A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays.

If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.

Method for manufacture and cryopreservation of cartilage microtissues.

The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function.

SP7 gene silencing dampens bone marrow stromal cell hypertrophy, but it also dampens chondrogenesis.

For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-ß1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-ß1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-ß1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies.

Spray nebulization enables polycaprolactone nanofiber production in a manner suitable for generation of scaffolds or direct deposition of nanofibers onto cells.

Spray nebulization is an elegant, but relatively unstudied, technique for scaffold production. Herein we fabricated mesh scaffolds of polycaprolactone (PCL) nanofibers via spray nebulization of 8% PCL in dichloromethane (DCM) using a 55.2 kPa compressed air stream and 17 ml h-1polymer solution flow rate. Using a refined protocol, we tested the hypothesis that spray nebulization would simultaneously generate nanofibers and eliminate solvent, yielding a benign environment at the point of fiber deposition that enabled the direct deposition of nanofibers onto cell monolayers. Nanofibers were collected onto a rotating plate 20 cm from the spray nozzle, but could be collected onto any static or moving surface. Scaffolds exhibited a mean nanofiber diameter of 910 ± 190 nm, ultimate tensile strength of 2.1 ± 0.3 MPa, elastic modulus of 3.3 ± 0.4 MPa, and failure strain of 62 ± 6%.In vitro, scaffolds supported growth of human keratinocyte cell epithelial-like layers, consistent with potential utility as a dermal scaffold. Fourier-transform infrared spectroscopy demonstrated that DCM had vaporized and was undetectable in scaffolds immediately following production. Exploiting the rapid elimination of DCM during fiber production, we demonstrated that nanofibers could be directly deposited on to cell monolayers, without compromising cell viability. This is the first description of spray nebulization generating nanofibers using PCL in DCM. Using this method, it is possible to rapidly produce nanofiber scaffolds, without need for high temperatures or voltages, yielding a method that could potentially be used to deposit nanofibers onto cell cultures or wound sites.

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-ß1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-ß)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-ß1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-ß1 (10 ng/mL). Like TGF-ß1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-ß1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-ß1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-ß1 was the most potent GF for inducing chondrogenesis in BMSCs.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

Bone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Collagenase treatment appears to improve cartilage tissue integration but damage to collagen networks is likely permanent.

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration.

Secreted frizzled related-protein 2 (Sfrp2) deficiency decreases adult skeletal stem cell function in mice.

In a previous transcriptomic study of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells"), SFRP2 was highly over-represented in a subset of multipotent BMSCs (skeletal stem cells, SSCs), which recreate a bone/marrow organ in an in vivo ectopic bone formation assay. SFRPs modulate WNT signaling, which is essential to maintain skeletal homeostasis, but the specific role of SFRP2 in BMSCs/SSCs is unclear. Here, we evaluated Sfrp2 deficiency on BMSC/SSC function in models of skeletal organogenesis and regeneration. The skeleton of Sfrp2-deficient (KO) mice is overtly normal; but their BMSCs/SSCs exhibit reduced colony-forming efficiency, reflecting low SSC self-renewal/abundancy. Sfrp2 KO BMSCs/SSCs formed less trabecular bone than those from WT littermates in the ectopic bone formation assay. Moreover, regeneration of a cortical drilled hole defect was dramatically impaired in Sfrp2 KO mice. Sfrp2-deficient BMSCs/SSCs exhibited poor in vitro osteogenic differentiation as measured by Runx2 and Osterix expression and calcium accumulation. Interestingly, activation of the Wnt co-receptor, Lrp6, and expression of Wnt target genes, Axin2, C-myc and Cyclin D1, were reduced in Sfrp2-deficient BMSCs/SSCs. Addition of recombinant Sfrp2 restored most of these activities, suggesting that Sfrp2 acts as a Wnt agonist. We demonstrate that Sfrp2 plays a role in self-renewal of SSCs and in the recruitment and differentiation of adult SSCs during bone healing. SFRP2 is also a useful marker of BMSC/SSC multipotency, and a factor to potentially improve the quality of ex vivo expanded BMSC/SSC products.

Human bone marrow-derived stromal cell behavior when injected directly into the bone marrow of NOD-scid-gamma mice pre-conditioned with sub-lethal irradiation.

BACKGROUND: Direct bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ-/- (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice. METHODS: One day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues. RESULTS: hBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities,  hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region. CONCLUSIONS: While significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time.

Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice.

