the species of primary antibody host: The species the primary antibody is raised in should be different from the species of your sample. This is to avoid cross-reactivity of the secondary anti- immunoglobulin antibody with endogenous immunoglobulins in the sample. For instance, if you are studying a mouse protein, choose a primary antibody that is raised in a species other than mouse. A primary antibody raised in rabbit would be an appropriate choice, followed by an anti-rabbit IgG secondary antibody. Choosing a secondary antibody: Secondary antibodies should be against the host species of the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary. Check the datasheet of the secondary antibody to ensure it is tested in the application you will be using. Choosing antibodies for dual staining: Double immunostaining of cell cultures or tissue requires the primary antibodies to be raised in different species, and that the secondary antibodies recognize one of the species exclusively. Choose from our range of secondary anti-Ig antibodies which have been pre-adsorbed against immunoglobulins from other species to remove cross-reactivity. Alternatively, our directly conjugated primary antibodies remove the need for secondary antibodies. MEEN40630 Biomaterials  Immunocytochemistry in physiological protein deposition Collagen I Collagen III Collagen IV Collagen V Collagen VI Decorin -CR +CR A. Satyam et al., Advanced Materials, 2014, 26, p.p. 3024-3034 75 µg/ml Carrageenan (CR); WS1 skin fibroblasts; 0.5 % Human Serum; Day 3 (2 days with CR) MEEN40630 Biomaterials Immunocytochemistry in physiological cell function Chinese hamster ovary cells labeled with Hoechst 33342 and BODIPY TR-X phallaoidin. The Hoechst stains the nuclear DNA blue and BOPIPY TR-X phallaoidin stains filamentous actin red. Chinese hamster ovary cells labeled with Hoechst 33342 to stain the nuclear DNA blue and by immunofluorescence with an antitubulin antibody and a FITC-conjugated secondary antibody microtubules are stained green. Chinese hamster ovary cells labeled with Hoechst 33342 and DiOC6. The Hoechst stains the nuclear DNA blue, and the DiOC6 stains the internal organelles green. MEEN40630 Biomaterials  Immunocytochemistry in non-physiological protein deposition Day 2 Day 5 Day 10 Col I Col II Col I Col II Col I Col II 0 µg/ml CR V. Graceffa et al., Journal of 50 µg/ml CR Tissue Engineering & Regenerative Medicine, 2019, 13, p.p. 217-231 Carrageenan (CR); human chondrocytes at passage 7 100 µg/ml CR 500 µg/ml CR MEEN40630 Biomaterials  Immunohistochemistry in skin pathophysiologies Ichthyosis – skin disorder Normal Skin MEEN40630 Biomaterials Immunohistochemistry for tissue graft / biomaterial composition verification Product Description & Name Collagen / Oxidised regenerated cellulose, Promogran (CORC- PG), Acelity , USA Ovine forestomach, Endoform (OF-EF), Hollister Wound Care, USA Porcine urinary bladder, MatriStem® (PUB-MS), ACell®, USA Porcine mesothelium, Meso Biomatrix® (PM-MB), DSM Biomedical, Netherlands Porcine mesothelium, Puracol® Ultra ECM (PM-PC), Medline Industries, USA BM: basement membrane; CT: Connective tissue; SR: Serosa; PL: Papillae MEEN40630 Biomaterials  Gene expression analysis; Polymerase chain reaction (PCR) Principle: Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. The temperature of the sample is repeatedly raised and lowered to help a DNA replication enzyme copy the target DNA sequence. The technique can produce a billion copies of the target sequence in just a few hours. How does it work? To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called Taq polymerase synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis. Steps (in brief): Identify gene of interest Find gene sequence on NCBI (PubMed) Design primers – Sections of 18 nucleotides that will bind to portions of sequence Extract DNA from cells / tissue (using chemicals) Do PCR reaction to amplify gene of interest Put amplified gene into agarose gel – Is there a band? MEEN40630 Biomaterials  Hall of fame – Kary B. Mullis Kary B. Mullis The Nobel Prize in Chemistry 1993 Born: 28 December 1944, Lenoir, NC, USA Died: 7 August 2019, Newport Beach, CA, USA Prize motivation: "for his invention of the