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Basic Principles of Cell Culture - Universidad de Granada

1 Basic Principles of Cell CultureR. Ian FreshneyCentre for Oncology and Applied Pharmacology, Cancer Research UK BeatsonLaboratories, Garscube Estate, Bearsden, Glasgow G61 1BD, Scotland, Types of Cell Primary Explantation Versus Proliferation Versus Organotypic Substrates and Isolation of cells for Tissue Collection and Biosafety and Record Disaggregation and Primary Life Growth Serial Characterization and Microbial of of cells for Tissue Engineering, edited by Gordana Vunjak-Novakovic and R. Ian FreshneyCopyright 2006 John Wiley & Sons, 1. INTRODUCTIONThe bulk of the material presented in this book assumes background knowledgeof the Principles and Basic procedures of cell and tissue Culture . However, it isrecognized that people enter a specializedfield, such as tissue engineering, frommany different disciplines and, for this reason, may not have had any formaltraining in cell Culture .

an extracellular matrix constituent, such as collagen, will usually give rise to an outgrowth of cells. This type of culture is known as a primary explant, and the cells migrating out are known as the outgrowth (Figs. 1.1, 1.2, See Color Plate 1). Cells in the outgrowth are selected, in the first instance, by their ability to migrate

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Transcription of Basic Principles of Cell Culture - Universidad de Granada

1 1 Basic Principles of Cell CultureR. Ian FreshneyCentre for Oncology and Applied Pharmacology, Cancer Research UK BeatsonLaboratories, Garscube Estate, Bearsden, Glasgow G61 1BD, Scotland, Types of Cell Primary Explantation Versus Proliferation Versus Organotypic Substrates and Isolation of cells for Tissue Collection and Biosafety and Record Disaggregation and Primary Life Growth Serial Characterization and Microbial of of cells for Tissue Engineering, edited by Gordana Vunjak-Novakovic and R. Ian FreshneyCopyright 2006 John Wiley & Sons, 1. INTRODUCTIONThe bulk of the material presented in this book assumes background knowledgeof the Principles and Basic procedures of cell and tissue Culture . However, it isrecognized that people enter a specializedfield, such as tissue engineering, frommany different disciplines and, for this reason, may not have had any formaltraining in cell Culture .

2 The objective of this chapter is to highlight those prin-ciples and procedures that have particularrelevance to the use of cell culturein tissue engineering. Detailed protocols for most of these Basic procedures arealready published [Freshney, 2005] and will not be presented here; the emphasiswill be more on underlying Principles and their application to three-dimensionalculture. Protocols specific to individualtissue types will be presented in TYPES OF CELL Primary Explantation Versus DisaggregationWhen cells are isolated from donor tissue, they may be maintained in a numberof different ways. A simple small fragment of tissue that adheres to the growthsurface, either spontaneously or aided bymechanical means, a plasma clot, oran extracellular matrix constituent, suchas collagen, will usually give rise to anoutgrowth of cells . This type of Culture is known as aprimary explant, and thecells migrating out are known as theoutgrowth(Figs.)

3 , , See Color Plate 1). cells in the outgrowth are selected, in the first instance, by their ability to migratefrom the explant and subsequently, if subcultured, by their ability to a tissue sample is disaggregated, either mechanically or enzymatically (SeeFig. ), the suspension of cells and small aggregates that is generated will con-tain a proportion of cells capable of attachment to a solid substrate, forming amonolayer. Those cells within the monolayer that are capable of proliferation willthen be selected at the first subculture and, as with the outgrowth from a primaryexplant, may give rise to acell line. Tissue disaggregation is capable of generatinglarger cultures more rapidly than explant Culture , but explant Culture may still bepreferable where only small fragments of tissue are available or the fragility of thecells precludes survival after disaggregation.

4 , Proliferation Versus DifferentiationGenerally, the differentiated cells in a tissue have limited ability to prolifer-ate. Therefore, differentiated cells do not contribute to the formation of a primaryculture, unless special conditions are used to promote their attachment and pre-serve their differentiated status. Usuallyit is the proliferating committed precursorcompartment of a tissue (Fig. ), such as fibroblasts of the dermis or the basalepithelial layer of the epidermis, that gives rise to the bulk of the cells in a4 Chapter 1. Freshney EXPLANT CULTUREDISSOCIATED CELL CULTUREORGANOTYPIC CULTUREORGAN CULTURET issue at gas-liquid interface; histologicalstructure maintainedTissue at solid-liquidinterface; cells migrateto form outgrowthDisaggregated tissue; cells form monolayer at solid-liquid interfaceDifferent cells co-cultured with or without matrix ; organotypic structure recreatedFigure of Culture .