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of "pieces" of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ-/- (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 µm achieving superior microtissue vascularisation relative to mesh with 36×36 µm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

A single day of TGF-ß1 exposure activates chondrogenic and hypertrophic differentiation pathways in bone marrow-derived stromal cells.

Virtually all bone marrow-derived stromal cell (BMSC) chondrogenic induction cultures include greater than 2 weeks exposure to transforming growth factor-ß (TGF-ß), but fail to generate cartilage-like tissue suitable for joint repair. Herein we used a micro-pellet model (5 × 103 BMSC each) to determine the duration of TGF-ß1 exposure required to initiate differentiation machinery, and to characterize the role of intrinsic programming. We found that a single day of TGF-ß1 exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-ß1 exposure. Despite cessation of TGF-ß1 exposure following 24 hours, intrinsic programming mediated further chondrogenic and hypertrophic BMSC differentiation. These important behaviors are obfuscated by diffusion gradients and heterogeneity in commonly used macro-pellet models (2 × 105 BMSC each). Use of more homogenous micro-pellet models will enable identification of the critical differentiation cues required, likely in the first 24-hours, to generate high quality cartilage-like tissue from BMSC.

Stromal cells cultivated from the choroid of human eyes display a mesenchymal stromal cell (MSC) phenotype and inhibit the proliferation of choroidal vascular endothelial cells in vitro.

Mesenchymal stromal cells (MSC), with progenitor cell and immunological properties, have been cultivated from numerous vascularized tissues including bone marrow, adipose tissue and the corneal-limbus of the eye. After observing mesenchymal cells as contaminants in primary cultures of vascular endothelial cells derived from the choroidal tunic of the human eye, we investigated whether the choroid might also provide a source of cultured MSC. Moreover, we examined the effect of the choroidal stromal cells (Ch-SC) on the proliferation of freshly isolated choroidal vascular endothelial cells (ChVEC) in vitro. The phenotype of cultures established from five choroidal tissue donors was examined by flow cytometry and immunocytochemistry. The potential for mesenchymal cell differentiation was examined in parallel with MSC established from human bone marrow. Additional cultures were growth-arrested by treatment with mitomycin-C, before being tested as a potential feeder layer for ChVEC. The five unique cultures established from choroidal stroma displayed a phenotype consistent with the accepted definition for MSC (CD34-, CD45-, HLA-DR-, CD73+, CD90+, and CD105+), including the capacity for mesenchymal differentiation when cultivated under osteogenic, adipogenic and chondrogenic conditions. Growth-arrested Ch-SC inhibited the proliferation of ChVEC derived from five separate donors. Cultures of Ch-SC secreted approximately 40-fold higher concentrations of the anti-angiogenic factor pigment epithelium derived factor (PEDF/serpin F1) compared to the pro-angiogenic factor, vascular endothelial growth factor (VEGF), regardless of normal or growth-arrested state. Our results provide first evidence of a resident MSC cell type within the choroid and encourage investigation of new mechanisms for altering the growth of ChVEC.

Intermittent parathyroid hormone (1-34) supplementation of bone marrow stromal cell cultures may inhibit hypertrophy, but at the expense of chondrogenesis.

BACKGROUND: Bone marrow stromal cells (BMSC) have promise in cartilage tissue engineering, but for their potential to be fully realised, the propensity to undergo hypertrophy must be mitigated. The literature contains diverging reports on the effect of parathyroid hormone (PTH) on BMSC differentiation. Cartilage tissue models can be heterogeneous, confounding efforts to improve media formulations. METHODS: Herein, we use a novel microwell platform (the Microwell-mesh) to manufacture hundreds of small-diameter homogeneous micro-pellets and use this high-resolution assay to quantify the influence of constant or intermittent PTH(1-34) medium supplementation on BMSC chondrogenesis and hypertrophy. Micro-pellets were manufactured from 5000 BMSC each and cultured in standard chondrogenic media supplemented with (1) no PTH, (2) intermittent PTH, or (3) constant PTH. RESULTS: Relative to control chondrogenic cultures, BMSC micro-pellets exposed to intermittent PTH had reduced hypertrophic gene expression following 1 week of culture, but this was accompanied by a loss in chondrogenesis by the second week of culture. Constant PTH treatment was detrimental to chondrogenic culture. CONCLUSIONS: This study provides further clarity on the role of PTH on chondrogenic differentiation in vitro and suggests that while PTH may mitigate BMSC hypertrophy, it does so at the expense of chondrogenesis.

