Navigating challenges: optimising methods for primary cell culture isolation

Authors

Oliwia Piwocka, Marika Musielak, Karolina Ampuła, Igor Piotrowski, Beata Adamczyk, Magdalena Fundowicz, Wiktoria Maria SuchorskaJulian Malicki

Abstract

Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines.

 

Background

Breast cancer (BC) is the most common neoplasm in women, responsible for 2,261,419 deaths in 2020, which makes it the highest mortality due to cancer in both developed and developing countries [1, 2]. Researchers predict decreasing mortality of breast cancer patients [3, 4] due to improvements in treatment methods, advancements in breast screening, and higher awareness among women [5]. During the last decades, the research on breast cancer was seminal for the development of innovative treatments for breast cancer. The elucidation of interpersonal genetic variations and functional characteristics with the help of primary cancer cell lines has been an important pre-requisite for this progress [6].

A primary cell line refers to a culture of cells directly derived from tissues or organs of an organism. These cells are typically used in laboratory research to study various aspects of cell biology, physiology, and disease. Unlike immortalized cell lines, which can divide indefinitely, primary cell lines have a limited lifespan in culture, reflecting the normal biological constraints of the cells [6]. Primary cancer cells can be obtained from surgically resected tissue samples, core biopsies, aspirates, pleural effusions, and autopsy materials. Cell populations can differ depending on the isolation method and the tissue composition of the collected material. Moreover, cells of the tumour microenvironment (TME) such as normal fibroblasts (NFs), cancer-associated fibroblasts (CAFs), endothelial cells, macrophages and lymphocytes, and endothelial cells are a crucial part of the experimental system [7].

Tumour resections and tumour biopsies are the favoured source for primary cultures [6, 8,9,10,11,12,13]. Primary cultures derived from tumour tissue preserve characteristics of the TME and the cells’ stem-like phenotype and display a specific cross-talk between healthy and malignant cells [14]. This intercellular cross-talk is critical during carcinogenesis, progression, and invasion and plays a vital role in response to therapy [15]. These features assure the validity of primary cultures as a model for preclinical studies or designing personalised therapy since patient-derived primary cells reflect tumour nature more accurately than immortalised cell lines [14]. Another advantage of primary cultures is a large transcriptomic and proteomic variety, which is important for personalized medicine research [16]. Moreover, primary cells preserve cellular markers and tissue functionality, while established cell lines often lose those properties [14]. Limitations of primary cell cultures are difficult isolation, short lifespan, and a finite ability to replicate [17]. Although established cell lines enable researchers to work on the same material worldwide, which guarantees replicability, and that feature has contributed to the formulation of many currently used therapies, primary cells are the better model according to individual requirements [14].

This article aimed to show the efficacy of different enzymatic and mechanical isolation methods for primary cancer cells applicable to breast cancer tumour biopsies. This paper systematises the current knowledge and protocols of isolation of primary BC cells and thus may serve as a compendium for this area of research. Moreover, the article addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations.

 

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