Publications by authors named "Michael Doran"

75 Publications

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.

Stem Cell Res Ther 2021 Apr 12;12(1):231. Epub 2021 Apr 12.

School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia.

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 × 10 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.
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http://dx.doi.org/10.1186/s13287-021-02297-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042930PMC
April 2021

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

Sci Rep 2021 Mar 4;11(1):5118. Epub 2021 Mar 4.

School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia.

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.
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http://dx.doi.org/10.1038/s41598-021-84154-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933425PMC
March 2021

Distal posterolateral corner injury in the setting of multiligament knee injury increases risk of common peroneal palsy.

Knee Surg Sports Traumatol Arthrosc 2021 Feb 9. Epub 2021 Feb 9.

New York University Langone Orthopedic Hospital, 333 East 38th Street, New York, NY, 10016, USA.

Purpose: The purpose of this study was to identify if the location of posterolateral corner (PLC) injury was predictive of clinical common peroneal nerve (CPN) palsy.

Methods: A retrospective chart review was conducted of patients presenting to our institution with operative PLC injuries. Assessment of concomitant injuries and presence of neurologic injury was completed via chart review and magnetic resonance imaging (MRI) review. A fellowship-trained musculoskeletal radiologist reviewed the PLC injury and categorized it into distal, middle and proximal injuries with or without a biceps femoral avulsion. The CPN was evaluated for signs of displacement or neuritis.

Results: Forty-seven operatively managed patients between 2014 and 2019 (mean age-at-injury 29.5 ± 10.7 years) were included in this study. Eleven (23.4%) total patients presented with a clinical CPN palsy. Distal PLC injuries were significantly associated with CPN palsy [9 (81.8%) patients, (P = 0.041)]. Nine of 11 (81.8%) patients with CPN palsy had biceps femoral avulsion (P = 0.041). Of the patients presenting with CPN palsy, only four (36.4%) patients experienced complete neurologic recovery. Three of 7 patients (43%) with an intact CPN had full resolution of their clinically complete CPN palsy at the time of follow-up (482 ± 357 days). All patients presenting with a CPN palsy also had a complete anterior cruciate ligament (ACL) rupture in addition to a PLC injury (P = 0.009), with or without a posterior cruciate ligament (PCL) injury. No patient presenting with an isolated pattern of PCL-PLC injury (those without ACL tears) had a clinical CPN palsy.

Conclusion: Distal PLC injuries have a strong association with clinical CPN palsy, with suboptimal resolution in the initial post-operative period. Specifically, the presence of a biceps femoris avulsion injury was highly associated with a clinical CPN palsy. Additionally, CPN palsy in the context of PLC injury has a strong association with concomitant ACL injury. Furthermore, the relative rates of involvement of the ACL vs. PCL suggest that specific injury mechanism may have an important role in CPN palsy.

Level Of Evidence: IV.
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http://dx.doi.org/10.1007/s00167-021-06469-zDOI Listing
February 2021

A survey of early-career researchers in Australia.

Elife 2021 Jan 11;10. Epub 2021 Jan 11.

School of Biomedical Sciences and Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia.

Early-career researchers (ECRs) make up a large portion of the academic workforce and their experiences often reflect the wider culture of the research system. Here we surveyed 658 ECRs working in Australia to better understand the needs and challenges faced by this community. Although most respondents indicated a 'love of science', many also expressed an intention to leave their research position. The responses highlight how job insecurity, workplace culture, mentorship and 'questionable research practices' are impacting the job satisfaction of ECRs and potentially compromising science in Australia. We also make recommendations for addressing some of these concerns.
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http://dx.doi.org/10.7554/eLife.60613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800379PMC
January 2021

A single day of TGF-β1 exposure activates chondrogenic and hypertrophic differentiation pathways in bone marrow-derived stromal cells.

Commun Biol 2021 Jan 4;4(1):29. Epub 2021 Jan 4.

National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), Department of Health and Human Services, Bethesda, MD, USA.

