carriers ins nd b suspension can be treated eed.mitotic cells can be orted that CHO rells ha sted fron Cvtodex iers by this nethod had a dex of up to 889 ent er the index of 41%obta ned when harvesting mito from a culture flask. Ne et al (74)treated expo nential cultures of CHO cells on microcarriers with Colcemid (100 mg/ml)for 2.5 h and then harvested mitotic cells by increasing stirring speed.The increased stirring speed dislodged the mitotic cells and harvests of mo re than 4x10mitotic cells/ml of microcarrier culture could be obtained. These cells had a mitotic index of 85-95%(74). 1.4.8 Transportation and storage of cells The large e are of microcarriers is advantageous when trans turing ce s or ce ransported the 吧oap ure e nique avo numbers of ayer culture v (e.g.n h 034 ag de s.The 0 attached o a cu surfa ra as ted g a tha ing fro st the prolifera are already attached to the culture surface and can continue to function and Procedu re s for storing most rage-dependent cells whilst still attached to ib 14.6. culture nsect cells can and pers.comm. 190) 27
27 Exponential cultures of cells growing on microcarriers in suspension can be treated with mitotic inhibitors (e.g. Colcemid) and by selecting the appropriate stirring speed, mitotic cells can be dislodged and collected in the medium. Mitchell and Wray (73) reported that CHO cells harvested from Cytodex microcarriers by this method had a mitotic index of up to 88%, a considerable improvement over the mitotic index of 41% obtained when harvesting mitotic cells by shaking from a culture flask. Ng et al (74) treated exponential cultures of CHO cells on microcarriers with Colcemid (100 mg/ml) for 2.5 h and then harvested mitotic cells by increasing stirring speed. The increased stirring speed dislodged the mitotic cells and harvests of more than 4x104 mitotic cells/ml of microcarrier culture could be obtained. These cells had a mitotic index of 85-95% (74). 1.4.8 Transportation and storage of cells The large surface area/volume ratio of microcarriers is advantageous when transporting and storing culturing cells. Large numbers of cells (up to approx. 107 cells/ ml) can be transported or stored whilst still attached to the culture substrate. This technique avoids transportation of large numbers of monolayer culture vessels (e.g. flasks) and also eliminates the need to store or transport suspensions of anchorage-dependent cells. The advantage of transporting or storing cells attached to a culture surface rather than a suspension is that loss of cells associated with harvesting and replating is avoided. After transportation or thawing from storage the cells are already attached to the culture surface and can continue to function and proliferate. Procedures for storing most anchorage-dependent cells whilst still attached to Cytodex microcarriers are described in section 4.6. and even cultured insect cells can be frozen and stored in liquid nitrogen when attached to microcarriers (E. Duda, pers. comm., 190)
2.Cytodex microcarriers 2.1 Requirements for an optimum microcarrier In order for a microcarrier to be suitable for animal cell culture at all scales it must fulfill certain basic criteria(16). Surface properties must be such that cells can adhere with a degree of spreading which permits proliferation.For homogeneous growth of cells the surface of the microcarrier must have an even,continuos contour.The surfaces of all microcarriers in the culture should have consistent properties. Density of the microcarriers should be slightly greater than that of the surrounding medium,thus facilitating easy separation of cells and medium.The density should also be sufficiently low to allow complete suspension of the microcarriers with only gentle stirring.Under standard culture conditions the optimum density for microcarriers is 1.030-1.045 g/ml. Size distribution should be narrow so that even suspension of all microcarriers is achieved and that confluence is reached at approximately the same stage on each microcarrier.Best growth of cells occurs when microcarriers have a size distribution which lies within limits of diameter in culture of 100-230 um. Optical properties should be such that routine observation of cells on microcarriers can be achieved using standard microscopy techniques.The microcarriers should also permit use of routine cytology procedures Non-toxic microcarriers are required not only for survival and good growth of the cells but also when cell culture products are used for veterinary or clinical purposes Non-rigid microcarrier matrices are required for good cell growth when the culture is stirred. Collisions between microcarriers occur in stirred cultures and compres ble matrix reduces the possibility of damage to the microcarriers anc the cells. All Cytodex r eet the above requirements and are nked xtran.Thi s mat ix was chosen becaus it provide r with suitable physi al properties and also because dextrar in non-toxic, g widespread use in clinical appl cations and for the preparation of pharmaceutically important materials(see ref.76 rocar iers are non-to kic and pro s whic ncan be used for the a wi d on6.1).These mi crocarrie】 dis on a Cytod with the re proce are suffici equi red fo ong to cul tions but dy str are non-rigid and ha 29
