Short-Term Cell Culture

We sought to identify culture conditions to meet several requirements. B cells should be maintained under conditions that 1) are standardized and reproducible between cell preparations; 2) minimally perturb their internal regulatory circuits; and 3) are convenient and easily transferable between AfCS and other laboratories. Thus, B cells should be cultured in a defined medium, without the use of serum or similar additives. We relied primarily on measures of B-cell viability to select such a medium. We also examined other variables, including functional integrity and recovery of B cells during experimental manipulations.

We tried to devise culture conditions that would be suitable for most assays and manipulations of splenic B cells. At the same time, we recognize that this will not always be possible; for instance, the presence of albumin promotes consistent availability of ligands to cells in medium (e.g., prevents trapping in plastic), but it may cause problems for short-term experiments better performed in the absence of added proteins (e.g., gel electrophoresis). We describe here the medium chosen for most culture conditions and assays.

Selection of a Base Medium. B-cell viability was measured in several commercial media including Iscove’s Modified Dulbecco’s Medium (IMDM), which was originally developed to support primary B lymphocytes in vitro (23). Other media tested included an Iscove’s-based medium developed by Mosier (24), which contains a 1:1 mixture of IMDM and Ham’s F-12 (Mosier Modified Medium, MMM); Dulbecco’s Modified Eagle’s Medium (DMEM); RPMI 1640, a common medium for immune cell culture; and Medium 199 (M199). Supplemental Table B lists the formulations for each of these media. All media contain 12.5 mM Hepes except IMDM, which contains 25 mM Hepes; except as noted, all media were initially supplemented with insulin (10 mg/ml), transferrin (5.5 mg/ml), and selenium (5 ng/ml) (ITS medium supplement), L-glutamine (2 mM), b-mercaptoethanol (55 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml). Additives other than b-mercaptoethanol were added to 500 ml of medium, which was which kept refrigerated for up to a week. b-mercaptoethanol was added immediately prior to use.

B-cell viability was first assessed at 0 and 24 hours using the Live/Dead Assay (Molecular Probes) . At 24 hours, B-cell viability in IMDM (65.5 ± 1.8%) and in MMM (59.8 ± 0.9%) was clearly better than in DMEM, RPMI, or M199 (Fig. 8A). IMDM and MMM were thus further tested for their capacity to maintain cell viability at 4 hours, since AfCS experiments will initially focus on short-term responses to ligands for various receptors. At 4 hours, B-cell viability was 86.5 ± 2.4% in IMDM and 77.4 ± 6.0% in MMM (Fig. 8B).

Several commercial media are designed specifically for cell culture in the absence of serum. We compared IMDM with two such formulations, GIBCO Hybridoma Serum-Free Medium (HSFM) and GIBCO CD medium, by assessing B-cell viability at 24 hours. B-cell viability was similar in IMDM and HSFM and only slightly higher in CD medium (Fig. 8D). Although HSFM and CD media are defined and readily available, their compositions are proprietary. Since IMDM proved similar in its capacity to support B-cell viability, we chose to continue using this nonproprietary formulation.

Effect of Cell Density. B cells were cultured in both IMDM and MMM at densities ranging from 1.25 to 20 x 10⁶ cells/ml, and viability was assessed at 24 hours. B-cell viability was greatest and most consistent at all densities in IMDM, whereas viability increased progressively with increasing density in MMM, eventually equaling that observed in IMDM (Fig. 8C).

Effect of Additives to IMDM. B-cell viability at 4 or 24 hours was not affected by removal of insulin, transferrin, or the ITS medium supplement (Table 2). Addition of bovine serum albumin (BSA, specifically shown to lack bacterial lipopolysaccharide) was tested at concentrations of 0.005 to 0.1 mg/ml with no observable effect on viability (data not shown). Other additives, including streptomycin, penicillin, dextran sulfate, polyethylene glycol (PEG), and cholesterol, had no effect on B-cell viability at 24 hours. Other additives were also tested in MMM and were similarly without effect. These included stearate, ethanolamine, Tween 80 (a lipid formulation found in M199), and taurine. Pluronic F-68 was tested at concentrations ranging from 0.0125% to 0.1%; this improved cell recovery during centrifugation when used at a concentration of 0.025% with no effect on viability.

Final Medium. The formulation selected for short-term culture of splenic B cells is termed Supplemented Iscove’s Modified Dulbecco’s Medium (SIMDM). Supplements in SIMDM include 0.025% Pluronic F-68, 2 mM L-glutamine, 55 mM b-mercaptoethanol and 100 mg/ml BSA (Supplemental Table B details the composition of base IMDM). BSA was included in the final medium because it improved survival and cell recovery. Pluronic F-68 was included because it improved cell recovery during experimental manipulation. In this report, however, functional studies of B cells were performed using this medium without BSA.

SIMDM has an osmolality of ~280 mOsm/kg. Plasma osmolality in the mouse is normally in the range of 310 to 325 mOsm/kg (25). Increasing the osmolality of SIMDM by addition of NaCl reduced B-cell viability (data not shown)