In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

Abstract

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

“…Currently, various spheroid culture methods have emerged to form cell aggregates and study their effect on MSCs, including the use of non-adherent plates or microfabrication-based non-adhesive surfaces, the hanging-drop method, spinner or rotary dynamic culture systems, or the use of agarose multi-well dishes from rubber micro-molds (Song et al, 2004; Nyberg et al, 2005; Bogdanova-Jatniece et al, 2014; Rettinger et al, 2014; Guo et al, 2015; Nakagawa et al, 2016). Compared to other methods, the hanging-drop method is relatively economical and convenient.…”

Digital microfluidic platform for dielectrophoretic patterning of cells encapsulated in hydrogel droplets

“…[4][5][6] High throughput procedures are traditionally executed in vitro using petri dishes or well plates. [7][8][9][10][11] Cell to cell communication and extracellular matrix (ECM) interaction is essential for cell viability and functional differentiation. [7][8][9][10][11] Cell to cell communication and extracellular matrix (ECM) interaction is essential for cell viability and functional differentiation.…”

“…[7][8][9][10][11] Cell to cell communication and extracellular matrix (ECM) interaction is essential for cell viability and functional differentiation. 10,11,14 The precision and control of microuidic devices has allowed for the creation of monodisperse microgels to serve as the ECM. 13 Thus, for two dimensional (2D) tissue array platforms, the ability to make predictions on in vivo performance is limited by the capacity to analyse the interaction between cells and their environment.…”

Mineral particles modulate osteo-chondrogenic differentiation of embryonic stem cell aggregates

“…Alternatively, a more physiologically relevant 3D microenvironment can be emulated through cell self-assembly, which has been applied to form multicellular aggregates in various cells types [31-33]. In particular, PSC aggregates are capable of recapitulating various morphogenetic processes associated with embryonic development and thus have been used to form various highly structured and functional tissues commonly referred to as “organoids” [34-38].…”