29 2. Cytodex microcarriers 2.1 Requirements for an optimum microcarrier In order for a microcarrier to be suitable for animal cell culture at all scales it must fulfill certain basic criteria (16). • Surface properties must be such that cells can adhere with a degree of spreading which permits proliferation. For homogeneous growth of cells the surface of the microcarrier must have an even, continuos contour. The surfaces of all microcarriers in the culture should have consistent properties. • Density of the microcarriers should be slightly greater than that of the surrounding medium, thus facilitating easy separation of cells and medium. The density should also be sufficiently low to allow complete suspension of the microcarriers with only gentle stirring. Under standard culture conditions the optimum density for microcarriers is 1.030-1.045 g/ml. • Size distribution should be narrow so that even suspension of all microcarriers is achieved and that confluence is reached at approximately the same stage on each microcarrier. Best growth of cells occurs when microcarriers have a size distribution which lies within limits of diameter in culture of 100-230 µm. • Optical properties should be such that routine observation of cells on microcarriers can be achieved using standard microscopy techniques. The microcarriers should also permit use of routine cytology procedures. • Non-toxic microcarriers are required not only for survival and good growth of the cells but also when cell culture products are used for veterinary or clinical purposes. • Non-rigid microcarrier matrices are required for good cell growth when the culture is stirred. Collisions between microcarriers occur in stirred cultures and a compressible matrix reduces the possibility of damage to the microcarriers and the cells. All Cytodex microcarriers are designed to meet the above requirements and are based on a spherical matrix of cross-linked dextran. This matrix was chosen because it provides a microcarrier with suitable physical properties and also because dextran in non-toxic, having widespread use in clinical applications and for the preparation of pharmaceutically important materials (see ref. 76) Cytodex microcarriers are non-toxic and provide surfaces which can be used for the cultivation of a wide variety of cell types (section 6.1). These microcarriers have a size distribution and density compatible with the culture procedures required for optimal cell growth. Furthermore Cytodex microcarriers are sufficiently strong to withstand normal culture procedures and conditions but are non-rigid and have excellent optical properties
Cytodex 1 CH2CH3 charges cross-linked dextran-O-CH,CH,-N CH,CH Cytodex3 OH cross-linked dextran-O-CH2-CH-CH2-NH-(Lys.COLLAGEN) Fig 18.Schematic representation of the three alternative types of Cydodex microcarriers. 2.2 Cytodex 1 Cytodex 1 microcarriers are basedon ich edN.N cross-linked dextra natrix which is vth(fig. thy DEAE) 4 ged groups are h e entre mat crocarrier(g.18)】 Published procedures for substitutin oss-linked dextra n with DEAE groups to form 35.55)can lead t oRortiond ch (see 24).Th ctio 1 that fo on such tanden 15%/of thu stability of the charged of cha nized.Win ged m the micr presence of leaked DEAE-dextr olysis mass to examine the possible p n in ated n olio vaccines of such g curre was found to be below the limits of d Cytodex 1.If tection,i.e. less than 20 Ppm (77 The physical characteristics of Cytodex 1 are summarized in Table 4. 2.3 Cytodex 3 Cytodex 3*microcarriers are based on an entirely different principle for microcarrier culture.While most surfaces used in cell culture posses a specific density of small charged molecules to promote attachment and growth of cells (e.g. glass,plastic.Cytodex 1.),certain proteins can also provide a surface for the growth patents pending 30
30 2.2 Cytodex 1 Cytodex 1 microcarriers are based on a cross-linked dextran matrix which is substituted with positively charged N,N-diethylaminoethyl (DEAE) groups to a degree which is optimal for cell growth (fig. 4). The charged groups are found throughout the entire matrix of the microcarrier (fig. 18). Published procedures for substituting cross-linked dextran with DEAE groups to form microcarriers for cell culture (3,22,35,55) can lead to the formation of a high proportion (up to 35%) of tandem charged groups (see 24). The chemical reaction conditions used to produce Cytodex 1 microcarriers are controlled so that formation of such tandem groups is minimized (only approx. 15% of groups); thus stability and homogeneity of the charged groups is enhanced and possible leakage of charged groups from the microcarriers is minimized. Windig et al (77) used pyrolysis mass spectroscopy to examine the possible presence of leaked DEAE-dextran in concentrated polio vaccines prepared from microcarrier cultures using Cytodex 1. If leakage of such groups occurred it was found to be below the limits of detection, i.e. less than 20 ppm (77). The physical characteristics of Cytodex 1 are summarized in Table 4. 2.3 Cytodex 3 Cytodex 3* microcarriers are based on an entirely different principle for microcarrier culture. While most surfaces used in cell culture posses a specific density of small charged molecules to promote attachment and growth of cells (e.g. glass, plastic, Cytodex 1,), certain proteins can also provide a surface for the growth *patents pending Fig. 18. Schematic representation of the three alternative types of Cydodex microcarriers. Cytodex 1 charges throughout matrix collagen layer coupled to surface Cytodex 3 cross-linked dextran cross-linked dextran - O - CH CH - N 2 2 CH CH 2 3 .HCL CH CH 2 3 - O - CH - CH - CH - NH - ( L 2 2 YS.COLLAGEN) OH
of cells in culture.The connective tissue protein,collagen,has proved to be a valuable cell culture substrate.Cytodex 3 microcarriers consist of a surface layer of denatured collagen covalently bound to a matrix of cross-linked dextran(fig.18). The amount of denatured collagen bound to the microcarrier matrix is approx. 60 ug/cm2 and results in maximum cell yields(24).The denatured collagen (MW 60.000-200.000)is derived from pig skin type I collagen which has been extracted and denatured by acid treatment,concentrated and purified by an ion exchange step and steam sterilized before being coupled to the microcarrier matrix. These microcarriers combine the advantages of collagen coated culture surfaces (see below)with the advantages and possibilities of microcarrier culture (section 1.1.1.4).Cytodex 3 microcarriers can also be used as a general purpose collagen-coated cell culture substrate. Most normal epithelial cells will attach more efficiently to collagen than to other cell to grow in culture (78.79).Collagen-coated surfaces are valuable eca use they permit differentiation of cells in vitro even at very sparse or colonel culture densities (79,80).Such surfaces are also adv tage ous when culturing for extended periods since they delay the detachment of the cell sheet that e lly occurs ong-term ma culture on d surfaces (81).A variety of diff crent types or routinely culture en-coa urfa and include roblasts chondrocytes,epiderm cells,myobla ts and mammary epith ro depends on the presence of to th ture surface (80 spread mor llagen than on star ture surfaces(81) nd growth Is also stimulated(83) Hepato e cult tured more succ um effices.The gen surfa lagen acy and rap an on any oth cu 4 on eq ent 01 lager 85)thi e most s to ture nepato 84).A ce media trate hepatocytes i vitro with of diore ex nction(86,87) et al (88)have described the culture of ndothelial c ary ex 3 (nlat /801 eppa etter tiat d al cell fu pred that t the cultur nd conp s(90.91)and Cer et al can also cultur th gen for the culture e of the pithelial kidney cel line.MDCK.Se ne emb kidney .a highe lating efficie ndex 3 tha n Cutodey 1 This i ads to highe n cell 31
31 of cells in culture. The connective tissue protein, collagen, has proved to be a valuable cell culture substrate. Cytodex 3 microcarriers consist of a surface layer of denatured collagen covalently bound to a matrix of cross-linked dextran (fig. 18). The amount of denatured collagen bound to the microcarrier matrix is approx. 60 µg/cm2 and results in maximum cell yields (24). The denatured collagen (MW 60,000-200,000) is derived from pig skin type I collagen which has been extracted and denatured by acid treatment, concentrated and purified by an ion exchange step and steam sterilized before being coupled to the microcarrier matrix. These microcarriers combine the advantages of collagen coated culture surfaces (see below) with the advantages and possibilities of microcarrier culture (section 1.1, 1.4). Cytodex 3 microcarriers can also be used as a general purpose collagen-coated cell culture substrate. Most normal epithelial cells will attach more efficiently to collagen than to other cell culture surfaces. Consequently, collagen-coated culture surfaces are used frequently for establishing primary cultures and for growing cells which are normally difficult to grow in culture (78, 79). Collagen-coated surfaces are valuable because they permit differentiation of cells in vitro even at very sparse or