Application of the 3R principles: Vertebrae as an additional source of murine bone-marrow cells

Abstract

Experimental murine models are an essential tool in the field of bone marrow (BM) transplantation research. Therefore, numerous mice are required to obtain a sufficient number of BM cells, which is in contrast with the Reduction principle of the 3R principles. The selection of the cell source and the isolation protocol are therefore critical in obtaining a sufficient yield of cells for experiments. Nowadays, the vertebrae are already used as an extra source of BM cells to enrich the number of isolated cells from the long bones and ilia (LBI), when needed. Yet, little is known if BM cells from LBI and vertebrae share the same characteristics and can be pooled together for further analysis. Therefore, in this study, we aimed to compare the quantity and characteristics of haematopoietic and stromal cell lines in the BM from the LBI and vertebrae. To count haematopoietic and mesenchymal stem/stromal progenitors, colony-forming unit assays were performed. To determine the expansion capacity of mesenchymal stem/stromal cells (MSCs), cultivation of MSCs and measurement of the expression of surface markers by flow cytometry was performed. The characterisation and enumeration of immune cell populations was also performed by flow cytometry. Here, we show that the vertebrae are a comparable source of BM cells to the LBI regarding the analysed parameters.

Transformation of primary murine peritoneal mast cells by constitutive KIT activation is accompanied by loss of Cdkn2a/Arf expression

“…Ultimately, alternatives to isolation of cells from and experiments with laboratory animals are needed to reduce the number of animals sacrificed for research. Thus, establishing new cell lines supports the 3R guidelines ( 24 ) and will help to achieve the future aim of ending the use of animals in research in the USA and the European Union ( 67 ). This new MC model will therefore sustain future studies on mechanisms and pharmacologic intervention of MC activation.…”

“…Challenging about PMCs is, however, that peritoneal lavages often yield only strictly limited amounts of enriched PMCs that cannot be cultured over a longer time period. As a consequence, to extensively characterize PMC biology, plenty of mice need to be sacrificed to yield validated scientific data, which is not in agreement with the 3R principles of animal welfare ( 24 ).…”

Transformation of primary murine peritoneal mast cells by constitutive KIT activation as a result of lostCdkn2a/Arfexpression

“…Ultimately, alternatives to isolation of cells from and experiments with laboratory animals are needed to reduce the number of animals sacrificed for research. Thus, establishing new cell lines supports the 3R guidelines (24) and will help to achieve the future aim of ending the use of animals in research in the USA and the European Union (67). This new MC model will therefore sustain future studies on mechanisms and pharmacologic intervention of MC activation.…”

“…The copyright holder for this preprint this version posted January 24, 2023. ; https://doi.org/10.1101/2023.01. 24.525344 doi: bioRxiv preprint and Arf was not detectable in PMC-306 cells. We demonstrated that these aberrations lead to an enhanced cell cycle activity, a strongly increased proliferative capacity and best explains the mechanism of PMC transformation.…”

“…The copyright holder for this preprint this version posted January 24, 2023. ; https://doi.org/10.1101/2023.01. 24.525344 doi: bioRxiv preprint carboxypeptidase A3 (CPA3) and heparin proteogylcans, whereas granules of mucosal MCs (MMCs) lack tryptase and instead of heparin predominantly contain chondroitin sulfate (21). In vitro, murine mucosal-like MCs can be obtained from multipotent bone marrow-derived precursor cells, which require a long-lasting differentiation process in the presence of IL-3 and (optionally) stem cell factor (SCF).…”