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BME 565 Exam 1 Study Guide

by: Hailey Rooney

BME 565 Exam 1 Study Guide BME 565

Marketplace > University of Miami > Biomedical Engineering > BME 565 > BME 565 Exam 1 Study Guide
Hailey Rooney
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Review of all material from PowerPoint presentations and class notes to prepare for the first exam
Principles of Cellular and Tissue Engineering
Study Guide
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This 5 page Study Guide was uploaded by Hailey Rooney on Wednesday September 28, 2016. The Study Guide belongs to BME 565 at University of Miami taught by in Fall 2016. Since its upload, it has received 6 views. For similar materials see Principles of Cellular and Tissue Engineering in Biomedical Engineering at University of Miami.

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Date Created: 09/28/16
BME 565 Exam 1 Study Guide  Tissue engineering purpose: develop biological substitutes for implantation to replace, repair,  maintain, or enhance function o Need: donor organ shortage, aging, difficulty using synthetic drugs or devices o REPLACE, REGENERATE, REPAIR  Max transport of nutrients for cells is about 200 microns via diffusion o Can be forced through further via agitation or perfusion  Modeling cell growth (N=N e0)kt  Matrix is substrate for attachment, multiplication, and differentiation toward desired tissue o Synthetic: reproducible, controllable shapes/properties, no pathogenic substances; foreign to  cells and have poor cell attachment and changes in cell morphology o Natural: familiar to cells; non­optimal control of properties/reproducibility, disease potential o Modular synthetic hydrogels: incorporate bioactive domains of natural ECM components,  provide cell­controlled degradation and permit migration/remodeling of hydrogel, possess  growth factor binding sites that contain a protease cleavage site for cell­mediated release  Tissue engineering objectives: o Alternative to gene therapy, drug therapy, organ transplantation o Control of natural processes of tissue repair/healing o Replace missing cells w/ functional organ or tissue o New ways to model human physiology  Transplant types: o Autogenic: patient’s own cells o Allograft: same species o Xenograft: different species o Synthetic: from synthetic materials  Stem cell types: o Hematopoietic for red blood cells o Epithelial for skin o Osteoclasts, osteoblasts, osteocytes for bone o Neural for CNS o Cardiomyocytes for heart  Epigenesis: process by which a simple initial structure becomes complex  Embryonic development: zygote => 8­cell stage => blastula  Gastrulation: establishing the three germ cell layers o Ectoderm: neural rest, neural tubes, lens, cornea, skin o Mesoderm: connective tissues, muscles, circulatory sytem o Endoderm: respiratory system, gut, endocrine  Cell potency: o Totipotent: can reproduce entire organism o Pluripotent: can generate all cells but embryonic tissue o Multipotent: limited number of lineages o Unipotent: only one type of cell fate  Self­renewal: ability of stem cell to divide and make identical copies of itself  All cells are equivalent; just regulated differently genetically  Induction of cell fate: o Conditional: environmentally deterministic (extrinsic)  Cellular cues, immobilized cues (matrix interactions), soluble cues (gradient fields) o Autonomous: internally endowed (intrinsic)  Stem cell niche: complex/dynamic structure that transmits & receives signals through cellular and  acellular mediators o The stem cell itself o Stromal cells o Soluble factors o Extracellular matrix o Neural inputs o Vascular network o Cell adhesion components  Cells retain a strong memory of their tissue or embryonic origin  De­differentiation (iPSC): reverting a differentiated cell to enhance cell fate potential  Trans­differentiation: changing cell from one lineage to another (fibroblast => cardiomyocyte)  Epigenetic regulation mechanisms: o DNA methylation: add methyl group to 5 position of cytosine o Histone modification o RNA­based mechanisms  Stem Cell Types: Advantages & Disadvantages o Embryonic: from blastocysts, unlimited self­renewal, totipotent  Cell colonies or embryoid bodies  Require feeder cells, lack of knowledge on cues for desired differentiation, low  conversion %, purity of final product, ethical issues, can form teratoma o Adult: multipotent, limited self­renewal  Easy sources (blood, bone marrow, fat, dental pulp, connective tissues) or hard  sources (liver, pancreas, brain, specialized organs) o Mesenchymal: pluripotent, slow self­renewal; both are variable  From all three germ layers  Serve as helper cells to promote positive remodeling & vascularization, homing of  cells to injury, secrete immunomodulatory proteins, no immunogenic markers,  secretors of cytokines, chemokines, etc.  Lack of stability of DNA and genome, teratoma formation, “helper” cells can  increase cancer growth o iPSC: multipotent, not as long as ESC, teratoma formation  organoids: collection of organ­specific cell types that develops from stem cells or organ progenitors  and self­organizes through cell sorting and spatially restricted lineage commitment similar to in vivo o multiple organ­specific cell types o can recapitulate specific function of the organ o spatially organized similarly to an organ  recreating stem cell niche: supplement with growth factors, signals, feeder layers of other cells  conditional specification of cell fate o support cells: secrete transmembrane cell­cell adhesion proteins, soluble factors, and the  surrounding ECM o important for differentiation and maintaining a supply of stem cells o orientation of the mitotic spindle determines cell fate  homeostatic conditions: asymmetric; one SC and one partially differentiated daughter  development/stress: symmetric; both daughter cells are SCs  symmetric differentiation: both daughters lose SC function  perfluorocarbon: dissolve oxygen (hemoglobin just binds the molecule), enabling them to load/unload oxygen twice as fast as hemoglobin  primary