A novel microplate 3D bioprinting platform for the engineering of muscle and tendon tissues

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases

Late-Gadolinium Enhancement Is Common in Older Pediatric Heart Transplant Recipients and Is Associated With Lower Ejection Fraction

BACKGROUND: Chronic graft failure and cumulative rejection history in pediatric heart transplant recipients (PHTR) are associated with myocardial fibrosis on endomyocardial biopsy (EMB). Cardiovascular magnetic resonance imaging (CMR) is a validated, non-invasive method to detect myocardial fibrosis via the presence of late gadolinium enhancement (LGE). In adult heart transplant recipients, LGE is associated with increased risk of future adverse clinical events including hospitalization and death. We describe the prevalence, pattern, and extent of LGE on CMR in a cohort of PHTR and its associations with recipient and graft characteristics. METHODS: This was a retrospective study of consecutive PHTR who underwent CMR over a 6-year period at a single center. Two independent reviewers assessed the presence and distribution of left ventricular (LV) LGE using the American Heart Association (AHA) 17-segment model. LGE quantification was performed on studies with visible fibrosis (LGE+). Patient demographics, clinical history, and CMR-derived volumetry and ejection fractions were obtained. RESULTS: Eighty-one CMR studies were performed on 59 unique PHTR. Mean age at CMR was 14.8 ± 6.2 years; mean time since transplant was 7.3 ± 5.0 years. The CMR indication was routine surveillance (without a clinical concern based on laboratory parameters, echocardiography, or cardiac catheterization) in 63% (51/81) of studies. LGE was present in 36% (29/81) of PHTR. In these LGE + studies, patterns included inferoseptal in 76% of LGE + studies (22/29), lateral wall in 41% (12/29), and diffuse, involving \u3e 4 AHA segments, in 21% (6/29). The mean LV LGE burden as a percentage of myocardial mass was 18.0 ± 9.0%. When reviewing only the initial CMR per PHTR (n = 59), LGE + patients were older (16.7 ± 2.9 vs. 12.8 ± 4.6 years, p = 0.001), with greater time since transplant (8.3 ± 5.4 vs. 5.7 ± 3.9 years, p = 0.041). These patients demonstrated higher LV end-systolic volume index (LVESVI) (34.7 ± 11.7 vs. 28.7 ± 6.1 ml/m CONCLUSIONS: LV LGE was present in approximately one third of PHTR, more commonly in older patients with longer time since transplantation. Grafts with LGE have lower LVEF. CMR-derived LGE may aid in surveillance of chronic graft failure in PHTR

TEDD annual meeting with 3D bioprinting workshop

Bioprinting is the technology of choice for realizing functional tissues such as vascular system, muscle, cartilage and bone. In the future, bioprinting will influence the way we engineer tissues and bring it to a new level of physiological relevance. That was the topic of the 2017 TEDD Annual Meeting at ZHAW Wädenswil on 8-9 November. In an exciting workshop, the two companies regenHU Ltd. and CELLINK gave us an insight into highly topical applications and collaborations in this domain

TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain stem tumor and the leading cause of pediatric cancer-related death. To date, these tumors remain incurable, underscoring the need for efficacious therapies. In this study, we demonstrate that the immune checkpoint TIM-3 (HAVCR2) is highly expressed in both tumor cells and microenvironmental cells, mainly microglia and macrophages, in DIPG. We show that inhibition of TIM-3 in syngeneic models of DIPG prolongs survival and produces long-term survivors free of disease that harbor immune memory. This antitumor effect is driven by the direct effect of TIM-3 inhibition in tumor cells, the coordinated action of several immune cell populations, and the secretion of chemokines/cytokines that create a proinflammatory tumor microenvironment favoring a potent antitumor immune response. This work uncovers TIM-3 as a bona fide target in DIPG and supports its clinical translation

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier–penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy