Cytodex

Cytodex 1 Pharmacia, Sweden 147-248 dextran, high positive charge... 

Cytodex 2 Pharmacia, Sweden 135-200 dextran, positive charge... 

Cytodex 3 Pharmacia, Sweden 141-211 coUagen-coated dextran... 

Contact inhibition is a characteristic of the growth of anchorage dependent cells grown on microcarriers as a monolayer. Hawboldt et al. (1994) reported data on MRC5 cells grown on Cytodex II microcarriers and they are reproduced here in Table 17.13. 

Table 17.13 Growth of MRC5 Cells Cell Density versus Time for MRC5 Cells Grown on Cytodex I Microcarriers in 250 mL Spinner Flasks Inoculated at 2.26 and 4.76 cells/bead...

Cytodex 1 Cross-linked dextran with DEAE groups GE Healthcare... 

Cytodex 3 Cross-linked dextran coated with a denatured collagen GE Healthcare... 

Beads may be supplied in suspension in PBS but more usually they are supplied dry and require swelling in PBS-A (100 ml/g) for 3 h and washing in PBS-A and autoclaving (15 min at 15 p.s.i.) before use. In the dry form they are stable for more than 2 years. One gram of Cytodex-1 (dryweight) will swell to about 18 ml containing 7 X 106 microcarriers with a surface area of 0.6 m2. 

Bio-Rad Laboratories supply Bio-Carriers . Instead of the cross-linked dextran of the Cytodex microcarriers these have a polyacrylamide matrix with the positively charged dimethylaminopropyl groups linked throughout the matrix. On swelling 1 g dry Bio-Car-riers provides 19 ml packed volume containing 7 X 106 beads with a total surface area of 0.5 m2. Each bead can accommodate 350 cells so that the theoretical value of 2 X 109 cells could grow on 1 g Bio-Carriers. 

Fig. 3.8. L929 cells were seeded in 100 ml GMEM with 10% calf serum at 3 cells per microcarrier bead onto 5 x10s Cl-(Cytodex 1), P(Biosilon) or G (Bioglas) beads. The cell number was estimated by releasing the cells with trypsin, briefly allowing the beads to settle and counting the cells with a Coulter counter. Difficulty was encountered releasing the cells from Cytodex, but cells attached only weakly to the glass and particularly to the plastic beads and many cells were free in suspension. The use of electronic cell counters is not recommended for counting cells released from microcarriers as the orifice occasionally becomes blocked by small microcarriers in the...

The following protocol can be used to isolate cells from polydex-tran (Cytodex-1) or polyacrylamide (Biosilon)microcarriers. 

In order to reduce costs the concentration of foetal bovine serum (FBS) can be reduced in growth media. Figure 5.3 shows the effect of lowering the serum concentration on the growth of a rat hy-bridoma cell line. Initially cells can be plated in 5% FBS and later this concentration can be reduced by using a combination of FBS (1.5%) and new bom calf serum. This leads to yields of diploid fibroblasts on Cytodex-1 microcarriers of 80% those obtained with 10% FBS (Clarke, 1983) ( 3.6). 

PHARMACIA (1978) Cytodex l — Beaded Microcarriers for Cell Culture (Pharmacia Fine Chemicals AB, Uppsala). 

The DON cells were cultured in DMEM/F-12 on Cytodex 1 microcarriers in a 3-1 perfusion bioreactor. 

Other examples for the successful employment of co-cultures are the interaction between muscle and nerve cells (Shahar et aL, 1985) and the co-cultivation of vascular endothelial and smooth-muscle cells using microcarrier techniques (Davies Kerr, 1982) co-cultivation of cerebellar granule cells, cerebral cortical neurons and cortical astrocytes on collagen-coated dextran beads (CytodexS) and the production and release of specific neurotransmitters and enzyme synthesis resembling in vivo interactions between neurons and astrocytes (Westergaard et aL, 1991). 

Decant PBS from sterilized stock solution of Cytodex 3 and replace with growth medium (1 g to 30-50 ml). Add Cytodex 3 to spinner vessel to give a final concentration of 2 g H (use in range 1-3 g H). 

Add cell inoculum obtained by trypsinization of late log phase cells (pre-stationary phase) at over five cells per bead (optimum to ensure cells on all beads is seven) inoculate at the same density per square centimetre as with other culture types, e.g. 5-10 x 10 cm". Cytodex 3 at 2 g H inoculated at six cells per bead gives ... 

As a guide to calculating settled volume and medium entrapment by microcarriers, 1 g of Cytodex, for example, has a volume of 15-18 ml... 

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