Chapter 13 - Clonal Cultures in vitro for Haemopoietic Cells Using Semisolid Agar Medium

Abstract

This chapter discusses how to create clonal cultures in vitro for hemopoietic cells using semisolid agar medium. The discovery of hemopoietic growth factors was facilitated greatly by the ability to grow hemopoietic cells in vitro. These culture systems enable the undifferentiated hemopoietic precursors to proliferate and differentiate into various hemopoietic cell lineages. Especially valuable has been the development of clonal cultures using semisolid agar or methylcellulose culture medium for hemopoietic precursor cells. The fundamental steps in establishing agar-medium cultures include mixing equal volumes of double-strength medium and double-strength agar solution and adding the cells to be cultured and mix. Fetal calf serum (FCS) is used as a source of nutrients in cell cultures. Batches of FCS should be tested extensively prior to purchase, for optimal colony formation in colony number and colony size, in semisolid cultures. Although specific stimuli may be required for many situations, and always for use as a positive control, a variety of conditioned media containing mixtures of hemopoietic growth factors can be prepared.