Iscove’s medium

Fusion is performed as in Section 5.4.3.1, and the cell pellet is taken up in 15 ml of Iscove s medium containing 53.3% FBS, 2.66% of HAT stock solution, 1.2 x 10 thymocytes and 2 mg EPS. To this, 25 ml of 0.2% (w/v) MC (Section 5.4.2.6) are added. The tube is then tipped several times and mixed for several seconds. Of this suspension 1 ml aliquots are distributed (with a syringe with an 18-gauge needle) into Petri dishes. Two of these dishes are placed in a 10 cm Petri dish, together with a third dish containing 1 ml distilled water and then placed in a 5% CO2 incubator at 37°C. 

RPMI, modified McCoy 5 A medium for cell culture HEPES, 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid IMDM, Iscov s modified Dulbecco s medium PCS, fetal calf serum... 

Use the type of medium the cells have been growing in as control. Test 3-4 commercially available balanced media, such as F12/DMEM (Ham s nutrient mixture/Dulbecco s modified Eagle s medium), DMEM, IMDM (Iscove s modified Dulbecco s medium), HB-CHO (Hana Biological Chinese hamster ovary medium) or RPMI-1640 (Roswell Park Memorial Institute). 

Low serum-containing medium Iscove s modified Dulbecco s medium (IMDM) (Sigma Chemical Company, St Louis, MO, USA) supplemented with 2% foetal bovine serum and 5 x 1(L M p-mercaptoethanol... 

One approach to implementing a fed-batch strategy is to feed cultures with medium concentrates. This can offer a rapid approach to increasing productivity, and can also be relatively simple to implement [17, 74]. Using GS-NSO cells producing an antibody, Bibila et al. [17] fed cultures with lOx basal medium concentrates (Iscove s Modified Dulbecco s medium) to increase productivity. Sodium chloride, potassium chloride and sodium bicarbonate were omitted from the medium concentrates in order to minimize the increases in osmo-larity caused by feeding. In their system, feeding basal medium concentrates did not result in an increase in the maximum viable cell concentration or the IVC. However, the final antibody concentration was increased 1.9-fold as a result of an increase in the specific production rate. 

Iscove s Modified Dulbecco s Medium (IMDM) (Life Technologies Cat. No. 12440)... 

FEE clone 745A was cultured at 37°C in Iscove s modified Dulbecco medium supplemented with 10% fetal calf serum (Gibco), penicillin (50 U ml ) and streptomycin (0.5 jUg nil ) in a humidified atmosphere with 5% CO2. Eogarithmically growing cells, seeded at 2 x 10 ml", doubled in approx. 11 h. 

A range of conventional media can be used but the commonest choices are Dulbecco s MEM (DMEM), Iscove s modified DMEM (IMDM), RPMI-1640, or a 1 1 mixture of DMEM and Ham s F12 medium. 

As an example, the main compounds of a 1 1 mixture of IMDM (Iscove s Modification of Dulbecco s Medium) and Ham s F12 are given as follows ... 

---