cells: isolated from tissues via biopsy o mixture of cells that requires purification to get cell of interest; variable culture methods  needed depending on type; variable cell characteristics  secondary cells (cell line): cells from in vitro cultures of primary cells o capable of 50­100 generations before terminating; easily expanded, grown, and frozen  continuous (immortalized): growth properties of cells altered to enable them to continue growing and  dividing indefinitely in culture if conditions are maintained o usually tumorgenic; transformation process can be genetic (DNA tumor virus) or conditional  cell death: o apoptosis: programmed cell death  cell shrinkage, loss of membrane asymmetry and attachment, gradual breakdown of  cellular components o necrosis: sudden cell death 1. cell swelling, chromatin digestion, disruption of plasma & organelle membranes 2. DNA hydrolysis, organelle breakdown, cell lysis (cell contents dumped)  controlling cell death: o soluble chemical factors:   exogenous (placed in media/matrix)  endogenous:  endocrine:  paracrine:  autocrine:  types: mitogens induce mitosis, cytokines (hormones) influence cell behavior, growth factors influence cell differentiation or growth  factors = unknown mechanism, hormones = known mechanism  nutritional factors: based on cell phenotype, variable needs of tissue, oxygen is  usually the dominating nutrient o cell­cell interactions:  homotypic: between stem cells through cell­cell contact  heterotypic: between stem and niche cell through soluble signaling  adherent cells and non­adherent cells: single cells (monolayers) or spheroids  monolayer: growth inhibited at critical surface density  spheroids: cell­cell interactions necessary for adequate function/interaction,  proliferating cells in outer 3­5 cell layers with necrotic cores at 50­300 micro  micropatterning creates tissues with defined architecture to control cell­cell  interactions and determine the effects of modulating it o cell­matrix interactions: define phenotype, growth, adhesion, and migration o surface receptors (cell adhesion molecules): mediate interactions b/w cell­cell & cell­matrix  selectivity of receptor/ligand interaction => specificity of adhesion characteristics for  cells and tissues  binding can be homotypic, heterotypic, linker mediated, or to ECM  affinity: binding strength  avidity: combined synergistic strength of bond affinities (# of receptor/ligand  interactions collected together)  cell adhesion molecules: o integrins: primarily responsible cell to matrix binding; heterodimer with alpha and beta  chains  low specificity, low affinity o cadherins: cell­cell binding; calcium dependent, usually hemophilic binding to cadherin on  another cell  high specificity, low affinity o selectins: cell­cell binding; single­chain transmembrane glycoprotein  low specificity, low affinity o  immunoglobulin (Ig) superfamily: cell­cell hemophilic binding, can regulate adhesion to the  ECM by binding integrins and inducing signaling o Transmembrane proteoglycans: cell­cell and cell­matrix; reduce non­specific binding  Cell migration/adhesion: mediated through expression of cell adhesion molecules o Chemotaxis: induced by secretion of cytokines or proteins by a cell or ECM by­product; cell  is polarized towards the source  Cell migration by cell type: o Neutrophils: phagocytosis of bacteria o Lymphocytes: destruction of infected cell o Macrophages: antigen presentation o Endothelial cells: angiogenesis o Epidermal cells & fibroblasts: wound healing o Tumor cells: metastasis o Neuron & axons: development and regeneration in nervous system o Embryonic cells: embryogenesis  Self­assembled protein monolayer: control cell­matrix interaction  Cell isolation: o Basic primary cell isolation: add tissue to buffer, add enzymes & incubate at optimum  temperature, resuspend cells in medium o Enzymes used:  Collagenase: highly specific, breaks triple helix collagen found in connective tissue  Trypsin: little selectivity for extracellular proteins => ineffective for tissue  dissociation; cleaves peptide chains at carboxyl site of the amino acid’s lysine or  arginine; MOST widely used for cell culture of adherent cells  Elastase: hydrolyze native elastin  Hyaluronidase: breaks hyaluronic acid down  Papain: wide specificity to degrade most protein substrates  Chymotrypsin: hydrolysis of peptide bonds o Mechanical disruption: agitation to release cells from ECM; yield vs. viability  Good to use if cell surface markers are susceptible to enzymatic digestion  Cell sorting: o Gradient: sort by cell density o MACS or FACS sort by target antibody  FACS advantages: high purity, don’t require a lot of antibodies, multicolor staining,  internal staining sorting o Positive selection: label desired cells o Depletion: label cells you are not interested in  Cell media: buffer, amino acids, vitamins, salts, glucose, hormones/growth factors; commonly  supplemented with serum and antibiotics  Cell proliferation & viability: o Cell counting using hemocytometer, Coulter counter o Mitosis indicator using BrdU o Cytoplasmic dyes pass through membrane for live/dead markers o Metabolic activity using MTT/MTS dyes (absorbance proportional to cell number), oxygen  consumption rate o Proliferation by radioactivity incorporated during replication, CFSE dilution o Cell death by propidium iodide (excluded from viable cell membranes, used for necrosis),  Annexin V used for detecting necrosis,  TUNEL detects DNA fragmentation from apoptosis  (membrane intact), Capase detects apoptotic cells  Adhesion: centrifugation, micropipette, or microfluidic assay  Mobility: scrape wound, agarose migration, fence assay, Boyden chamber  Histology: use hematoxylin or eosin, TriChrome staining  Immunohistochemistry: primary antibody, secondary antibody, fluorescent or colormetric tag o Weston blot shows presence but not location, ELISA is quantitative, PCR (quantitative for  assessment of gene expression), in situ hybridization (qualitative) detects complementary  DNA sequence


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