An osteosarcoma-on-a-chip model for studying osteosarcoma matrix-cell interactions and drug responses.

Marrow niches in osteosarcoma (OS) are a specialized microenvironment that is essential for the maintenance and regulation of OS cells. However, existing animal xenograft models are plagued by variability, complexity, and high cost. Herein, we used a decellularized osteosarcoma extracellular matrix (dOsEM) loaded with extracellular vesicles from human bone marrow-derived stem cells (hBMSC-EVs) and OS cells as a bioink to construct a micro-osteosarcoma (micro-OS) through 3D printing. The micro-OS was further combined with a microfluidic system to develop into an OS-on-a-chip (OOC) with a built-in recirculating perfusion system. The OOC system successfully integrated bone marrow niches, cell‒cell and cell-matrix crosstalk, and circulation, allowing a more accurate representation of OS characteristics in vivo. Moreover, the OOC system may serve as a valuable research platform for studying OS biological mechanisms compared with traditional xenograft models and is expected to enable precise and rapid evaluation and consequently more effective and comprehensive treatments for OS.

Detection of lineage-reprogramming efficiency of tumor cells in a 3D-printed liver-on-a-chip model.

Background: The liver metastasis accompanied with the loss of liver function is one of the most common complications in patients with triple-negative breast cancers (TNBC). Lineage reprogramming, as a technique direct inducing the functional cell types from one lineage to another lineage without passing through an intermediate pluripotent stage, is promising in changing cell fates and overcoming the limitations of primary cells. However, most reprogramming techniques are derived from human fibroblasts, and whether cancer cells can be reversed into hepatocytes remains elusive. Methods: Herein, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Then the virus was transduced into MDA-MB-231 cells to generated human induced hepatocyte-like cells (hiHeps) and single-cell sequencing was used to analyze the fate for the cells after reprogramming. Furthermore, we constructed a Liver-on-a-chip (LOC) model by bioprinting the Gelatin Methacryloyl hydrogel loaded with hepatocyte extracellular vesicles (GelMA-EV) bioink onto the microfluidic chip to assess the metastasis behavior of the reprogrammed TNBC cells under the 3D liver microenvironment in vitro. Results: The combination of the genes HNF4A, FOXA2, FOXA3, ATF5, PROX1 and HNF1A could reprogram MDA-MB-231 tumor cells into human-induced hepatocytes (hiHeps), limiting metastasis of these cells. Single-cell sequencing analysis showed that the oncogenes were significantly inhibited while the liver-specific genes were activated after lineage reprogramming. Finally, the constructed LOC model showed that the hepatic phenotypes of the reprogrammed cells could be observed, and the metastasis of embedded cancer cells could be inhibited under the liver microenvironment. Conclusion: Our findings demonstrate that reprogramming could be a promising method to produce hepatocytes and treat TNBC liver metastasis. And the LOC model could intimate the 3D liver microenvironment and assess the behavior of the reprogrammed TNBC cells.

Lentiviral Capsid-Mediated Streptococcus pyogenes Cas9 Ribonucleoprotein Delivery for Efficient and Safe Multiplex Genome Editing.

Transient expression of the CRISPR-Cas9 machinery is desirable to reduce the risks of off-targets and immune responses. Electroporation of Cas9 ribonucleoproteins (RNPs) is the most common delivery method to achieve transient Cas9 expression. Recently, retroviral capsids have been used for delivering Streptococcus pyogenes Cas9 RNPs, in which Cas9 was fused to the viral proteins. The fusion strategy may cause relative low capsid assembly efficiency. We recently developed virus-like particles (VLPs) consisting of lentiviral capsid and Staphylococcus aureus Cas9 RNPs using the specific interactions between aptamer and aptamer-binding protein (ABP), and obtained near-normal capsid assembly efficiency. Here we test whether highly active Streptococcus pyogenes Cas9 (SpCas9) RNP VLPs can be generated with high efficiency by aptamer/ABP interaction. We found that by optimizing the locations and types of aptamer used for single guide RNA modification, highly active SpCas9 RNP VLPs can be generated efficiently. VLP-delivered SpCas9 generated lower off-target insertions and deletions than SpCas9 RNPs delivered by electroporation. VLPs containing Cas9 from different species and targeting multiple genes can be efficiently prepared in single-particle preparation. Multiple-target VLPs were more efficient than the combination of single-target VLPs for simultaneous targeting of multiple genes. Thus, in addition to better safety features, the Cas9 VLPs are especially suited for multiplex genome editing. In summary, our VLPs offer safe, efficient, and flexible multiplex genome editing.

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities.

Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.

3D scaffold-free microlivers with drug metabolic function generated by lineage-reprogrammed hepatocytes from human fibroblasts.

Generating microliver tissues to recapitulate hepatic function is of increasing importance in tissue engineering and drug screening. But the limited availability of primary hepatocytes and the marked loss of phenotype hinders their application. Human induced hepatocytes (hiHeps) generated by direct reprogramming can address the shortage of primary hepatocytes to make personalized drug prediction possible. Here, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Transducing human fetal and adult fibroblasts with low vector dosage generated human induced hepatocyte-like cells (hiHeps) displaying characteristics of mature hepatocytes and capable of drug metabolism. To mimic the physiologic liver microenvironment and improve hepatocyte function, we prepared 3D scaffold-free microliver spheroids using hiHeps and human liver nonparenchymal cells through self-assembly without exogenous scaffolds. We then introduced the microliver spheroids into a two-organ microfluidic system to examine interactions between hepatocytes and tumor cells. The hiHeps-derived spheroids metabolized the prodrug capecitabine into the active metabolite 5-fluorouracil and induced toxicity in downstream tumor spheroids. Our results demonstrate that hiHeps can be used to make microliver spheroids and combined with a microfluidic system for drug evaluation. Our work could make it possible to use patient-specific hepatocyte-like cells to predict drug efficacy and side effects in various organs from the same patient.

An oxygen-releasing device to improve the survival of mesenchymal stem cells in tissue engineering.

Supplying oxygen to inner areas of cell constructs to support cell proliferation and metabolism is a major challenge in tissue engineering involving stem cells. Developing devices that incorporate oxygen release materials to increase the availability of the localized oxygen supply is therefore key to addressing this limitation. Herein, we designed and developed a 3D-printed oxygen-releasing device composed of an alginate hydrogel scaffold combined with an oxygen-generating biomaterial (calcium peroxide) to improve the oxygen supply of the microenvironment for culturing adipose tissue-derived stem cells. The results demonstrated that the 3D-printed oxygen-releasing device alleviated hypoxia, maintained oxygen availability, and ensured proliferation of the embedded cells, whilst also reducing hypoxia-induced apoptosis. The introduction of this 3D-printed oxygen-releasing device could enhance the survival of embedded stem cells.