Virtually all bone marrow-derived stromal cell (BMSC) chondrogenic induction cultures include greater than 2 weeks exposure to transforming growth factor-β (TGF-β), but fail to generate cartilage-like tissue suitable for joint repair. Herein we used a micro-pellet model (5 × 10 BMSC each) to determine the duration of TGF-β1 exposure required to initiate differentiation machinery, and to characterize the role of intrinsic programming. We found that a single day of TGF-β1 exposure was sufficient to trigger BMSC chondrogenic differentiation and tissue formation, similar to 21 days of TGF-β1 exposure. Despite cessation of TGF-β1 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 × 10 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.
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http://dx.doi.org/10.1038/s42003-020-01520-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782775PMC
January 2021

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.

Exp Eye Res 2020 11 2;200:108201. Epub 2020 Sep 2.

School of Environment and Science Griffith University Nathan, Queensland, 4111, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.exer.2020.108201DOI Listing
November 2020

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

Stem Cell Res Ther 2020 07 29;11(1):321. Epub 2020 Jul 29.

School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia.

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.
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http://dx.doi.org/10.1186/s13287-020-01820-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7389809PMC
July 2020

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.

Biofabrication 2019 12 4;12(1):015015. Epub 2019 Dec 4.

School of Chemistry, Physics and Mechanical Engineering (CPME), Science and Engineering Faculty (SEF), Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Brisbane, Australia. Doran Laboratory, School of Biomedical Sciences, Faculty of Health, Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Brisbane, Australia. Translational Research Institute (TRI), Brisbane, Australia.

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.
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http://dx.doi.org/10.1088/1758-5090/ab4053DOI Listing
December 2019

CD27, CD201, FLT3, CD48, and CD150 cell surface staining identifies long-term mouse hematopoietic stem cells in immunodeficient non-obese diabetic severe combined immune deficient-derived strains.

Haematologica 2020 01 9;105(1):71-82. Epub 2019 May 9.

Mater Research Institute - The University of Queensland, Woolloongabba

Staining for CD27 and CD201 (endothelial protein C receptor) has been recently suggested as an alternative to stem cell antigen-1 (Sca1) to identify hematopoietic stem cells in inbred mouse strains with low or nil expression of SCA1. However, whether staining for CD27 and CD201 is compatible with low fms-like tyrosine kinase 3 (FLT3) expression and the "SLAM" code defined by CD48 and CD150 to identify mouse long-term reconstituting hematopoietic stem cells has not been established. We compared the C57BL/6 strain, which expresses a high level of SCA1 on hematopoietic stem cells to non-obese diabetic severe combined immune deficient NOD.CB17-/Sz (NOD-) mice and NOD.CB17-/Sz (NSG) mice which both express low to negative levels of SCA1 on hematopoietic stem cells. We demonstrate that hematopoietic stem cells are enriched within the linage-negative C-KIT CD27 CD201 FLT3 CD48-CD150 population in serial dilution long-term competitive transplantation assays. We also make the novel observation that CD48 expression is up-regulated in Lin KIT progenitors from NOD- and NSG strains, which otherwise have very few cells expressing the CD48 ligand CD244. Finally, we report that unlike hematopoietic stem cells, SCA1 expression is similar on bone marrow endothelial and mesenchymal progenitor cells in C57BL/6, NOD- and NSG mice. In conclusion, we propose that the combination of Lineage, KIT, CD27, CD201, FLT3, CD48, and CD150 antigens can be used to identify long-term reconstituting hematopoietic stem cells from mouse strains expressing low levels of SCA1 on hematopoietic cells.
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http://dx.doi.org/10.3324/haematol.2018.212910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939540PMC
January 2020

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

PeerJ 2018 6;6:e6072. Epub 2018 Dec 6.

Therapeutics Research Centre, The University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane, Australia.

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 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 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 kinetics of intravenously infused human MSCs in mouse blood and liver. A mathematical model-based system is developed to characterize 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 attractant for MSCs through the SDF-1/CXCR4 axis and reveal the significance of the SDF-1/CXCR4 chemotaxis on homing of MSCs. This 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 homing patterns.
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http://dx.doi.org/10.7717/peerj.6072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286806PMC
December 2018

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

BMC Cancer 2018 May 24;18(1):592. Epub 2018 May 24.