5 Different modes of Culture are represented from left to right. First, an organculture on a filter disk on a triangular stainless steel grid over a well of medium, seen in section in thelower diagram. Second, explant cultures in a flask, withsection below and with an enlarged detail in sectionin the lowest diagram, showing the explant and radialoutgrowth under the arrows. Third, a stirred vesselwith an enzymatic disaggregation generating a cell suspension seeded as a monolayer in the lower , a filter well showing an array of cells , seen insection in the lower diagram, combined with matrixand stromal cells . [From Freshney, 2005.](a)(b)Figure explant and outgrowth. Microphotographsof a Giemsa-stained primary explant fromhuman non-small cell lung carcinoma. a) Low-power (4 objective) photograph of explant (top left) andradial outgrowth. b) Higher-power detail (10 objective) showing the center of the explant to the right andthe outgrowth to the left.

6 (See Color Plate 1.)primary Culture , as, numerically, these cells represent the largest compartmentof proliferating, or potentially proliferating, cells . However, it is now clear thatmany tissues contain a small population of regenerative cells which, given thecorrect selective conditions, will also provide a satisfactory primary Culture , whichmay be propagated as stem cells or mature down one of several pathways towardBasic Principles of Cell Culture5 Totipotent stem cell; embryonal, bone marrow, or otherTissue stem cell; uni-, pluri-, or multipotentTransit amplifying progenitor, or precursor (TAP), cellsDifferentiationRestricted in propagated cell lines in favor of cell proliferationSource of bulk of cell mass in cultured cell linesMay be present in primary cultures and cell lines as minority; may self-renew or progress to TAP cellsNeed enrichment (>107?) and inhibition of progression to create cell lineAttenuation: LIF, TGF- , MIP-1 Amplification: EGF, FGF, PDGF Figure of cell lines.

7 Diagrammatic representation of progression from totipotent stem cell,through tissue stem cell (single or multiple lineage committed) to transit amplifying progenitor cell com-partment. Exit from this compartment to the differentiated cell pool (far right) is limited by the pressure onthe progenitor compartment to proliferate. Italicized text suggests fate of cells in Culture and indicates thatthe bulk of cultured cells probably derive from the progenitor cell compartment, because of their capacityto replicate, but accepts that stem cells may be present butwill need a favorable growth factor environmentto become a significant proportion of the cells in the Culture . [From Freshney, 2005.]differentiation. This implies that not only must the correct population of cells beisolated, but the correct conditions must be defined to maintain the cells at anappropriate stage in maturation to retain their proliferative capacity if expansionof the population is required.

8 This was achieved fortuitously in early Culture offibroblasts by the inclusion of serum that contained growth factors, such as platelet-derived growth factor (PDGF), that helped tomaintain the proliferative precursorphenotype. However, this was not true of epithelial cells in general, where serumgrowth factors such as transforming growth factor (TGF- ) inhibited epithelialproliferation and favored differentiation. It was not until serum-free media weredeveloped [Ham and McKeehan, 1978, Mather, 1998, Karmiol, 2000] that thiseffect could be minimized and factors positive to epithelial proliferation, such asepidermal growth factor and cholera toxin, used to maximum undifferentiated precursors may give the best opportunity for expansionin vitro, transplantation may require thatthe cells be differentiated or carry thepotential to differentiate. Hence, two sets of conditions may need to be used, one forexpansion and one for differentiation.

9 The factors required to induce differentiationwill be discussed later in this chapter (See Section ) and in later chapters. Ingeneral, it can be said that differentiation will probably require a selective mediumfor the cell type, supplemented with factors that favor differentiation, such as6 Chapter 1. Freshney retinoids, hydrocortisone, and planar-polar compounds, such as sodium butyrate(NaBt). In addition, the correct matrix interaction, homotypic and heterotypic cellinteraction, and, for epithelial cells , the correct cellular polarity will need to beestablished, usually by using an organotypic Culture . This assumes, of course, thattissue replacement will require the graft tobe completely or almost completelydifferentiated, as is likely to be the case where extensive tissue repair is carriedout. However, there is also the option that cell Culture will only be required toexpand a precursor cell type and the process of implantation itself will then inducedifferentiation, as appears to be the case with stem cell transplantation [Greco andLecht, 2003].

10 Organotypic CultureDispersed cell cultures clearly lose their histologic characteristics after disag-gregation and, although cells within a primary explant may retain some of thehistology of the tissue, this will soon be lost because of flattening of the explantwith cell migration and some degree of central necrosis due to poor of histologic structure, and its associated differentiated properties, maybe enhanced at the air/medium interface, where gas exchange is optimized and cellmigration minimized, as distinct from thesubstrate/medium interface, where dis-persed cell cultures and primary outgrowths are maintained. This so-calledorganculture(See Fig. ) will survive for up to 3 weeks, normally, but cannot bepropagated. An alternative approach, with particular relevance to tissue engineer-ing, is the amplification of the cell stock by generation of cell lines from specificcell types and their subsequent recombination inorganotypic Culture .


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