Since British scientist Robert Hooke discovered the building block of life and named them “cells” in 1665, and with the development of cell theory by German biologists Matthias Jakob Schleiden and Theodor Schwann, humans have developed a clearer understanding of cells, the relatively independent units that make up life.

To better understand the essence of life and uncover the basic rules governing cellular activities, scientists have conducted a series of studies on processes such as cell proliferation, movement, metabolism, and death. The concept of isolating living cells from tissues was proposed as early as the 19th century. However, it wasn’t until the 1950s that animal cell culture research began. Initially, cells were merely observed, but later, primary cells were isolated from tissues, and eventually, scientists obtained immortalized cell lines capable of indefinite propagation. Cells have transitioned from a vague concept to a crucial experimental material for in vitro studies, playing a central role in the history of biological development.

There are two main types of cells commonly used in research: primary cells isolated directly from tissues and commercially available cell lines. Typically, cells from the first to tenth generations of isolation are considered primary cells. Most primary cells will eventually cease to grow, age, and die after several generations. In rare cases, cells that survive beyond fifty generations experience genetic changes and become immortal or continuous cell lines. Most immortal cell lines exhibit only the characteristic of indefinite growth, while some also lose contact inhibition, allowing for multilayered growth. Some of these cells even exhibit tumorigenicity when transplanted into other organisms.

Due to their immortal nature and rapid division, immortal cell lines are often the first choice for cellular research. They are easy to culture, come in a variety of types, grow quickly, and are cost-effective, allowing for rapid research progress. For example, the HeLa cell line, the first human cell line capable of indefinite propagation, has greatly facilitated research on human cell diseases and the mechanisms of drug activity. It is widely used in fields such as oncology, immunology, and virology.

However, recent studies have revealed that despite the many advantages of immortal cell lines, there are some limitations to their use.

Limitation 1: Changes in Biological Characteristics of Cells

In research, large numbers of samples are often needed, which means immortal cell lines must rapidly proliferate and expand. During this continuous process, mutations may occur. Continuous passaging without limit can lead to changes in both the genotype and phenotype of the cell lines, which can impact experimental results.

Moreover, immortalized cell lines inherently differ significantly from cells in living tissues in terms of biological characteristics. They cannot fully represent the real environmental conditions inside an organism. Primary cells, on the other hand, are considered more representative of in vivo tissue conditions. In some countries/regions, the use of primary cells has gained legal recognition (e.g., the UK’s Human Tissue Act 2004), especially in early drug toxicity testing. Compared to immortal cell lines, primary cells isolated directly from patient tissues are often seen as a more suitable experimental model.

Limitation 2: Cross-Contamination of Cell Lines

When we think of cell contamination, the first thought is usually microbial contamination leading to experimental failure. However, what we’re talking about here is cross-contamination between cell lines. In other words, other types of cells might inadvertently mix into your experimental cell line!

This problem can arise when cell lines are established or through improper handling, such as co-culturing multiple cell types simultaneously, reusing materials, or contamination through cell culture products. A small mistake in handling can cause an entire experiment to fail. In 2011, the U.S. established national standards for cell line identification using STR analysis, but many researchers are still unaware of the severity of this issue. Relevant organizations have sounded the alarm:

In December 2014 and February 2015, *Science* magazine published articles highlighting the seriousness of cross-contamination and mis-identification of cell lines, emphasizing the consequences of contamination. It not only wastes time and effort, but also leads to irreproducible results, severely impacting research outcomes. Authorities such as the NIH and ATCC have repeatedly called for researchers to authenticate their cell lines. In April 2015, *Nature* announced that all journals under its umbrella would require authors to authenticate the cell lines used in their papers. In June of the same year, it was reported that a scientist had retracted a *Nature* paper due to cell line issues. A 2017 *PLoS ONE* article found that over 30,000 papers used incorrect cell lines contaminated by HeLa cells, rendering their research results invalid【1】. A 2019 study showed that at least 24% of human cell lines were contaminated by HeLa cells【2】.

Limitation 3: Limited Use Case

Many cell types, such as neurons, skeletal muscle cells, cardiomyocytes, pericytes, and terminally differentiated hepatocytes, cannot proliferate in culture. When conducting experiments with these cell types, primary cells must be isolated from fresh tissues each time.

Primary cells most accurately mimic the physiological environment in vivo, retaining specific tissue characteristics. Therefore, they are the most suitable model for evaluating drug efficacy and toxicity, and they are much less likely to be cross-contaminated with other cell lines. In oncology and immunology research, primary cells are also essential for creating in vivo animal models. However, culturing primary cells is more difficult than working with ordinary cell lines, and the isolation and preparation of primary cells remains a significant challenge. How can we efficiently prepare highly active single-cell suspensions while ensuring reproducibility?

The RWD Single-Cell Suspension Preparation Instrument easily addresses this challenge. With self-developed tissue processing tubes, enzyme digestion kits, and built-in optimized tissue processing programs, it can prepare highly active single-cell suspensions or tissue homogenates.

This instrument enables the quick preparation of high-activity, uniform single-cell suspensions, greatly enhancing experimental reproducibility. It features independent four-channel operation and a heating jacket to improve tissue processing efficiency. It is widely used in immunology, oncology, and neurobiology research.

After isolating primary cells, the next steps involve counting and viability analysis of the single-cell suspension, followed by placing the cells in a culture system or appropriate buffer.

Accurate live-cell counting is essential, and automated cell counters can precisely count primary cells. The RWD C100 Automated Cell Counter, equipped with multiple fluorescence channels, provides quantitative analysis of cells in suspension, displaying both brightfield and fluorescence images to clearly present counting results and cell morphology. This instrument is ideal for cell analysis in immunology, vaccine development, cell therapy, cancer research, stem cell research, and metabolic studies.

The final step in the primary cell isolation process is cell culture. The Reward CO₂ Incubator precisely controls temperature and CO₂ concentration while maintaining a high-humidity environment. Its HEPA filters effectively remove airborne particulate contaminants, and the 140°C dry heat sterilization function ensures regular maintenance and sterilization. This incubator provides a stable and clean environment for culturing primary cells.

References:

1. Serge P. J. M. Horbach, Willem Halffman. "The Ghosts of HeLa: How Cell Line Misidentification Contaminates the Scientific Literature." *PLoS ONE*, October 12, 2017, doi:10.1371/journal.pone.0186281.
2. Lin J, Chen L, Jiang W, Zhang H, Shi Y, Cai W. "Rapid Detection of Low-Level HeLa Cell Contamination in Cell Culture Using Nested PCR." *J Cell Mol Med*, 2019;23(1):227–236. doi:10.1111/jcmm.13923.