Mechano-responsive hydrogel for direct stem cell manufacturing to therapy

Abstract

Bone marrow-derived mesenchymal stem cell (MSC) is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties. Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers, but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo. To overcome these challenges, in this work, we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy. With our technology, we found that dynamic mechanical stimulation (DMS) enhanced matrix-integrin β1 interactions which induced MSCs spreading and proliferation. In addition, DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities (e.g., growth factor secretion) through YAP nuclear localization and FAK-ERK pathway. With our magnetic hydrogel, complex procedures from MSC manufacturing to final clinical use, can be integrated into one single platform, and we believe this 'all-in-one' technology could offer a paradigm shift to existing standards in MSC therapy.

Keywords: Cell therapy; DMS, Dynamic Mechanical Stimulation; Dynamic mechanical stimulation; MP, Magnetic Particle; MSC, Mesenchymal Stem Cell; Magnetic hydrogel; Mesenchymal stem cell; Stem cell manufacturing.

Graphical abstract

Scheme 1

Schematic illustration of GelMA/PEGDA-MP (GPM) magnetic hydrogel to provide dynamic mechanical stimulation (DMS) to influence mesenchymal stem cell (MSC) morphology, proliferation, stemness, differentiation, and secretome profile.

Fig. 1

Characterization of GelMA/PEGDA-MP (GPM) hydrogel. (a) Photographs of GPM liquid precursor becoming hydrogel after UV-mediated photo-crosslinking. (b) Storage modulus of GPM hydrogel with different polymer and MP concentrations. (c) Flow cytometric analysis showing negligible leakage of magnetic particles (MPs) from the hydrogel with/without mechanical stimulation. (d) Dynamic Light Scattering (DLS) revealed size of magnetic particle (MP) to be ∼4.1 μm. Inserted image: Transmission Electron Microscopy (TEM) image of MP with a spherical morphology. (e) M − H curves of GPM hydrogels with different MP concentrations via superconducting quantum interference device (SQUID) analysis, showing that GPM hydrogels were magneto-responsive. (f) Scanning Electron Microscopy (SEM) images of porous network in GPM hydrogel with varying MP concentrations. As the MP amount increased, the hydrogel network was gradually perturbed with decreased network crosslinking rate and enhanced pore sizes. (g) Average pore diameter of GPM hydrogel network with different MP concentrations. All box plots indicate median (middle line), 25th, 75th percentile (box) and the lowest (respectively highest) data point (whiskers). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig. 2

Cell growth and morphology of bone marrow-derived mesenchymal stem cells (MSCs) in GPM hydrogel. (a) Live-dead staining of MSCs encapsulated in hydrogel from 0 to 14 days, indicating high biocompatibility of GPM hydrogel. (b) Profile of magnetic force applied to GPM hydrogel from the dynamic magnetic device with the magnet movement ranging from 20 to 66 mm. (c) Multiphysics modelling of DMS-induced force distribution within cylinder hydrogel (magnet-hydrogel distance are 20 and 66 mm), suggesting that the force is uniform within hydrogel construct. (d) Fluorescent images of MSC morphology and cytoskeletons under different culture conditions, showing that dynamic mechanical stimulation (DMS) promoted MSC spreading. Violin plot of MSC (e) spreading area and (f) cell aspect ratio under DMS. Internal box plots show medians with interquartile range and whiskers. (g) Fluorescent images of MSC cytoskeleton and integrin protein level showing that DMS promoted higher surface expressions of integrin-β1 linked to MSC spreading. (h) Box and whisker plots of MSC spreading area upon siRNA inhibition indicating that integrin β1 plays a key role in cell spreading in DMS. All box plots indicate median (middle line), 25th, 75th percentile (box) and the lowest (respectively highest) data point (whiskers). Error bars represent standard deviations based on four biological replicates, in which at least 10 MSC single cells were analyzed for each replicate. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s. is not significant.

