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.

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.

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 × 103 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 × 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.