Abstract
The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm3 cubes termed explants, which are cultured 1–3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.
Highlights
Nowadays, the great majority of knowledge related to tumour biology stems from or was verified in established tumour cell lines, which have at times been generated long ago and have been passaged innumerable times
Thereafter, cells were gently centrifuged at 0.3 relative centrifugal force (RCF) for 2 minutes and pellets were resuspended in PBS or cell culture medium, depending on further treatment
Functional analyses of molecules involved in processes of cancer comprises three types of experimental systems: animal models of cancer [11], primary short-term cultures of tumour cells ex situ, and permanent cancer cell lines [12]
Summary
The great majority of knowledge related to tumour biology stems from or was verified in established tumour cell lines, which have at times been generated long ago and have been passaged innumerable times. For the case of genomic analyses, divergences between primary cells and permanent lines might be of great importance and are controversially discussed [2]. This is even more pronounced in the case of tumours with phenotypically and molecularly heterogeneous subtypes, such as for example breast cancer. Primary tumour cells obviously reflect the in vivo situation in the human being more closely, but might contain contaminating non-tumour cells and are commonly more difficult to obtain in sufficient amounts. The study of primary tumour cells is of utmost importance to obtain insights in molecular processes occurring in situ as closely as possible
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