《食品分析》课程实验室操作参考手册（Amersham Biosciences）07 微载体细胞培养手册 Microcarrier cell culture principles & methods.pdf_大学文库

New opportunities and applications for animal cell culture Cytodex provides convenient surfaces for the growth of animal cells and can be used in suspension culture systems or to increase the yield of cells from standard monolayer culture vessels and perfusion chambers.Applications include production of large quantities of cells,viruses and cell products,studies on differentiation and cell function.perfusion culture systems,microscopy studies,harvesting mitotic cells, isolation of cells,membrane studies,storage and transportation of cells,assays involving cell transfer and studies on uptake of labelled compounds(see section 1.4. for a description of these applications). Increased production capacity eig"56emecsggngCoewoGcgSgayoatiout types of monolayer cultures.The possibility to culture cells in small compact culture systems is especially important when working with pathogenic organisms. Improved control systems provide excellent opportunities ers (e.g.pH.gas tensions etc).The que prov des a method lent cells for growing anchorage-derhe improvedca system ha advantages of suspens n c tur ntrol poss microcarrier culture allo w for a ho us culture system h naving a wide variety of process d oring an sampling mi cultures is simpl r technique for producing large numbers chorage-depen ent c cells nts for culture medium ique sstirr micr or a given v ne of di superio ave ng chicken ster ovary ce lls (6) pig kidney ts(2,3) ce. 7),prin dney ce ells (8) and trans mous (9).This re cost 9 m means en expensive serum supplements such oetal alf serum are used. Reduced requirem ents for labour cells cells/ can be cutured in smal volu uired wh with ore than 10 cultures.For e can handle a vaccine oduction mple nt to 900 roller bottles pe week (10).One litr e can as man cells as up to 50 r bottles (490 cme bottles 2) carrier edu the labour for rou dction and s on cle aration of Separation of cells from the culture medium is simple:when the stirring is stopped the microcarriers with cells attached settle under the influe ence of gray and the supernatant can be removed.Unlike true suspension cell culture s nc entrifugation step ar necessary

8 New opportunities and applications for animal cell culture Cytodex provides convenient surfaces for the growth of animal cells and can be used in suspension culture systems or to increase the yield of cells from standard monolayer culture vessels and perfusion chambers. Applications include production of large quantities of cells, viruses and cell products, studies on differentiation and cell function, perfusion culture systems, microscopy studies, harvesting mitotic cells, isolation of cells, membrane studies, storage and transportation of cells, assays involving cell transfer and studies on uptake of labelled compounds (see section 1.4. for a description of these applications). Increased production capacity The very large culture surface area to volume ratio offered by the microcarrier system (e.g. 30 cm2 in 1 ml using 5 mg Cytodex 1) provides high cell yields without having to resort to bulky equipment and tedious methodology. For a given quantity of cells or their products microcarrier cultures demand much less space than other types of monolayer cultures. The possibility to culture cells in small compact culture systems is especially important when working with pathogenic organisms. Improved control Suspension culture systems provide excellent opportunities for the control of culture parameters (e.g. pH, gas tensions etc). The microcarrier technique provides a method for growing anchorage-dependent cells in a system having all the advantages of suspension culture. The improved control possibilities with microcarrier culture allow for a homogenous culture system having a wide variety of process designs (1). Monitoring and sampling microcarrier cultures is simpler than with any other technique for producing large numbers of anchorage-dependent cells. Reduced requirements for culture medium When compared with other monolayer culture techniques stirred microcarrier cultures yield 2-4 times as many cells for a given volume of medium. The superior yields with microcarrier culture have been reported for a wide variety of systems including chicken fibroblasts (2,3), pig kidney cells (4), fish cells (5), Chinese hamster ovary cells (6), human fibroblasts (7), primary monkey kidney cells (8) and transformed mouse fibroblasts (9). This reduction in requirement for medium means considerable savings in cell culture costs (6,9), particulary when expensive serum supplements such as foetal calf serum are used. Reduced requirements for labour Because large numbers of cells can be cultured in small volumes (more than 109 cells/litre) fewer culture vessels are required when working with microcarrier cultures. For example, with microcarrier culture one technician can handle a vaccine production equivalent to 900 roller bottles per week (10). One litre of microcarrier culture can yield as many cells as up to 50 roller bottles (490 cm2 bottles, 2). The simplified procedures required with microcarriers reduce the labour necessary for routine production and save on cleaning and preparation of glassware. Separation of cells from the culture medium is simple; when the stirring is stopped the microcarriers with cells attached settle under the influence of gravity and the supernatant can be removed. Unlike true suspension cell culture systems, no centrifugation steps are necessary