Stem Cell Therapies Laboratory, Queensland University of Technology (QUT), Translational Research Institute (TRI), 37 Kent Street, Brisbane, QLD, Australia.

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.
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http://dx.doi.org/10.1186/s12885-018-4473-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968610PMC
May 2018

Liquid chromatography/mass spectrometry for the detection of ash tree metabolites following Emerald Ash Borer infestation.

Rapid Commun Mass Spectrom 2018 03;32(5):385-392

Water Quality Centre, Trent University, 1600 West Bank Drive, Peterborough, ON, K9L 0G2, Canada.

Rationale: The Emerald Ash Borer (EAB), Agrilus planipennis, an invasive insect detected in the USA and Canada in 2002, is a threat to ash trees with both ecological and economic implications. Early detection of EAB-infestation is difficult due to lack of visible signs and symptoms in the early stages of attack, but is essential to prevent ash mortality. An efficient and reliable tool for the early detection of EAB-infestation would be advantageous.

Methods: A mass spectrometry based metabolomics approach, using liquid chromatography/mass spectrometry (LC/MS), has been used to investigate the leaf metabolites of both healthy and EAB-infested trees.

Results: Leaves from 40 healthy and 40 EAB-infested trees were extracted and analyzed using LC/MS. Resulting data were examined to differentiate between foliage from healthy and EAB-infested trees. Possible biomarkers of EAB attack have been detected. Twenty-one metabolites with increased average ion intensity in EAB-infested ash tree samples and nine metabolites with increased average ion intensity in healthy ash tree samples were identified.

Conclusions: Results of this study indicate that metabolomic screening of leaf samples using LC/MS can be useful as a potential tool for the early detection of EAB-infestation.
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http://dx.doi.org/10.1002/rcm.8057DOI Listing
March 2018

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

Stem Cells Transl Med 2018 01 6;7(1):78-86. Epub 2017 Dec 6.

Therapeutics Research Centre, The University of Queensland Diamantina Institute, Brisbane, Australia.

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.
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http://dx.doi.org/10.1002/sctm.17-0209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746161PMC
January 2018

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

Biointerphases 2017 Aug 29;12(5):05G602. Epub 2017 Aug 29.

Future Industries Institute, University of South Australia, Mawson Lakes Blvd, Mawson Lakes, SA 5095, Australia and School of Agriculture, Food and Wine, University of Adelaide, Adelaide, SA 5005, Australia.

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.
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http://dx.doi.org/10.1116/1.4986054DOI Listing
August 2017

ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.

Nature 2017 06 14;546(7660):671-675. Epub 2017 Jun 14.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2-ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.
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http://dx.doi.org/10.1038/nature22820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576182PMC
June 2017

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

Exp Hematol 2017 08 17;52:50-55.e6. Epub 2017 May 17.

Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia. Electronic address:

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 LinCD34CD38 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.
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http://dx.doi.org/10.1016/j.exphem.2017.05.004DOI Listing
August 2017

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.

Tissue Eng Part C Methods 2017 04;23(4):200-218

1 Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology , Brisbane, Australia .

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 CD34CD38 cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34CD38 cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34CD38 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.
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http://dx.doi.org/10.1089/ten.tec.2016.0329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397247PMC
April 2017

Internalization of secreted antigen-targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis.

Sci Transl Med 2016 11;8(367):367ra167

Urology Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.
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http://dx.doi.org/10.1126/scitranslmed.aaf2335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568040PMC
November 2016

A computer program to simplify analysis of mass scan data of organometallic compounds from high-resolution mass spectrometers.

Rapid Commun Mass Spectrom 2016 Dec;30(23):2561-2567

Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, Canada, K9L 0G2.

Rationale: Software accompanying high-resolution mass spectrometers, particularly that used for the analysis of organometallic compounds, has lagged the technology of the instruments themselves. We have developed a computer program that partially fills this gap.