colonel culture densities (79,80). Such surfaces are also advantageous when culturing for extended periods since they delay the detachment of the cell sheet that eventually occurs in long-term mass culture on uncoated surfaces (81). A variety of different types of cells are routinely cultured on collagen-coated surfaces and include hepatocytes, fibroblasts, chondrocytes, epidermal cells, myoblasts and mammary epithelial cells (82). Differentiation of myoblasts at sparse densities in vitro depends on the presence of collagen bound to the culture surface (80, 81). Myoblasts attach and spread more satisfactorily on collagen than on standard cell culture surfaces (81) and growth is also stimulated (83). Hepatocytes can be cultured more successfully on collagen surfaces. The collagen permits freshly isolated hepatocytes to attach with maximum efficiency and spreading is more rapid than on any other cell culture surface (84). Since exogenous fibronectin is not required for attachment of hepatocytes to collagen (85) this culture surface is the most suitable surface for culture of hepatocytes in protein-free media (84). A collagen culture substrate also allows for more extended studies of hepatocytes in vitro with improved retention of differentiated function (86,87). Folkman et al (88) have described the culture of capillary endothelial cells from a variety of sources on collagen and excellent growth of endothelial cells on Cytodex 3 has been demonstrated (plate 2). Geppart et al (89) reported that there was better maintenance of differentiated alveolar type II cell function when using collagen as the culture substrate. Surface-bound collagen can also be used for the culture of fibroblasts (90, 91) and Ceriejido et al (92) described the use of cross-linked collagen for the culture of the epithelial kidney cell line, MDCK. Secondary bovine embryo kidney cells have a higher plating efficiency when grown on Cytodex 3 than when grown on Cytodex 1. This improvement in plating efficiency leads to higher cell yields (fig. 19)
10 Fg19.Thegowhofgcoadan todex 1( 3 (Ce 10 the had 100 200 Hours Cytodex 3 microcarriers are coated with denatured Type I collagen because this is the most generally useful form of collagen for cell culture.Although certain types of cells may show a specificity for attachment to a particular form o native collagen. this specificity is much less apparent when denatu ed collagen is used.Adsorption of the attachment glycoprotein,fibronectin,to the culture su ace is known to be important in th hesion of many cells(section 1.2)and fibronectin is believed to be also invol d in the attachment of many ce to oth native and dena tured forms of collagen (93) Fibronectin binds equally s of collagen(94)but y to phac with native collagen (97). Table 4.Some physical characteristics of Cytodex microcarrier Cytodex 1 Cytodex 3 Suem 103 1.04 ar (um) 131-220 133-215 ng dry) Size is based on diamete 0%of the eof a sample of microcarriers (dor the range between d)calculated from cumulative volume distributions. *In 0.9%NaCl. 品
32 Cytodex 3 microcarriers are coated with denatured Type I collagen because this is the most generally useful form of collagen for cell culture . Although certain types of cells may show a specificity for attachment to a particular form o native collagen, this specificity is much less apparent when denatured collagen is used. Adsorption of the attachment glycoprotein, fibronectin, to the culture surface is known to be important in the adhesion of many cells (section 1.2) and fibronectin is believed to be also involved in the attachment of many cells to both native and denatured forms of collagen (93). Fibronectin binds equally well to all types of collagen (94) but shows a preference for denatured forms (82,95,96) and binds more rapidly to Sephadex® beads coated with denatured collagen than to beads coated with native collagen (97). Table 4. Some physical characteristics of Cytodex microcarriers. Cytodex 1 Cytodex 3 Density* (g/ml) 1.03 1.04 Size* d50 (µm) 180 175 d5-95 (µm) 131-220 133-215 Approx. area* (cm2/g dry weight) 4,400 2,700 Approx. no microcarriers /g dry weight 6.8x106 4.0x106 Swelling* (ml/g dry weight) 18 14 Size is based on diameter at 50 % of the volume of a sample of microcarriers (d50), or the range between the diameter at 5 % and 95 % of the volume of a sample of microcarriers (d5-95). Thus size is calculated from cumulative volume distributions. *In 0,9% NaCl. Cells/ml 106 105 100 200 Hours Fig. 19. The growth of secondary bovine embryo kidney cells on Cytodex 1 (—●—) and Cytodex 3 (—❍—) microcarriers. The cells were a mixed population with predominantly epithelial morphology. (Gebb, Ch., Clark, J.M., Hirtenstein, M.D. et al, Develop. Biol. Standard. (1981), in press, by kind permission of the authors and the publisher)