3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Tsinghua University

Blowflies (Calliphoridae) are recognized as a powerful tool for estimating the minimum postmortem interval (PMImin). The times for blowflies to develop from oviposition to eclosion is mainly controlled by temperature, which can differ between even closely related species. Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) is a blowfly distributed throughout Asia and Australia. However, a systematic determination of the developmental times of H. ligurriens under constant temperature, necessary for estimating the PMImin, is lacking. Such an examination would broaden the forensic importance of the species. Thus, this study explored the growth curves of larval H. ligurriens at 7 constant temperatures (16, 19, 22, 25, 28, 31, and 34°C). Isomegalen and isomorphen diagrams were successfully constructed, depicting the time of larval length or developmental event, respectively, at different temperatures. A thermal summation model was also constructed via regression analysis, by estimating the developmental threshold temperature t and thermal summation constant K. The thermal summation model indicated that t at 8.3°C and K at 5747.5 degree-hours (°Ch) are required for complete development from oviposition to eclosion, and suggested an optimum temperature range of 16-28°C for the development of H. ligurriens. These data establish for the first time the temperature-dependent developmental time of H. ligurriens for forensic entomology application. The 3 developmental models are provided.

Technical note: Development of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) at constant temperatures: Applications in estimating postmortem interval.

Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9Δ cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.

Sch9 regulates intracellular protein ubiquitination by controlling stress responses.

Phenotypic screening has regained momentum in the pharmaceutical industry owing to its success over target-based screening. Most phenotypic screening relies on nonspecific biochemical readouts regarding cellular viability, which hampers the discovery of novel drug mechanisms of action (MOAs). Here we present a Contractility-based bi-Content micro-Collagen Chip (3CChip), which establishes cellular contractility as a biomechanics-related phenotype for drug screening. Bi-content analysis of cell contractility (imaged by iPhone) and viability suggests that the label-free contractility-based analysis exhibits superior sensitivity to compounds targeting contractile elements (e.g. focal adhesion, cytoskeleton), resulting in a enlarged target pool for drug assessment. Six typical readout patterns of drug response are summarized according to the relative positions of the contraction/viability curves, and drug targets are profiled into three categories (biomechanical, biochemical and housekeeping) by 3CChip, which will benefit subsequent target identification. The simple-to-use and effective 3CChip offers a robust platform for micro-tissue-based functional screening and may lead to a new era of mechanism-informed phenotypic drug discovery.

Bi-content micro-collagen chip provides contractility-based biomechanical readout for phenotypic drug screening with expanded and profiled targets.

Examining the succession pattern of carrion insects on vertebrate carcasses is widely accepted as an effective method to estimate the postmortem interval (PMI) of decayed bodies. Investigation of the community of sarcosaprophagous insects, especially flies and beetles, is the foundation of the succession pattern study. This study aimed to investigate the sarcosaprophagous beetles succession on animal carcasses in the suburban area of southwestern China and to establish a basic catalog for forensic application. The present study was conducted in 2013 in a mountain in Chongqing municipality with modified Schoenly traps. Carcasses of miniature pig were used to simulate human bodies. For most carcasses, five decomposition stages were observed. A total of 2108 adult coleopterans belonging to at least 61 species and 18 families were collected in the study, and most of the specimens occurred at the advanced decay stage. Omosita colon (Linnaeus, 1758), Necrodes nigricornis (Harold, 1875), Necrobia ruficollis (Fabricius, 1775) and Neosilusa ceylonica (Kraatz, 1857) were the dominant species.

A checklist of beetles (Insecta, Coleoptera) on pig carcasses in the suburban area of southwestern China: A preliminary study and its forensic relevance.

The characteristic life stages of infesting blowflies (Calliphoridae) such as Chrysomya megacephala (Fabricius) are powerful evidence for estimating the death time of a corpse, but an established reference of developmental times for local blowfly species is required. We determined the developmental rates of C. megacephala from southwest China at seven constant temperatures (16-34°C). Isomegalen and isomorphen diagrams were constructed based on the larval length and time for each developmental event (first ecdysis, second ecdysis, wandering, pupariation, and eclosion), at each temperature. A thermal summation model was constructed by estimating the developmental threshold temperature D0 and the thermal summation constant K. The thermal summation model indicated that, for complete development from egg hatching to eclosion, D0 = 9.07 ± 0.54°C and K = 3991.07 ± 187.26 h °C. This reference can increase the accuracy of estimations of postmortem intervals in China by predicting the growth of C. megacephala.

Developmental Times of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) at Constant Temperatures and Applications in Forensic Entomology.

Recent breakthrough in stroma-reprogrammed combinatorial therapy (SRCT) for pancreatic tumor opens a new route for improving conventional chemotherapeutic efficacy, which utilizes VDR ligand to reprogram activated stromal cells in stiffened microenvironment, leading to reduced 'barrier effects' and increased tissue-infiltration of the chemotherapy drug. As a novel therapeutic strategy and mechanism of action, the progress of SRCT relies on tailored in vitro drug assessment platforms to further optimize its efficacy and extend to applications in other tumor types. Here, a high-throughput mechanopharmacological drug screening platform for SRCT was established based on biomechanically primed hepatic stromal stellate cells to recapitulate state-specific liver microtumors with barrier effects. Fifteen generic chemotherapy drugs co-administered with VDR ligand were screened to obtain optimal SRCT formulations (e.g. carboplatin + calcipotriol), which efficacy was successfully verified in xenograft tumor models. Overall, this platform provides a powerful tool for discovery and optimization of tissue-specific SRCT and realizes 'mechanopharmacology' to translate insights of stromal mechanobiology to pharmaceutical applications.

Biomechanically primed liver microtumor array as a high-throughput mechanopharmacological screening platform for stroma-reprogrammed combinatorial therapy.

Lyu Zhou

Department of Biomedical Engineering,
School of Medicine,
School of Life Sciences,
Department of Biomedical Engineering, School of Medicine

Tsinghua University

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Lyu Zhou

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering,School of Medicine,School of Life Sciences,Department of Biomedical Engineering, School of Medicine

Tsinghua University

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Department of Biomedical Engineering,
School of Medicine,
School of Life Sciences,
Department of Biomedical Engineering, School of Medicine