polymerase chain reaction (PCR) method.” Work: An organism\'s genome is stored inside DNA molecules, but analyzing this genetic information requires quite a large amount of DNA. In 1985, Kary Mullis invented the process known as polymerase chain reaction (PCR), in which a small amount of DNA can be copied in large quantities over a short period of time. By applying heat, the DNA molecule\'s two strands are separated and the DNA building blocks that have been added are bonded to each strand. With the help of the enzyme DNA polymerase, new DNA chains are formed and the process can then be repeated. PCR has been of major importance in both medical research and forensic science. MEEN40630 Biomaterials  Real-time PCR in action Cells: Human adipose derived stem cells TCP: Tissue culture plastic Collagen type II sponges fabricated from collagen extracted from: ❑ MARC: Male articular cartilage ❑ FARC: Female articular cartilage ❑ MTRC: Male tracheal cartilage ❑ FTRC: Female tracheal cartilage ❑ MAUC: Male auricular cartilage ❑ FAUC: Female auricular cartilage MEEN40630 Biomaterials  Gene array Gene array of tenocytes on PLGA substrates to variable groove depth (2 um groove width and distance between grooves) MEEN40630 Biomaterials  PCR MEEN40630 Biomaterials  Omics era Genomics is the new science that deals with the discovery and noting of all the sequences in the entire genome of a particular organism. The genome can be defined as the complete set of genes inside a cell. Genomics, is, therefore, the study of the genetic make-up of organisms. Transcriptomics is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome, under specific circumstances or in a specific cell—using high-throughput methods, such as microarray analysis. Comparison of transcriptomes allows the identification of genes that are differentially expressed in distinct cell populations, or in response to different treatments. Proteins are responsible for an endless number of tasks within the cell. The complete set of proteins in a cell can be referred to as its proteome and the study of protein structure and function and what every protein in the cell is doing is known as proteomics. The proteome is highly dynamic and it changes from time to time in response to different environmental stimuli. The goal of proteomics is to understand how the structure and function of proteins allow them to do what they do, what they interact with, and how they contribute to life processes. Metabolomics is one of the newest ‘omics’ sciences. The metabolome refers to the complete set of low molecular weight compounds in a sample. These compounds are the substrates and by-products of enzymatic reactions and have a direct effect on the phenotype of the cell. Thus, metabolomics aims at determining a sample’s profile of these compounds at a specified time under specific environmental conditions. MEEN40630 Biomaterials  Omics data Figure: Single-cell RNA sequencing highlights transcriptomic heterogeneity in glioblastoma and glioblastoma stem cells. a. tSNE of location-averaged transcriptome for all tumor cells colored by patient. Cancer cells cluster by patient, whereas normal cells from all patients cluster together (encircled clusters indicated by arrows). GSC corresponds to glioma stem cell samples, W corresponds to whole samples. b. Enriched glioblastoma stem cell (GSC) gene expression heatmaps showing relative gene expression (raw data) sorted by PC1 per patient. These maps are separated into three rows: top row—100 genes with the lowest value for PC1 loading; bottom row—100 genes with the highest value for PC1 loading; middle row—100 genes with the highest value for PC2 loading. These gene signatures correspond to neuronal, astrocytic, and progenitor signatures, respectively. The TCGA subtype is also shown for each GSC. c. Mean and actual rank of genes by PC1 correlation. The actual gene rank (y axis, one point per sample) correlates strongly with the mean gene rank (x axis) in all patients. d. Flow cytometry analysis of GSCs and whole-tumor, demonstrating mutually exclusive expression of CD24 and CD44. e. Heatmap of gene expression by cNMF signature with associated cell cycle scores and TCGA subtype (right). The most characteristic