Methods: Given the user's expectation for the number of atoms of a target element likely to be in an ion, the program calculates isotopologue mass differences for combinations of that element's isotopes and their expected intensity ratios relative to the most abundant isotopologue. These values are compared with mass differences and intensity ratios found in the experimental mass scan data and these metrics feed into a four-factor scoring model which ranks the ions as to the likelihood of each containing the specified number of the target atoms. The program was tested using experimental data obtained for selenomethionine.

Results: Across a broad range of sample concentrations, the program consistently ranked selenomethionine at or near the top of the list of ions that passed the screening and ranking process. Mass scan data files in excess of 24,000 records were analyzed in less than one second.

Conclusions: The program is quick and efficient at scanning voluminous experimental data files for the presence of ions containing the expected number of atoms of a target element. Best results were obtained the scarcer the target element and the more isotopes it comprised. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/rcm.7748DOI Listing
December 2016

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue.

J Vis Exp 2016 06 6(112). Epub 2016 Jun 6.

Translational Research Institute, Queensland University of Technology; Translational Research Institute, Mater Medical Research - University of Queensland;

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.
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http://dx.doi.org/10.3791/54204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927767PMC
June 2016

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice.

Sci Rep 2016 Apr 11;6:23886. Epub 2016 Apr 11.

Queensland University of Technology (QUT) at the Translational Research Institute (TRI), 37 Kent Street, Brisbane, Queensland, Australia 4102.

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.
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http://dx.doi.org/10.1038/srep23886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827391PMC
April 2016

Polydimethylsiloxane (PDMS) modulates CD38 expression, absorbs retinoic acid and may perturb retinoid signalling.

Lab Chip 2016 Apr;16(8):1473-83

Stem Cell Therapies Laboratory, Queensland University of Technology at the Translational Research Institute, 37 Kent Street Brisbane, QLD 4102, Australia. and Mater Medical Research - University of Queensland, Translational Research Institute, 37 Kent Street Brisbane, QLD 4102, Australia.

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.
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http://dx.doi.org/10.1039/c6lc00269bDOI Listing
April 2016

Applying ⁸⁹Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins.

Mol Pharm 2016 Feb 12;13(2):683-8. Epub 2016 Jan 12.

Department of Radiology and Biomedical Imaging, University of California San Francisco , 185 Berry Street, Lobby 6 Suite 350, San Francisco, California 94143, United States.

Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4738321PMC
February 2016

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells.

PLoS One 2015 14;10(12):e0144941. Epub 2015 Dec 14.

Institute of Health and Biomedical Innovation, Queensland University of Technology at the Translational Research Institute, Brisbane, Queensland, Australia.

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144941PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687640PMC
July 2016

Androgen Receptor Upregulation Mediates Radioresistance after Ionizing Radiation.

Cancer Res 2015 Nov 2;75(22):4688-96. Epub 2015 Oct 2.

Human Oncology Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651750PMC
November 2015

The rapid manufacture of uniform composite multicellular-biomaterial micropellets, their assembly into macroscopic organized tissues, and potential applications in cartilage tissue engineering.

PLoS One 2015 28;10(5):e0122250. Epub 2015 May 28.

Stem Cell Therapies Laboratory, Institute of Health and Biomedical Innovation, Queensland University of Technology at the Translational Research Institute, Brisbane, Australia; Mater Medical Research-University of Queensland, Brisbane, Australia.

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix "cartilage dust (CD)". Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122250PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447443PMC
April 2016

The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues.

Biomaterials 2015 Sep 20;62:1-12. Epub 2015 May 20.

Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia; Mater Medical Research - University of Queensland, Brisbane, QLD, Australia. Electronic address:

Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10(3) bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10(5) MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.
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http://dx.doi.org/10.1016/j.biomaterials.2015.05.013DOI Listing
September 2015

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

Cell Tissue Res 2015 Sep 30;361(3):755-68. Epub 2015 Apr 30.

Stem Cell Therapies Laboratory, Institute of Health and Biomedical Innovation, Queensland University of Technology at the Translational Research Institute, Brisbane, Australia.

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.
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http://dx.doi.org/10.1007/s00441-015-2159-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550660PMC
September 2015