Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

Abstract

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

Hematopoietic Niche – Exploring Biomimetic Cues to Improve the Functionality of Hematopoietic Stem/Progenitor Cells

“…Moreover, although MSC cultivated as 2‐D adherent monolayer cells or as 3‐D spheroids supported similar expansion levels of total BM hematopoietic cells, higher expansion of more primitive HSC was observed in the presence of a 3‐D HSPC/MSC organization. However, contradictory results were obtained by Schmal and colleagues, who stated that, despite enhanced expression of niche ECM components by 3‐D MSC spheroids, expansion of primitive UCB CD34 + ‐isolated hematopoietic progenitors was favored by a 2‐D monolayer arrangement of MSC . These conflicting results might be, at least in part, explained by differences in MSC isolation protocols, culture media and species variation since the former study used murine MSC and HSPC populations whereas the latter obtained UCB HSPC and BM MSC from human donors.…”

Mass Spectrometry Analyses of Multicellular Tumor Spheroids

“…The simplest, and most widely used, means of generating MCTS consist of suspensions of homotypic cancer cells which are encouraged to aggregate by a range of methods. The best characterized include culturing on non‐adhesive surfaces, hanging drop, and spinner flask (reviewed in).…”

Spheroid model for functional osteogenic evaluation of human adipose derived stem cells

“…Also, unlike in the 2D monolayer, the spheroids do not have a firm attachment to the TCPS surface. This may further reduce their expansion potential, as observed by Schmal et al in spheroids formed with MSCs of umbilical origin …”

“…This may further reduce their expansion potential, as observed by Schmal et al in spheroids formed with MSCs of umbilical origin. 31 Considering the reciprocal relationship between proliferation and differentiation, we then expected to observe a superior osteogenic differentiation in our 3D spheroid culture. To study the osteogenic differentiation and maturation capacity of hASCs cultured on ELP-PEI coated and uncoated TCPS surfaces, we selected two of the well-researched early and late markers of differentiation, namely, ALP activity and OCN production.…”