genes for each signature group are depicted on the x axis. Signatures (y axis) are ordered according to hierarchical clustering (left tree). Left color bar represents the patient sample that generated each signature—patient colors match those in Fig. 1a. Red represents high expression; blue represents low expression. Gene signatures groupings correspond to progenitors, astro-glia (mesenchymal and classical), and neurons, with the addition of cell cycle and hypoxia signatures. cNMF—clustered non-negative matrix factorization. f. Heatmap of gene expression by signature ordered by patient as shown by the left color bar. Genes (x axis) are in the same order as Fig. 1e. Patient colors in the color bar match those in Fig. 1a, e. Each patient contains signatures from multiple groups. Couturier CP, Ayyadhury S, Le PU, Nadaf J, Monlong J, Riva G, Allache R, Baig S, Yan X, Bourgey M, Lee C, Wang YCD, Wee Yong V, Guiot MC, Najafabadi H, Misic B, Antel J, Bourque G, Ragoussis J, Petrecca K. Single- cell RNA-seq reveals that glioblastoma recapitulates a normal neurodevelopmental hierarchy. Nat Commun. 2020 Jul 8;11(1):3406. doi: 10.1038/s41467-020-17186-5. Erratum in: Nat Commun. 2020 Aug 7;11(1):4041 MEEN40630 Biomaterials  Advantages and disadvantages of in vitro culture In vitro testing gives information regarding cytotoxicity, cell proliferation and differentiation and is more easily standardised and quantifiable than in vivo testing. Does that make sense? Why? In vitro studies are also useful for screening new materials for product quality and the release of potentially harmful additives incorporated during the manufacturing process. In vitro characterisation is not able to demonstrate the tissue response to materials. Cellular responses can vary between cell lines and passage number. In vitro tests could overestimate the level of material toxicity and are limited to acute studies of the effects of toxicity due to the relatively short lifespan of cultured cells. In vitro organ culture maintains tissue or organs, which may allow some differentiation and preservation of architecture and function. However systemic factors are absent, the lack of vascularisation (the formation of blood vessels) limits nutrient and oxygen supply and waste removal and therefore extrapolation of results to the in vivo situation limits the model. In vitro cells may suffer from phenotypic drift, which may be due to dissociation of cells from their three-dimensional geometry and/or growth on a two-dimensional surface (e.g. keratocytes; tenocytes; chondrocytes). It is difficult to recreate in vitro the appropriate cell interactions found in vivo. One major limitation to bone culture is the lack of controlled physiological loading since without load bone will increase resorption, as is seen in patients after prolonged bed rest. The same applies for tenocytes. It is impossible to recreate in vitro the in vivo biological environment. MEEN40630 Biomaterials  References / Further reading Ethics in immortalisation: https://www.nature.com/articles/d41586-020-03042-5 https://www.nature.com/articles/d41586-020-02494-z The Immortal Life of Henrietta Lacks (2010), Rebecca Skloot Immortalisation: Nakamura H. hTERT-immortalized cells useful for analyzing effects of low-dose-rate radiation on human cells. J Radiat Res. 2008 Jan;49(1):9-15 Hubbard K, Ozer HL. Mechanism of immortalization. Age (Omaha). 1999;22(2):65-69. doi:10.1007/s11357-999-0008-1 Electrophoresis: Chevallet M, Luche S, Rabilloud T. Silver staining of proteins in polyacrylamide gels. Nat Protoc. 2006;1(4):1852-1858. doi:10.1038/nprot.2006.288 Kurien BT, Scofield RH. Common artifacts and mistakes made in electrophoresis. Methods Mol Biol. 2012;869:633-640. doi:10.1007/978-1-61779-821-4_58 The eukaryotic cell cycle: https://www.ncbi.nlm.nih.gov/books/NBK9876/ Immunocytochemistry: Dey P. (2018) Immunocytochemistry in Histology and Cytology. In: Basic and Advanced Laboratory Techniques in Histopathology and Cytology. Springer, Singapore. https://doi.org/10.1007/978-981-10-8252-8_16 Lovchik, R.D., Taylor, D. & Kaigala, G. Rapid micro-immunohistochemistry. Microsyst Nanoeng 6, 94 (2020). https://doi.org/10.1038/s41378-020-00205-2 MEEN40630 Biomaterials