Integration of an ultra-strong poly(lactic-co-glycolic acid) (PLGA) knitted mesh into a thermally induced phase separation (TIPS) PLGA porous structure to yield a thin biphasic scaffold suitable for dermal tissue engineering.

We aimed to capture the outstanding mechanical properties of meshes, manufactured using textile technologies, in thin biodegradable biphasic tissue-engineered scaffolds through encapsulation of meshes into porous structures formed from the same polymer. Our novel manufacturing process used thermally induced phase separation (TIPS), with ethylene carbonate (EC) as the solvent, to encapsulate a poly(lactic-co-glycolic acid) (PLGA) mesh into a porous PLGA network. Biphasic scaffolds (1 cm × 4 cm × 300 µm) were manufactured by immersing strips of PLGA mesh in 40 °C solutions containing 5% PLGA in EC, supercooling at 4 °C for 4 min, triggering TIPS by manually agitating the supercooled solution, and lastly eluting EC into 4 °C Milli-Q water. EC processing was rapid and did not compromise mesh tensile properties. Biphasic scaffolds exhibited a tensile strength of 40.7 ± 2.2 MPa, porosity of 94%, pore size of 16.85 ± 3.78 µm, supported HaCaT cell proliferation, and degraded in vitro linearly over the first ∼3 weeks followed by rapid degradation over the following three weeks. The successful integration of textile-type meshes yielded scaffolds with exceptional mechanical properties. This thin, porous, high-strength scaffold is potentially suitable for use in dermal wound repair or repair of tubular organs.

Modelling of the SDF-1/CXCR4 regulated in vivo homing of therapeutic mesenchymal stem/stromal cells in mice.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are a promising tool for cell-based therapies in the treatment of tissue injury. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis plays a significant role in directing MSC homing to sites of injury. However in vivo MSC distribution following intravenous transplantation remains poorly understood, potentially hampering the precise prediction and evaluation of therapeutic efficacy. METHODS: A murine model of partial ischemia/reperfusion (I/R) is used to induce liver injury, increase the hepatic levels of SDF-1, and study in vivo MSC distribution. Hypoxia-preconditioning increases the expression of CXCR4 in human bone marrow-derived MSCs. Quantitative assays for human DNA using droplet digital PCR (ddPCR) allow us to examine the in vivo kinetics of intravenously infused human MSCs in mouse blood and liver. A mathematical model-based system is developed to characterize in vivo homing of human MSCs in mouse models with SDF-1 levels in liver and CXCR4 expression on the transfused MSCs. The model is calibrated to experimental data to provide novel estimates of relevant parameter values. RESULTS: Images of immunohistochemistry for SDF-1 in the mouse liver with I/R injury show a significantly higher SDF-1 level in the I/R injured liver than that in the control. Correspondingly, the ddPCR results illustrate a higher MSC concentration in the I/R injured liver than the normal liver. CXCR4 is overexpressed in hypoxia-preconditioned MSCs. An increased number of hypoxia-preconditioned MSCs in the I/R injured liver is observed from the ddPCR results. The model simulations align with the experimental data of control and hypoxia-preconditioned human MSC distribution in normal and injured mouse livers, and accurately predict the experimental outcomes with different MSC doses. DISCUSSION: The modelling results suggest that SDF-1 in organs is an effective in vivo attractant for MSCs through the SDF-1/CXCR4 axis and reveal the significance of the SDF-1/CXCR4 chemotaxis on in vivo homing of MSCs. This in vivo modelling approach allows qualitative characterization and prediction of the MSC homing to normal and injured organs on the basis of clinically accessible variables, such as the MSC dose and SDF-1 concentration in blood. This model could also be adapted to abnormal conditions and/or other types of circulating cells to predict in vivo homing patterns.

Using high throughput microtissue culture to study the difference in prostate cancer cell behavior and drug response in 2D and 3D co-cultures.

