Hours of Low - High - Density Exogenous AP High - Density Density Plating Plating

We report development of a model of retroviral gene transduction in high-density limb bud cell micromass culture. The replication competent avian retrovirus RCAS BP (A) carrying the human placental alkaline phosphatase gene (RCAS AP) was used as a marker for retroviral infection and spread. The final protocol balances the need to allow time for retroviral integration and gene transduction against loss of chondrogenic potential when limb bud cells are plated at low density. It includes: (i) incubation of the dissociated limb bud cells with RCAS virus for 2 h followed by lowdensity culture for 48 h to allow retroviral gene expression; and (ii) secondary replating as high-density micromass culture to initiate chondrogenesis. The pattern and level of chondrogenesis in the retrovirustransduced micromass cultures is similar to regular micromass cultures. At least 40%–50% of cells express the retroviraltransduced genes 24 h after high-density plating. This new approach facilitates ectopic gene expression in micromass culture, enabling molecular dissection of chondrogenesis and serves as a model for gene transduction in other organotypic cultures. INTRODUCTION Understanding how single cells collaborate to form a complex organism with many different organs is a central issue in biological research. Organotypic cultures, which mimic the process of organogenesis and can be experimentally manipulated, have been helpful research tools in studying how cells interact with each other and their environment to achieve a complex three-dimensional structure. However, it has not been easy to incorporate genetic approaches into these cultures. Many of these cultures involve the growth of primary cells over a short period of time where stable transfection is non-applicable and where transient transfection may be either too toxic or transfect too few cells. Application of some of the knowledge gained from gene therapy technology (13,29) to organotypic cultures to alter gene expression would make these models much more powerful. In this report, we describe our efforts to adopt such an approach in one wellestablished organotypic culture, highdensity limb bud micromass culture (1,6,9,40). In this culture system, dissociated epithelium-free mesenchymal cells derived from the distal third of the chick limb bud, embryonic stage E23/24 (14), are plated in 10–15-μL drops at a density of 2 × 107 cells/mL. The cells initially appear homogenous, but within 24 h, small cell aggregates are formed. An extracellular matrix rich in chondroitin sulfate and proteoglycans accumulates, and by four days, cartilage nodules are present that are positive for type II collagen and Alcian Blue staining, both markers of chondrocyte phenotype. The sequence of cell differentiation in micromass culture parallels that seen in in vivo cartilage formation, making this a good model for the study of chondrogenesis (5). Micromass culture has been used to study the role of growth factors and adhesion molecules in chondrogenesis (6,21,37,43). These studies have been facilitated by the development of serum-free media (33,34). Progress has also been made in dissecting out some of the signaling pathways involved in chondrogenesis (23,25,41). However, it has been difficult to use this model to dissect the molecular pathways that control chondrogenic differentiation. We have used electroporation to introduce exogenous genes into micromass culture (43); however, the high cell death rate during this procedure and the transient nature of the gene expression make it difficult to achieve a high level of gene expression. Retroviral-mediated gene delivery has several advantages over transient transfection: (i) the initial infection is not toxic to the cell; (ii) the gene expression is stable; (iii) and the percentage of infected cells will increase over time if the virus is replicationcompetent. In this report, we devised a two-stage micromass culture, with a low-density plating window to allow retroviral gene transduction in primary limb bud cells before high-density plating. We used the replication competent avian retroviral vector (RCAS) (A) (17,31,35), as this produces high levels of exogenous gene expression in infected chick limb buds in ovo (30) and chick chondrocytes in long-term culture (20). We demonstrate that the viral exposed cell cultures retain the capacity to undergo chondrogenesis in high-density culture after 48 h of low-density plating. The low-density plating period provides a window for viral replication and gene expression before the onset of 660 BioTechniques Vol. 24, No. 4 (1998) Retroviral Gene Transfer in Chondrogenic Limb Bud Micromass Cultures BioTechniques 24:660-666 (April 1998) chondrogenesis. This new strategy provides a new approach to analyze the molecular cascade in chondrogenesis. MATERIALS AND METHODS