9 Lower risk of contamination In cell culture the risk of contamination is related to the number of handling steps (opening and closing of culture vessels) required to produce a given quantity of cells or their products. Microcarrier culture provides a method for reducing the number of handling steps. There is a much reduced risk of contamination when the production of large quantity of cells is from a single microcarrier culture rather than several hundred roller bottles (6). The principles and methods necessary to achieve the best results with microcarrier culture are described in this book. Although this technique is one of the most advanced in animal cell culture it need not be restricted to experienced cell culturist. Since cell culture is being used by a wide variety of scientists this book is written for both beginners and those experienced in cell culture and only a basic knowledge of cell culture is assumed. This book aims at describing the principles and techniques of cell culture with Cytodex so that the reader is able to deduce optimum procedures with a minimum of effort. The principles aim at a flexible and systematic approach. They are essential to making the most off microcarrier culture and to achieving consistent results with high yields. All methods described her have been developed for use with Cytodex and are not necessary suitable for use with other surfaces for cell culture. 1.2 Adhesion of cells to culture surfaces The adhesion of cells to culture surfaces is fundamental to both traditional monolayer culture techniques and microcarrier culture. Since the proliferation of anchorage-dependent cells can only occur after adhesion to a suitable culture surface (11), it is important to use surfaces and culture procedures which enhance all of the steps involved in adhesion. Adhesion of cells in culture is a multistep process and involves a) adsorption of attachment factors to the culture surface, b) contact between the cells and the surface, c) attachment of the cells to the coated surface and finally d) spreading of the attached cells (11, fig. 2). Substrate Cell Adsorption Substrate Cell Contact -CIG Substrate Cell Attachment -CIG MHS Substrate Cell Spreading -CIG MHSFig. 2. Simplified outline of steps involved in adhesion of animal cells to culture sufaces. The whole process involves divalent cations and glycoproteins adsorbed to the culture surface. Under usual culture conditions the attachment proteins vitronectin and fibronectin originates from the serum supplement in the medium. MHS is synthesized by the cells. CIG - fibronectin or vitronectin. MHS – multivalent heparan sulphate. (Adapted from refs. 11, 17, 30)

