Major limitation discovered in cellular biology analysis tool

Australian researchers have revealed that specialised immune cells called macrophages are being fragmented during the extraction process to generate single-cell suspensions from bone marrow and other haematopoietic tissues.

Published in the journal Cell Reports, their discovery indicates that data collected using this scientific method are often skewed — which will force the reassessment of a large amount of already-published scientific literature to determine if the reported observations are accurate or incorrectly interpreted. It is also expected to change how researchers approach analysis of these tissues in the future.

Led by Mater Research, the scientists examined a specific subtype of macrophages, known as tissue-resident macrophages, that are present in each tissue of the body and adapt to where they are located to support the tissue’s development, function, health, protection from infection and regeneration after injury. Previous research using single-cell preparations from hematopoietic tissues has often had unexplained, and often ignored, under-representation of tissue-resident macrophages in the generated datasets — even though these cells represent up to 15% of the tissue.

Lead investigator Professor Allison Pettit, Mater Research Director of Biomedical Research, said her team’s examination of tissue-resident macrophages in bone marrow, spleen and lymph nodes discovered that all the macrophages were breaking up during the extraction process for single-cell preparation.

“It was a big shock as it has significant implications for the type of research we do, and it will likely cause controversy in the field, but the first priority of academic research is the integrity of the data, as this is integral to the reliability of translating knowledge gains into real-world impacts,” Prof Pettit said.

Many biological fields rely on tissue disruption techniques to release individual cells for more detailed study. In the case of haematopoietic tissues, it is a simple procedure in which the whole organ is subjected to a gentle mechanical disruption procedure to release the individual cells within the tissue.

“The most surprising aspect of our discovery is that during fragmentation, the macrophages leave remnants of themselves behind on other cells within the preparation, creating a ‘Trojan horse’ effect,” Prof Pettit said.

“This means fragmentation of the macrophages during this procedure creates a great deal of complexity and confounding information in the analysis of the data.

“We have exposed that despite the use of state-of-the-art technical approaches, cells that were thought to be macrophages are actually other cells that had macrophage remnants stuck to them. Consequently, a lot of what we think we know about these cells from decades of research will include a lot of inaccuracies.”

Prof Pettit said many studies that focused on other types of cells within these tissues have also been unknowingly co-detecting macrophage molecules, potentially creating misleading information about the cell types.

“Accurate knowledge relating to the cellular and molecular biology of these tissues is critical to understanding blood cell production and turnover and immune function, as well as informing discovery of causes and cures for leukaemia and other diseases,” she said.

Dr Susan Millard, first author on the study, said the team discovered macrophage fragmentation and the problems it caused when they used imaging flow cytometry as part of their quality control assessment of tissue cell suspension analysis.

“We have been working with these tissues for decades and we always combine both intact whole tissue analysis with the tissue cells suspension analysis,” Dr Millard said. “However, these separate analyses would often provide differing results and we weren’t sure why.

“When the Translational Research Institute, where Mater Research is partly based, acquired the capability to perform imaging flow cytometry to visualise complex multidimensional molecular analysis, we were able to see our data in a whole new light that revealed the macrophage fragmentation issue.

“We know that tissue-resident macrophages in these tissues play incredibly important roles in many aspects of red and white blood cell production, education of effector immune cells to fight off disease, regeneration of these tissues after injury and adaption of these tissues in response to stress. We’ve had a poor track record of harnessing these functions to improve human health and fight disease — and this has likely been contributed to by the flawed analysis methods we uncovered.

“Our study exposes an opportunity for a renewed frontier of discovery empowered by precision knowledge.”

The researchers intend to expand their work to investigate if tissue-resident macrophage fragmentation also occurs when other tissues are similarly studied using single-cell suspension strategies. They are also working to develop new ways to create single-cell suspension that preserves the macrophages.

Image credit: ©stock.adobe.com/au/PRB ARTS

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