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Tissue Engineering

Tissue Engineering - Poster Session D

Poster L1 - Plasma cell motility and homing is modulated by various niche factors in the bone marrow-on-a-chip model

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  In order to leverage B cells for antibody-based therapeutics, it is important to understand their organization, motility, and interactions in the human bone marrow (hBM). Antibody-secreting cells (ASCs) are terminally differentiated from activated B cells and they secrete antigen-specific antibodies as part of the adaptive immune system. Our previous work has shown that ASCs can mature and survive in our human-bone-marrow-on-a-chip (BMOC) devices, which provide an innovative platform for in vitro B cell studies. By encapsulating the hBM endosteal, central marrow, and perivascular niches using a specially designed 96-well plate, the microfluidic BMOC is able to uniquely capture the microenvironment’s complexities. As a 3D microtissue, it can provide novel spaciotemporal information about the BM microenvironment, which has previously only been possible to attain in vivo from invasive procedures on transgenic mice. ASC movement and homing in the hBM niche have not been studied. Previous work using mice and other in vitro models has indicated that CXCL12-CXCR4 interactions, VCAM-1/VLA-4 interactions, and survival signals from APRIL are important for ASC survival, migration, and adhesion in the BM1. Furthermore, mouse models have shown that ASCs are uniquely motile in the BM, exhibiting “stop-and-go” movement that has not been seen in ASC niches outside of the BM. We found that ASCs localize in the perivascular space in clusters along the vessels adjacent to the aforementioned survival and retention factors. A population of ASCs was found to be motile, exhibiting signs of “stop-and-go” movement, and their motility was modulated by these same factors.

Materials and

Methods:  The BMOCs were manufactured as previously described2. The microenvironment was created by first differentiating mesenchymal stromal cells (MSCs) into osteoblasts (OBs) for 21 days. Then, human umbilical vein endothelial cells (HUVECs) and additional MSCs were loaded into a fibrin-collagen I hydrogel atop the OB layer and normal human lung fibroblasts were seeded in the outer channels. Following BMOC creation, cells were loaded into the devices via the media channel. Devices were rested for 4 hours to allow the cells time to enter the central channel. Then, inhibitors or cytokines were diluted in media and added to the media channel(s): 1000 ng/mL AMD3100 (added to both channels) or 100 ng/mL CXCL12 (added to only one channel, creating a gradient). Images were taken every 3 minutes for 10 hours using an incubated and heated Lionheart FX epifluorescence microscope. The images were analyzed for cell motility using ImageJ. First, images were stacked and movement-corrected using ImageJ plugin Template_Matching.jar. Then, individual cell motility information was gathered using ImageJ plugin TrackMate. The data output from TrackMate was then filtered and sorted based on track displacement into low, medium, and high-motility groups in MATLAB. From there, the data was analyzed and graphed, looking at both all tracks and only high displacement tracks in GraphPad Prism. Statistical analysis used Kruskal-Wallis with Dunn’s post hoc test for nonparametric datasets. A significance level of p < 0.05 was used to indicate statistical significance for all tests.

Results, Conclusions, and Discussions: It was found that the ASCs localize perivascularly in clusters around the vessels. It was further hypothesized that the ASCs would be in close contact with important survival and retention factors upon localization. CXCL12, VCAM-1, and APRIL are highly expressed in the vasculature and ASCs were found to localize in the same z-plane location as the vessels next to these factors. A population of ASCs was found to be motile in the BM, with 62% ± 4.6 of cells having low motility, 12.6% ± 1.6 having medium motility, and 24% ± 3.3 having high motility. Additionally, qualitative analysis of track morphology revealed that motile cells exhibit the “stop-and-go” movement previously noted in mouse studies1. Niche components that impact ASC motility and homing were then tested by creating niche variations: no OB and HUVEC only (2 no endosteal conditions), no perivascular MSCs, and full niche. The no endosteal conditions saw increased ASC motility, with higher average track displacements and higher confinement (CFN) ratios (track displacement/total distance traveled). The impact of a CXCL12 gradient on motility was also analyzed in the full niche. Time-lapse imaging data revealed an increase in the CFN ratio and linearity of forward progression (LFP) compared to the no gradient condition. To further confirm this, treatment with the CXCR4 inhibitor AMD3100 caused the ASCs to no longer respond to the CXCL12 gradient. The CFN ratio and LFP returned to baseline values. Our results used the BMOC to analyze human ASC spatial organization and movement in the hBM, for which there are limited studies in a human-specific environment. The new information about ASC migration and homing point toward factors important for lifelong humoral immunity and that may be implicated in various pathologies. Future work should look at how age may impact these results by using cells from donors of different ages.

Acknowledgements (Optional):  1Benet Z, et al. Cell Rep. 2021;34(6):108733. 2Nelson MR, et al. Biomaterials. 2021;270:120683.