Fig. 3

The gene expression of potential activated integrin-related pathways under dynamic mechanical stimulation (DMS). Heatmap showing changed gene expression profile of bone marrow-derived mesenchymal stem cells (MSCs) in different DMSs. The dynamic force is conducive to MSC spreading, proliferation, and secretion in the standard medium. DMS has a positive effect on cell osteogenesis while it impeded cell chondrogenesis. Each row represents the gene expression profile for MSCs from rat donors in different conditions, and each column represents a gene. Hierarchical clustering dendrogram showing Pearson correlation between different genes.

Fig. 4

Cell proliferation of bone marrow-derived mesenchymal stem cells (MSCs) in GPM hydrogel. (a) Parametric analysis of MSCs proliferation condition found that stronger and longer dynamic mechanical stimulation (DMS) better promoted MSC proliferation. (b) Strong positive correlation between cell proliferation and DMS (ρ, Pearson correlation coefficient). Magneto-stimulation index is obtained by multiplying ‘magnetic force amplitude’ with ‘stimulation duration’ which shows the DMS strength. (c) Western blot analysis that dynamic environment promotes MSC proliferation via YAP pathway that downregulates LATS-driven YAP phosphorylation and facilitates YAP nuclear localization rate. (d) Fluorescent images of YAP nuclear localization under different DMS conditions. (e) Flow cytometry of CD90 expression on MSC surface under different culture conditions. Data show that static and 3D environment better maintained MSC stemness. The red dashed outlines represent cell cluster with relatively lower CD90 expression, and this cell subpopulation was highest in 2D culture and 3D culture under strong DMS. (f) Bar graph showing that strong DMS (3Day/20 mm) improved cell proliferation while compromising MSC stemness, similar to 2D culture. (g) Graph of MSC manufacturing under different DMS conditions suggesting that 3D culture with gentle dynamic stimulation (low magneto-stimulation index) could produce MSCs with a better balance between cell quantity and quality (i.e., PROLIFERATION*STEMNESS). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s. is not significant.

Fig. 5

Osteogenic and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) in GPM hydrogel. (a) Parametric analysis of MSC osteogenic differentiation indicating that stronger and longer dynamic mechanical stimulation (DMS) favored osteogenesis. (b) Strong positive correlation between osteogenesis and external stimulation (ρ, Pearson correlation coefficient). (c) Western blot analysis showing that DMS promoted MSC osteogenesis via FAK pathway. (d) Parametric analysis of MSC chondrogenic differentiation indicating that stronger and longer DMS disfavored chondrogenesis. (e) Strong negative correlation between chondrogenesis and external stimulation (ρ, Pearson correlation coefficient). (f) Western blot analysis showing reduced chondrogenic marker proteins under dynamic conditions. (g) Schematic illustration of osteochondral interface on the knee joint. (h) Photos and microscopic images of bilayer hydrogel for MSC osteo-chondrogenesis. (i) 3D stack image of confocal microscopy showing that MSCs preferentially underwent osteogenesis under DMS and chondrogenesis under static microenvironment. (j,k) The MSC differentiation preferences are represented by the intensity of secreted collagen type I and II in different hydrogel layers, which is related to osteogenesis and chondrogenesis, respectively. Data are expressed as mean ± SD, n = 4.

Fig. 6

Secretome of bone marrow-derived mesenchymal stem cells (MSCs) in GPM hydrogel. (a) Parametric analysis of MSC-derived vascular endothelial growth factor (VEGF) indicating that stronger and longer dynamic mechanical stimulation (DMS) favored VEGF production. (b) Strong positive correlation between VEGF secretion and external stimulation (ρ, Pearson coefficient). (c) Western blot analysis showing DMS promoted MSC VEGF secretion via FAK-ERK pathway. (d) Contrast brightfield images monitoring of angiogenic sprouting from fibrin gel beads in 0 and 6 h incubated in MSC conditioned medium under different culture conditions. (e) Tube formation bioassay of umbilical vein endothelial cells incubated with MSC conditioned medium from different culture conditions, showing that MSC conditioned medium under stronger DMS (i.e., 3Day/20 mm) had better angiogenic sprouting and vascularization network formation. Yellow frames represent the zone of below fluorescent images. Data are expressed as mean ± SD, n = 3.