Guide for the initiation of primary tumor cell cultures


Cancer cell lines are used to study tumor biology in vitro and as models for testing novel anticancer therapeutics. To date, the vast majority of studies in this field was done based on established cell lines that were cultured for decades. However, upon extensive in vitro propagation, cancer cell lines can undergo changes as they potentially acquire multiple genetic and epigenetic alterations, lose the heterogeneity present in the parental tumor, and tend to lack tumor-initiating as well as multilineage differentiation capacity. Consequently, classical tumor cell lines only insufficiently represent the characteristics of a tumor (PMID: 22068913). To generate improved models for cancer research, novel cell lines are derived from primary tissues and used at low passage numbers. However, this process is very inefficient for most tumor entities. In addition, most of the media used include large amounts of mostly undefined serum, which has been shown to exert a selective pressure leading to a reduction of clonal heterogeneity in primary tumor cell cultures. Moreover, serum drives primary tumor cells to a more differentiated state when used over multiple passages. (PMID: 17122771, 18371377, 16697959) Taken together, thus far there is a lack of methods and reagents allowing for an unbiased, non-selective culture of primary tumor cells. For this reason, Miltenyi Biotec developed instruments and reagents, including specialized media, to provide an optimized workflow for the initiation and maintenance of primary tumor cell lines with retained heterogeneity. 

Workflow for initiating primary tumor cell cultures

Overview of the procedure

Figure 1 illustrates the workflow for the generation and culture of tumor cells, encompassing sample collection, automated tumor tissue dissociation, tumor cell isolation, flow cytometric analysis of isolated tumor cells, and ultimately the cultivation of tumor cells. A workflow for a pancreatic tumor cell line is shown by way of example. The workflow offers options to standardize various process steps such as tissue dissociation, seeding of purified tumor cells, and tumor cell propagation. One of the most crucial components within the workflow is the culture medium because it contributes eminently to the maintenance of cell culture stability and heterogeneity of the tumor cells.

Figure 1: Workflow for the generation of a tumor cell line. 

TumorMACS Media

Miltenyi Biotec designed specialized media, the TumorMACS Media, which enable the initiation and expansion of primary tumor cell cultures from epithelial tumors (PMID: 26855150). These media are serum free and have been optimized with regard to formulation, stability, and usability. To streamline the process of tumor cell culture, TumorMACS Media offer the flexibility to seed tumor pieces, single cells from bulk tumor, or pre-sorted populations of tumor cells. Notably, TumorMACS Media enable the unbiased growth of epithelial tumor cells, thus offering the gentle selection of cancerous cells while preserving the initial heterogeneity of the bulk tumor with regard to tumor cell subtypes. To address the diverse needs of different tumor entities, TumorMACS Media are optimized for different tumor types.



Important considerations when initiating primary tumor cell cultures

Examples of results