Bioprintable, Stiffness-Tunable Collagen-Alginate Microgels for Increased Throughput 3D Cell Culture Studies.

3D culture platforms with tunable stiffness have the potential to improve many applications, such as drug discovery, organoid studies, and stem cell differentiation. Both dimensionality and stiffness regulate crucial and relevant cellular processes. However, 3D culture models are often limited in throughput and difficult to adopt for widespread use. Here, we demonstrate an accessible 3D, stiffness-tunable tissue culture platform, based on an interpenetrating network of collagen-1 and alginate. When blended with polymers that induce phase separation, these networks can be bioprinted at microliter volumes, using standard liquid handling infrastructure. We demonstrate robust reproducibility in printing these microgels, consistent tunability of mechanical properties, and maintained viability of multiple printed cell types. To highlight the utility and importance of this system, we demonstrate distinct morphological changes to cells in culture, use the system to probe the role of matrix mechanics and soluble factors in a collagen contraction assay, and perform a prototype viability screen against a candidate chemotherapeutic, demonstrating stiffness-dependent responses.

Disentangling the fibrous microenvironment: designer culture models for improved drug discovery.

INTRODUCTION: Standard high-throughput screening (HTS) assays rarely identify clinically viable 'hits', likely because cells do not experience physiologically realistic culture conditions. The biophysical nature of the extracellular matrix has emerged as a critical driver of cell function and response and recreating these factors could be critically important in streamlining the drug discovery pipeline. AREAS COVERED: The authors review recent design strategies to understand and manipulate biophysical features of three-dimensional fibrous tissues. The effects of architectural parameters of the extracellular matrix and their resulting mechanical behaviors are deconstructed; and their individual and combined impact on cell behavior is examined. The authors then illustrate the potential impact of these physical features on designing next-generation platforms to identify drugs effective against breast cancer. EXPERT OPINION: Progression toward increased culture complexity must be balanced against the demanding technical requirements for high-throughput screening; and strategies to identify the minimal set of microenvironmental parameters needed to recreate disease-relevant responses must be specifically tailored to the disease stage and organ system being studied. Although challenging, this can be achieved through integrative and multidisciplinary technologies that span microfabrication, cell biology, and tissue engineering.

Emerging Strategies for Stem Cell Lineage Commitment in Tissue Engineering and Regenerative Medicine.

Stem cells have transformed the fields of tissue engineering and regenerative medicine, and their potential to further advance these fields cannot be overstated. The stem cell niche is a dynamic microenvironment that determines cell fate during development and tissue repair following an injury. Classically, stem cells were studied in isolation of their microenvironment; however, contemporary research has produced a myriad of evidence that shows the importance of multiple aspects of the stem cell niche in regulating their processes. In the context of tissue engineering and regenerative medicine studies, the niche is an artificial environment provided by culture conditions. In vitro culture conditions may involve coculturing with other cell types, developing specific biomaterials, and applying relevant forces to promote the desired lineage commitment. Considerable advance has been made over the past few years toward directed stem cell differentiation; however, the unspecific differentiation of stem cells yielding a mixed population of cells has been a challenge. In this review, we provide a systematic review of the emerging strategies used for lineage commitment within the context of tissue engineering and regenerative medicine. These strategies include scaffold pore-size and pore-shape gradients, stress relaxation, sonic and electromagnetic effects, and magnetic forces. Finally, we provide insights and perspectives into future directions focusing on signaling pathways activated during lineage commitment using external stimuli.

Loss of the mammalian DREAM complex deregulates chondrocyte proliferation.

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes.