Primary human marrow cultures for erythroid bursts in a serum-substituted system

To determine environmental requirements for erythroid burst formation in primary culture, we added human bone marrow cells to serum-depleted methylcellulose, agar, or fibrin clot cultures. Optimal BFU-E proliferation was present in cultures prepared with Iscove's modified Dulbecco medium, 248 micrograms/ml transferrin, 1.63 micrograms/ml ferric chloride, 117 micrograms/ml bovine serum albumin, and each of seven preparations of erythropoietin. Burst number was comparable to that in serum containing culture. Reducing sodium dodecyl sulfate electrophoresis of commercial albumin preparations showed them to contain abundant lipoproteins. Results of experiments with human plasma albumin found to be greater than 98% pure by one- and two-dimensional gel electrophoresis and delipidated albumin indicate that an albumin source is needed for burst formation to occur. Together with albumin, exogenous phosphatidylcholine but not phosphatidylserine augmented burst number. Bursts routinely appeared in serum-depleted culture without added burst-promoting activity (BPA). However, leukocyte-conditioned medium (LCM) and its high-speed supernatant and pellet fractions enhanced burst formation. Antimembrane IgG capable of neutralizing BPA reduced burst number to a level below that achieved in LCM-depleted culture, suggesting that endogenous BPA was inactivated. We conclude that human marrow BFU-E proliferation requires iron-saturated transferrin, albumin, and erythropoietin. Exogenous BPA and phospholipids enhance but are not essential for burst formation to proceed in primary culture.