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3881 Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Program: Oral and Poster Abstracts
Session: 506. Bone Marrow Microenvironment: Poster III
Hematology Disease Topics & Pathways:
Fundamental Science, Research
Monday, December 12, 2022, 6:00 PM-8:00 PM

Frances E Lock, PhD1*, Alannah Wilson, BSc2*, Amanda Amoah, PhD2*, Sharon Gao2*, Carolina Tropini, PhD3,4*, Arefeh Rouhi, PhD2,5* and Florian Kuchenbauer, MD PhD1,6,7

1Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, BC, Canada
2Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, Canada
3School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada
4Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada
5Department of Medicine, University of British Columbia, Vancouver, BC, Canada
6The Leukemia/Bone Marrow Transplant Program of British Columbia, British Columbia Cancer Agency, Vancouver, BC, Canada
7Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada


BM-MSC are a collection of primitive cell populations, a major stromal component of the BM niche and are essential to maintain tissue homeostasis. 2D culture protocols are popular, though not standardized, and effective for BM-MSC expansion, but display limited niche function in co-culture systems. 3D co-culture leverages the importance of cell signaling networks activated by cell–cell and cell–matrix interactions in native tissues. Here, we establish a superior and easy-to-use 3D culture protocol and compare to conventional 2D approaches.

Methods, Results and Discussion

First, we compared commercially available expansion media to the most widely cited investigator-defined media for optimal 2D expansion and 3D culture of ex vivo murine BM-MSC.

Based on this, we developed a five-marker flow cytometry panel to accurately quantify BM-MSC culture purity. To test BM-MSC niche function in 2D vs 3D cultures, we generated 3D spheroid hanging drop (HD) cultures from 2D expanded BM-MSC. In HD spheroids, cells are in direct contact with each other as well as extracellular matrix components and can be used to co-culture multiple different cell types. To assess the niche-like function of 3D HD cultures compared to 2D adherent cultures, we assessed Nestin protein expression since Nestin is a marker of HSC niches within mouse BM in vivo. BM-MSC were cultured in 2D or HD and Nestin protein expression assessed by immunofluorescence imaging, showing a significant increase in percent of Nestin-positive cells in HD-cultured BM-MSC, compared to matched 2D controls, in agreement with previous reports in human BM-MSC 3D culture. To assess 3D in vitro BM-MSC niche (IVBMN) function, we assayed stemness markers and survival of FACS isolated primary HSPC (Lineage-Sca1+cKit+, LSK) from mouse BM. LSK cells were cultured as standard (alone) or seeded as co-cultures with BM-MSC within HD with specific cytokine support, in low oxygen (6.5%) conditions (IVBMN) (Figure 1). LSK cells cultured within IVBMN exhibited superior survival as well as retention of stemness markers, demonstrating the benefit of BM-MSC support.

In vivo, the BM niche also includes BM niche resident macrophages (BM-Mɸ), which were recently shown to be a limiting factor for engraftment and HSC reconstitution in irradiated mice, and are part of long term and short term HSC niches. To further extend our IVBMN, BM-Mɸ [F4/80+ Ly6G- VCAM1+ CD169+] were collected by FACS and added to IVBMN cultures (IVBMN-Mɸ). Notably, the IVBMN-Mɸ co-culture model further enhanced survival and stemness of primary LSK cells, when compared to LSK cultured alone or within IVBMN. Based on these preliminary data, we demonstrate that our IVBMN-Mɸ culture system is a unique tool which mimics niche properties in vitro and is easy-to-use for HSPC manipulation.


Here we describe a standardized flow panel for reliable murine BM-MSC immunophenotyping, and characterize a simple but effective 3D BM-MSC co-culture system which allows niche-like function in vitro.

Fig. 1. In vitro BM niche (IVBMN) experimental workflow: A) Ex vivo expanded primary BM-MSC (20x) (i) -/+ FACS isolated BM niche resident Mɸ (ii) in co-culture with primary BM HPSC (LSK) (iii). B) Cells were cultured in a 3D mixed culture system with specific cytokine support, in 6.5% low oxygen. C) IVBMN-Mɸ [MSC+ BM-Mɸ +LSK] co-culture promotes maintenance of primary viable LSK compared to IVBMN [MSC+LSK] or LSK standard culture conditions alone, after 5 days culture.

Disclosures: No relevant conflicts of interest to declare.

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