Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo.

Bone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. OBJECTIVE: The objective of this study was to characterise ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. DESIGN: oBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation capacity. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. RESULTS: Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. CONCLUSION: The sensitivity of oBMSC, compared to human BMSC, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oACh were more effective than micro-pellets manufactured from oBMSC in the repair of osteochondral defects in sheep. While oBMSC can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration.

Constraints to counting bioluminescence producing cells by a commonly used transgene promoter and its implications for experimental design.

It is routine to genetically modify cells to express fluorescent or bioluminescent reporter proteins to enable tracking or quantification of cells in vitro and in vivo. Herein, we characterized the stability of luciferase reporter systems in C4-2B prostate cancer cells in mono-culture and in co-culture with bone marrow-derived mesenchymal stem/stromal cells (BMSC). An assumption made when employing the luciferase reporter is that the luciferase expressing cell number and bioluminescence signal are linearly proportional. We observed instances where luciferase expression was significantly upregulated in C4-2B cell populations when co-cultured with BMSC, resulting in a significant disconnect between bioluminescence signal and cell number. We subsequently characterized luciferase reporter stability in a second C4-2B reporter cell line, and six other cancer cell lines. All but the single C4-2B reporter cell population had stable luciferase reporter expression in mono-culture and BMSC co-culture. Whole-genome sequencing revealed that relative number of luciferase gene insertions per genome in the unstable C4-2B reporter cell population was lesser than stable C4-2B, PC3 and MD-MBA-231 luciferase reporter cell lines. We reasoned that the low luciferase gene copy number and genome insertion locations likely contributed to the reporter gene expression being exquisitely sensitive BMSC paracrine signals. In this study, we show that it is possible to generate a range of stable and reliable luciferase reporter prostate- and breast- cancer cell populations but advise not to assume stability across different culture conditions. Reporter stability should be validated, on a case-by-case basis, for each cell line and culture condition.

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis.

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

Sheep as a model for evaluating mesenchymal stem/stromal cell (MSC)-based chondral defect repair.

Osteoarthritis results from the degradation of articular cartilage and is one of the leading global causes of pain and immobility. Cartilage has a limited capacity for self-repair. While repair can be enhanced through surgical intervention, current methods often generate inferior fibrocartilage and repair is transient. The development of tissue engineering strategies to improve repair outcomes is an active area of research. While small animal models such as rodents and rabbits are often used in early pre-clinical work, larger animals that better recapitulate the anatomy and loading of the human joint are required for late-stage preclinical evaluation. Because of their physiological similarities to humans, and low cost relative to other large animals, sheep are routinely used in orthopedic research, including cartilage repair studies. In recent years, there has been considerable research investment into the development of cartilage repair strategies that utilize mesenchymal stem/stromal cells (MSC). In contrast to autologous chondrocytes derived from biopsies of articular cartilage, MSC offer some benefits including greater expansion capacity and elimination of the risk of morbidity at the cartilage biopsy site. The disadvantages of MSC are related to the challenges of inducing and maintaining a stable chondrocyte-like cell population capable of generating hyaline cartilage. Ovine MSC (oMSC) biology and their utility in sheep cartilage repair models have not been reviewed. Herein, we review the biological properties of MSC derived from sheep tissues, and the use of these cells to study articular cartilage repair in this large animal model.

The Microwell-mesh: A high-throughput 3D prostate cancer spheroid and drug-testing platform.

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.