Nội dung

Introduction to Animal Tissue culture What is tissue culture?  In vitro culture (maintain and/or proliferate) of cells, tissues or organs.  Types of tissue culture • Cell culture • Primary explant culture • Organ culture 2 Three major categories of tissue culture  Cell culture: Adherent monolayer on a solid substrate (various cell types) suspension in the culture medium (few cell types)  Primary explant culture: A fragment of tissue attachment and migration occurs in the plane of the solid substrate  Organ culture: Expla nt cultur e Cell culture A spherical or three-dimensional shape specific histological interaction Explant: living cells, tissues, or organs from animals or plants that transfer to a nutrient medium. 3 Cell culture & Enzymatic Dissociation Tissue from an explant is dispersed, mostly enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture. 4 Advantages & Disadvantages Advantages  Development of a cell line over several generations  Scale-up is possible  Absolute control of physical environment  Homogeneity of sample  Less compound needed than in animal models Disadvantages  Cells may lose some differentiated characteristics.  Hard to maintain  Only grow small amount of tissue at high cost  Dedifferentiation  Instability, aneuploidy 5 Tissue Culture Is the growth of tissues or cells separate from the organism.  This is typically facilitated via use of a liquid, semisolid, or solid growth medium, such as broth or agar.  6 Advantages & Disadvantages Advantages  Some normal functions may be maintained.  Better than organ culture for scale-up but not ideal. Disadvantages  Original organization of tissue is lost. 7 Organ culture  The entire embryos or organs are excised from the body and culture  Advantages  Normal physiological functions are maintained.  Cells remain fully differentiated.  Disadvantages  Scale-up is not recommended.  Growth is slow.  Fresh explantation is required for every experiment. 8 EMP04 9 Why do we need Cell culture? Research ◦ To overcome problems in studying cellular behavior such as:  confounding effects of the surrounding tissues  variations that might arise in animals under experimental stress ◦ Reduce animal use Commercial or large-scale production ◦ Production of cell material: vaccine, antibody, hormone 10 Initiation of culture Animal Plant Tissue Primary culture Subculture Stored Stored Cell line Finite numbers Continuous cell line Indefinite numbers 11 Types of Cell culture 1. Primary Cultures  Derived directly from excised tissue and cultured either as:  Outgrowth of excised tissue in culture  Dissociation into single cells (by enzymatic digestion or mechanical dispersion). Primary Culture Preparat ion 12 Characteristics of Primary Cultures  Characteristics:  Morphologically similar to the parent tissue  Limited number divisions of Primary Culture Preparat ion cell  Best experimental models for in vivo situations 13 Advantages & Disadvantages ◦ Advantages:  usually retain many of the differentiated characteristics of the cell in vivo ◦ Disadvantages:  initially heterogeneous but later become dominated by fibroblasts.  the preparation of primary cultures is labor intensive  can be maintained in vitro only for a limited period of time.  Difficult to obtain  Relatively short life span in culture  Very susceptible to contamination  May not fully act like tissue due to complexity of media 14 Types of Cell culture 2. Continuous Cultures  derived from subculture (or passage, or transfer) of primary culture  Subculture = the process of dispersion and re-culture the cells after they have increased to occupy all of the available substrate in the culture  usually comprised of a single cell type  can be serially propagated in culture for several passages  There are two types of continuous cultures  Cell lines  Continuous cell lines 15 Types of continuous culture 1) Cell lines  Cell lines derived from primary cultures have a limited life span  After the first subculture, the primary culture becomes cell line  finite life, senesce after approximately thirty cycles of division  usually diploid and maintain some degree of differentiation  it is essential to establish a system of Master and Working banks in order to maintain such lines for long periods 16 Types of continuous culture 2) Continuous cell lines   can be propagated indefinitely generally have this ability because they have been transformed by:  tumor cells.  viral oncogenes  chemical treatments  Spontaneously  the disadvantage of having retained very little of the original in vivo characteristics 17 Transformation VS Transfection Transformation ◦ Spontaneous or induced permanent phenotypic changes resulting from change in DNA and gene expression that result and effect in:  growth rate  mode of growth (loss of contact inhibition)  specialized product formation  longevity  loss of need for adhesion Transfection ◦ Introduction of DNA into a cell (like viral DNA) 18 Cell Culture Morphology  Morphologically cell cultures take one of two forms: ◦ growing in suspension (as single cells or small free-floating clumps)  cell lines derived from blood (leukemia, lymphoma) ◦ growing as a monolayer that is attached to the tissue culture flask.  Cells from solid tissue (lungs, kidney, breast), endothelial, epithelial, neuronal, fibroblasts Hela-Epithelial HT1080- kidney MRC5-Fibroblast BAE1-Endothelial SHSY5Y-Neuronal MCF-7 breast 3LL - lungs 19 Cell culture application • Excellent model systems for studying:  The normal physiology and biochemistry of cells  The effects of drugs and toxic compounds on the cells  Mutagenesis and carcinogenesis • Used in drug screening and development • Large scale manufacturing of biological compounds (vaccines, insulin, interferon, other therapeutic protein)