BACKGROUND: There is increasing appreciation that non-cancer cells within the tumour microenvironment influence cancer progression and anti-cancer drug efficacy. For metastatic prostate cancer (PCa), the bone marrow microenvironment influences metastasis, drug response, and possibly drug resistance. METHODS: Using a novel microwell platform, the Microwell-mesh, we manufactured hundreds of 3D co-culture microtissues formed from PCa cells and bone marrow stromal cells. We used luciferase-expressing C42B PCa cells to enable quantification of the number of PCa cells in complex microtissue co-cultures. This strategy enabled us to quantify specific PCa cell growth and death in response to drug treatment, in different co-culture conditions. In parallel, we used Transwell migration assays to characterize PCa cell migration towards different 2D and 3D stromal cell populations. RESULTS: Our results reveal that PCa cell migration varied depending on the relative aggressiveness of the PCa cell lines, the stromal cell composition, and stromal cell 2D or 3D geometry. We found that C42B cell sensitivity to Docetaxel varied depending on culture geometry, and the presence or absence of different stromal cell populations. By contrast, the C42B cell response to Abiraterone Acetate was dependent on geometry, but not on the presence or absence of stromal cells. CONCLUSION: In summary, stromal cell composition and geometry influences PCa cell migration, growth and drug response. The Microwell-mesh and microtissues are powerful tools to study these complex 3D interactions.

Concise Review: Quantitative Detection and Modeling the In Vivo Kinetics of Therapeutic Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) present a promising tool in cell-based therapy for treatment of various diseases. Currently, optimization of treatment protocols in clinical studies is complicated by the variations in cell dosing, diverse methods used to deliver MSCs, and the variety of methods used for tracking MSCs in vivo. Most studies use a dose escalation approach, and attempt to correlate efficacy with total cell dose. Optimization could be accelerated through specific understanding of MSC distribution in vivo, long-term viability, as well as their biological fate. While it is not possible to quantitatively detect MSCs in most targeted organs over long time periods after systemic administration in clinical trials, it is increasingly possible to apply pharmacokinetic modeling to predict their distribution and persistence. This Review outlines current understanding of the in vivo kinetics of exogenously administered MSCs, provides a critical analysis of the methods used for quantitative MSC detection in these studies, and discusses the application of pharmacokinetic modeling to these data. Finally, we provide insights on and perspectives for future development of effective therapeutic strategies using pharmacokinetic modeling to maximize MSC therapy and minimize potential side effects. Stem Cells Translational Medicine 2018;7:78-86.

Caspofungin on ARGET-ATRP grafted PHEMA polymers: Enhancement and selectivity of prevention of attachment of Candida albicans.

There is a need for coatings for biomedical devices and implants that can prevent the attachment of fungal pathogens while allowing human cells and tissue to appose without cytotoxicity. Here, the authors study whether a poly(2-hydroxyethylmethacrylate) (PHEMA) coating can suppress attachment and biofilm formation by Candida albicans and whether caspofungin terminally attached to surface-tethered polymeric linkers can provide additional benefits. The multistep coating scheme first involved the plasma polymerization of ethanol, followed by the attachment of α-bromoisobutyryl bromide (BiBB) onto surface hydroxyl groups of the plasma polymer layer. Polymer chains were grafted using surface initiated activators regenerated by electron transfer atom transfer radical polymerization with 2-hydroxyethylmethacrylate, yielding PHEMA layers with a dry thickness of up to 89 nm in 2 h. Hydroxyl groups of PHEMA were oxidized to aldehydes using the Albright-Goldman reaction, and caspofungin was covalently immobilized onto them using reductive amination. While the PHEMA layer by itself reduced the growth of C. albicans biofilms by log 1.4, the addition of caspofungin resulted in a marked further reduction by >4 log units to below the threshold of the test. The authors have confirmed that the predominant mechanism of action is caused by antifungal drug molecules that are covalently attached to the surface, rather than out-diffusing from the coating. The authors confirm the selectivity of surface-attached caspofungin in eliminating fungal, not mammalian cells by showing no measurable toxicity toward the myeloid leukaemia suspension cell line KG-1a.

HIF-1α-stabilizing agent FG-4497 rescues human CD34+ cell mobilization in response to G-CSF in immunodeficient mice.

Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxia-inducible factor (HIF)-1α, synergize with G-CSF in vivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg-/- mice by transplanting them with human umbilical cord blood CD34+ HSPCs and then treating them with G-CSF with and without co-administration of the PHD inhibitor FG-4497. We observed that combination treatment with G-CSF and FG-4497 resulted in significant mobilization of human lineage-negative (Lin-) CD34+ HSPCs and more primitive human Lin-CD34+CD38- HSPCs into blood and spleen, whereas mice treated with G-CSF alone did not mobilize human HSPCs significantly. These results suggest that the PHD inhibitor FG-4497 also increases human HSPC mobilization in a xenograft mouse model, suggesting the possibility of testing PHD inhibitors to boost HSPC mobilization in response to G-CSF in humans.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38- cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38- cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38- cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.