Size-Based Microfluidic-Enriched Mesenchymal Stem Cell Subpopulations Enhance Articular Cartilage Repair.

BACKGROUND: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing. HYPOTHESIS: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair. STUDY DESIGN: Controlled laboratory study. METHODS: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis. RESULTS: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone. CONCLUSION: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs. CLINICAL RELEVANCE: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes.

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties.

BACKGROUND: Mesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD. METHODS: Human bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination. RESULTS: As compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls. CONCLUSION: Our study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence.

Perspective in Achieving Stratified Articular Cartilage Repair Using Zonal Chondrocytes.

Articular cartilage is composed of superficial, medial, and deep zones, which endow the tissue with biphasic mechanical properties to withstand shearing force and compressional loading. The tissue has very limited self-healing capacity once it is damaged due to its avascular nature. To prevent the early onset of osteoarthritis, surgical intervention is often needed to repair the injured cartilage. Current noncell-based and cell-based treatments focus on the regeneration of homogeneous cartilage to achieve bulk compressional properties without recapitulating the zonal matrix and mechanical properties, and often oversight in aiding cartilage integration between host and repair cartilage. It is hypothesized that achieving zonal architecture in articular cartilage tissue repair could improve the structural and mechanical integrity and thus the life span of the regenerated tissue. Engineering stratified cartilage constructs using zonal chondrocytes have been hypothesized to improve the functionality and life span of the regenerated tissues. However, stratified articular cartilage repair has yet to be realized to date due to the lack of an efficient zonal chondrocyte isolation method and an expansion platform that would allow both cell propagation and phenotype maintenance. Various attempts and challenges in achieving stratified articular cartilage repair in a clinical setting are evaluated. In this review, different perspectives on achieving stratified articular cartilage repair using zonal chondrocytes are described. The effectiveness of different zonal chondrocyte isolation and zonal chondrocyte phenotype maintenance methodologies during expansion are compared, with the focus on recent advancements in zonal chondrocyte isolation and expansion that could present a possible strategy to overcome the limitation of applying zonal chondrocytes to facilitate zonal architecture development in articular cartilage regeneration. Impact Statement The zonal properties of articular cartilage contribute to the biphasic mechanical properties of the tissues. Recapitulation of the zonal architecture in regenerated articular cartilage has been hypothesized to improve the mechanical integrity and life span of the regenerated tissue. This review provides a comprehensive discussion on the current state of research relevant to achieving stratified articular cartilage repair using zonal chondrocytes from different perspectives. This review further elaborates on a zonal chondrocyte production pipeline that can potentially overcome the current clinical challenges and future work needed to realize stratified zonal chondrocyte implantation in a clinical setting.

A Pre-Clinical Animal Study for Zonal Articular Cartilage Regeneration Using Stratified Implantation of Microcarrier Expanded Zonal Chondrocytes.

OBJECTIVE: The zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. In this study, the efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a porcine chondral defect model. DESIGN: Autologous chondrocytes extracted from articular cartilage in the non-weight bearing trochlea region of the knee were subjected to an expansion-sorting strategy, integrating dynamic microcarrier (dMC) culture, and spiral microchannel size-based zonal chondrocyte separation. Zonal chondrocytes were then implanted as bilayered fibrin hydrogel construct in a porcine knee chondral defect model. Repair efficacy was compared with implantation with cell-free fibrin hydrogel and full thickness (FT) cartilage-derived heterogenous chondrocytes. Cartilage repair was evaluated 6 months after implantation. RESULTS: Sufficient numbers of zonal chondrocytes for implantation were generated from the non-weight bearing cartilage. Six-month repair outcomes showed that bilayered implantation of dMC-expanded zonal chondrocytes resulted in substantial recapitulation of zonal architecture, including chondrocyte arrangement, specific Proteoglycan 4 distribution, and collagen alignment, that was accompanied by healthier underlying subchondral bone. CONCLUSION: These results demonstrate that with appropriate expansion and isolation of zonal chondrocytes, the strategy of stratified zonal chondrocyte implantation represents a significant advancement to Autologous Chondrocyte Implantation-based cartilage regeneration, with the potential to improve the long-term integrity of the regenerated tissues.

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells.

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.

Label-free separation of mesenchymal stem cell subpopulations with distinct differentiation potencies and paracrine effects.

Mesenchymal stem cells (MSCs) have the capability to differentiate into multiple cell lineages, and produce trophic factors to facilitate tissue repair and regeneration, and disease regression. However, the heterogeneity of MSCs, whether inherent or developed during culture expansion, has a significant impact on their therapeutic efficacy. Therefore, the ability to identify and select an efficacious subpopulation of MSCs targeting specific tissue damage or disease holds great clinical significance. In this study, we separated three subpopulations from culture expanded human bone marrow derived MSCs according to cell size, using a high-throughput label-free microfluidic cell sorting technology. The size-sorted MSC subpopulations varied in tri-lineage differentiation potencies. The large MSCs showed the strongest osteogenesis, medium-size MSCs were advantageous in chondrogenesis and adipogenesis, and the small MSCs showed the weakest tri-lineage differentiation. The size-sorted MSC subpopulations also exhibited different secretome profiles. The large MSC secretome possessed highest levels of osteogenic promotor proteins and senescence-associated factors, but lower levels of osteogenic inhibitor proteins compared to the medium-size MSC secretome. The medium-size MSC secretome had high levels of chondrogenic promotor proteins, and contained lower levels of chondrogenic inhibitor proteins compared to the large MSC secretome. The secretome of size-sorted MSC subpopulations showed differences in paracrine effects. We found that the secretome of large MSCs enhanced osteogenic and adipogenic potencies during MSC culture expansion, but also induced cell senescence; and the secretome of medium-size MSCs promoted chondrogenesis. This study demonstrates size-dependent differentiation potency and secretome profile of MSC subpopulations, and provides an effective and practical technology to isolate the respective subpopulations, which may be used for more targeted tissue repair and regeneration.

Improved zonal chondrocyte production protocol integrating size-based inertial spiral microchannel separation and dynamic microcarrier culture for clinical application.

The zonal property of articular cartilage endows the tissue with biphasic mechanical properties to withstand shearing force and compressional loading. Current treatments for articular cartilage damage are not able to efficiently restore the zonal organisation and functionality. Size-based sorting of freshly isolated chondrocytes from full thickness (FT) cartilage using a spiral microfluidic device was shown to efficiently separate and enrich zonal chondrocytes. The translational application of this sorting protocol is challenging in the clinical setting due to the limited number of autologous chondrocytes from a patient. It is thus essential to explore the practicability of this sorting protocol on expanded chondrocytes. In this study, we first show that size-sorted zonal chondrocytes expanded on microcarriers in dynamic condition (dMC) were able to support comparable proliferation, while maintaining cell morphology, and the zonal cell size-phenotype relation, in contrast to expansion on a tissue culture plate. We further show that post-expansion size-based sorting can be applied on dMC-expanded FT chondrocytes, generating enriched zonal subpopulations that form phenotypically distinct cartilage constructs in the 3D hydrogel. This study demonstrates a novel scale-up zonal chondrocyte production protocol, incorporating size-based zonal chondrocyte separation and dMC platform, to maintain zonal chondrocytes' phenotypes better to support zonal repair of articular cartilage.

Characterization and application of size-sorted zonal chondrocytes for articular cartilage regeneration.

Current clinical approaches for articular cartilage repair have not been able to restore the tissue with zonal architecture, and its biomechanical and functional properties. Mimicking the zonal organization of articular cartilage in neo-tissue by implanting zonal chondrocyte subpopulations in multilayer construct could enhance the functionality of the graft, engineering of stratified tissue has not yet been realized due to lack of efficient and specific zonal chondrocyte isolation protocol. We show that by using a spiral microchannel device, the superficial, middle and deep zone chondrocytes can be separated based on cell size, and enriched from full thickness porcine cartilage in a high-throughput, label-free manner. The size-sorted cells show zone-specific characteristics in RT-PCR analysis of zonal cartilage markers. Both freshly sorted and two-passage expanded zonal chondrocytes formed cartilage tissue in 3D hydrogel, bearing respective zonal characteristics, indicated by RT-PCR, histology, extracellular matrix proteins, and mechanical compression test. In the proof-of-concept in vivo study using a rodent cartilage defect model, the size-sorted zonal chondrocytes when delivered in bi-layered hydrogel construct, facilitated better cartilage repair with mechanically enhanced cartilage tissue, in comparison to conventional chondrocytes implantation. This study provides an effective approach to obtain large numbers of zonal chondrocytes, and demonstrates the translational potential of stratified zonal chondrocyte implantation for clinical repair of critical size cartilage defects.

Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential in vitro.

Mesenchymal stem cells (MSCs) have been shown as potential candidates for cell-based therapies for a diverse range of tissue regenerative applications. Therapeutic use of MSCs usually requires culture expansion, which increases the heterogeneity of MSCs in vitro, thus affecting the potency of the MSCs for more specific indications. The capacity for identifying and isolating special subsets of MSCs for treatment of specific diseases therefore holds great clinical significance. An important therapeutic application of MSC is for the regeneration of cartilage tissue. We and others have previously developed label-free microfluidic means to isolate subpopulations of culture expanded MSCs based on distinct biophysical characteristics. Here we utilize a spiral micro-channel device to separate culture expanded MSCs into five subgroups according to cell size, and study their proliferation and chondrogenesis at early, middle and late passages. Results show that in all passages, the medium-size subpopulation (cell size of 17-21 µm), compared to other subpopulations, displays significantly higher proliferation rate and chondrogenic capacity in terms of cartilage extracellular matrix formation. Also, the small cell subpopulation (average cell size of 11-12 µm) shows lower viability, and large cell subpopulation (average cell size 23-25 µm) expresses higher level of senescence-associated ß-galactosidase. Finally, we show that repeated microfluidic exclusion of MSCs larger than 21 µm and smaller than 17 µm at every passage during continuous culture expansion result in selected MSCs with faster proliferation and better chondrogenic potential as compared to MSC derived from conventional expansion approach. This study demonstrates the significant merit and utility of size-based cell selection for the application of MSCs in cartilage regeneration.