The culture surface must be hydrophilic and correctly charged before adhesion of cells can occur(11).All vertebrate cells possess unevenly distributed negative surface charged(12)and can be cultured on surfaces which are either negatively or positively charged(11.13-16).Examples of suitable culture surfaces bearing charges of different polarities are glass and plastic (negatively charged)and polylysine coated surfaces or Cytodex 1 microcarriers(positively charged).Since cells can adhere and grow on all of these surfaces,the basic factor governing adhesion and growth of cells is the density of the charges on the culture surface tather than the polarity of the charges(15.17). Two factors in culture medium are essential for adhesion of cells to culture surfaces divalent cations and protein(s)in the medium or adsorbed to the culture surface (11).In the absence of protein and divalent cations cells attach to a culture surface only by non-specific adsorption(11,18).The protein molecule essential for full adhesion of cells to a culture surface is now known to be a glycoprotein(19-21).The "critical charge densities"noted for microcarriers (16,22-24.fig 4)and other culture surfaces(14)are more likely to be related to interactions between attachn ent glycoprotein(s)and th charged surface rather than direct electrostatic interaction between the cells and the culture surface (17). Attachment glycoproteins found in the serum in culture medium are fib ronectin and vitronectin.secreted from certain cells(19.25-27 Vitronectin and/c must be e culture surface bet re they can pr and spre ey are corporate d into the spr norm sulphate pro eparan teoglycans me e a n of cells o cu sby co-o dinate he CIG adsorbed on the culture surfac In order to achieve good adhesion of the cells to ormed cell or an atla nt glycoprotein is satis Ma secret 0 h esion occu (1825).C n types of co as diploid ts of fi can secre cant n and require an exogenous source atta hment s glycoprotein 10 When initiating a culture it is usual pr actice to let the culture ome into 1096 ture ectin/ml(27 p oximately 23 次 ith: fe s (18)propor d a lara n of the fibronectin /mD befor ttach to P8 n2 is of cell.BHK (25).Therefore.standard ng usually ensure that the culture surface (plastic.glass or Cytodex micr carrier)is coated with ade equate nts of glycoproteins involved in cell attachment. 10

10 The culture surface must be hydrophilic and correctly charged before adhesion of cells can occur (11). All vertebrate cells possess unevenly distributed negative surface charged (12) and can be cultured on surfaces which are either negatively or positively charged (11,13-16). Examples of suitable culture surfaces bearing charges of different polarities are glass and plastic (negatively charged) and polylysine coated surfaces or Cytodex 1 microcarriers (positively charged). Since cells can adhere and grow on all of these surfaces, the basic factor governing adhesion and growth of cells is the density of the charges on the culture surface tather than the polarity of the charges (15,17). Two factors in culture medium are essential for adhesion of cells to culture surfaces - divalent cations and protein(s) in the medium or adsorbed to the culture surface (11). In the absence of protein and divalent cations cells attach to a culture surface only by non-specific adsorption (11,18). The protein molecule essential for full adhesion of cells to a culture surface is now known to be a glycoprotein (19-21). The “critical charge densities” noted for microcarriers (16,22-24, fig 4) and other culture surfaces (14) are more likely to be related to interactions between attachment glycoprotein(s) and the charged surface rather than direct electrostatic interaction between the cells and the culture surface (17). Attachment glycoproteins found in the serum in culture medium are fibronectin and vitronectin, secreted from certain cells (19,25-27). Vitronectin and/or fibronectin must be adsorbed on the culture surface before they can promote cell attachment and spreading (28) and they are subsequently incorporated into the extracellular matrix of the spread cells (27). Under normal culture conditions multivalent heparan sulphate proteoglycans mediate adhesion of cells to culture surfaces by co-ordinate binding to glycoproteins on the cell surfaces and the CIG adsorbed on the culture surface (20). In order to achieve good adhesion of the cells to culture surfaces it is necessary that the requirement for an attachment glycoprotein is satisfied. Many established and transformed cell types secrete only very small amounts of fibronectin and require a fibronectin or serum supplement in the culture medium before adhesion occurs (18,25). Certain types of cells such as diploid fibroblasts can secrete significant quantities of fibronectin and do not require an exogenous source of this glycoprotein for attachment (19,29). When initiating a culture it is usual practice to let the culture surface come into contact with medium containing serum before cells are added to the culture. Culture medium supplemented with 10% (v/v) foetal calf serum contains approximately 2-3 µg fibronectin/ml (27) and a large proportion of the fibronectin adsorbs to culture surfaces within a few minutes (18). Serum-free media often require addition of fibronectin (1-50 µg/ml) before many cells can attach to culture surfaces. A minimum of 15 ng of adsorbed fibronectin/cm2 is required for spreading of an established type of cell, BHK (25). Therefore, standard culture procedures usually ensure that the culture surface (plastic, glass or Cytodex microcarrier) is coated with adequate amounts of glycoproteins involved in cell attachment