Biofabrication of size-controlled liver microtissues incorporated with ECM-derived microparticles to prolong hepatocyte function

Multicellular microtissues of primary human hepatocytes(PHHs)co-cultured with other supporting cell types are a promis-ing model for drug screening and toxicological studies.However,these liver microtissues(LMs)rapidly lose their functions during ex vivo culture.Here,in order to mimic the cellular and structural hepatic microenvironment,we co-cultured PHHs with human mesenchymal stromal cells(MSCs)and human umbilical vein endothelial cells(HUVECs)in the presence of cell-sized microparticles(MPs)derived from liver extracellular matrix(LEMPs).The microwell culture platform enabled biofabrication of size-controlled multicellular microtissues(PHH:HUVEC:MSC=3:2:1)with efficient LEMP incorporation(about 70%at a 2:1 ratio of cells:MP).The biofabricated liver microtissues(BLMs)were cultured ex vivo for 14 days and compared to the cell-only LM in terms of gene and protein expression,functional activity,cytochrome P450(CYP450)enzyme inducibility,and drug sensitivity.The results supported superior hepatic-related gene expression,functional activity,and polarity for PHH in BLM compared to LM.CYP450 enzyme inducibility and dose-responsive sensitivity to toxic drugs were significantly higher in the BLM group.In conclusion,microtissue engineering by incorporation of tissue-specific microparticles within a multicellular microtissue can offer some advantages for drug discovery studies and cell transplantation applications.In the near future,this approach could generate a scalable platform of several functional biofabricated microtissues representing different organs.

Generation of ring-shaped human iPSC-derived functional heart microtissues in a M?bius strip configuration

Although human-induced pluripotent stem cell-derived cardiomyocytes(hi PSC-CMs) have been used for disease modeling and drug discovery, clinically relevant three-dimensional(3D) functional myocardial microtissues are lacking. Here, we developed a novel ring-shaped cardiac microtissue comprised of chamber-specific tissues to achieve a geometrically non-orientable ventricular myocardial band, similar to a M?bius loop. The ring-shaped cardiac tissue was constructed of hi PSC-CMs and human cardiac fibroblasts(h CFs) through a facile cellular self-assembly approach. It exhibited basic anatomical structure,positive cardiac troponin T(c Tn T) immunostaining, regular calcium transients, and cardiac-like mechanical strength. The cardiac rings can be self-assembled and scaled up into various sizes with outstanding stability, suggesting their potential for precise therapy, pathophysiological investigation, and large-scale drug screening.

Fabricating 3-dimensional human brown adipose microtissues for transplantation studies

Transplanting cell cultured brown adipocytes(BAs)represents a promising approach to prevent and treat obesity(OB)and its associated metabolic disorders,including type 2 diabetes mellitus(T2DM).However,transplanted BAs have a very low survival rate in vivo.The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells.There is a critical need for novel methods that can avoid cell death during cell preparation,transplantation,and in vivo.Here,we reported that preparing BAs as injectable microtissues could overcome the problem.We found that 3D culture promoted BA differentiation and UCP-1 expression,and the optimal initial cell aggregate size was 100μm.The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved.Fabricated microtissues could survive in vivo for long term.They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet(HFD)-induced OB and T2DM mice.Transplanted microtissues impacted multiple organs,secreted protein factors,and influenced the secretion of endogenous adipokines.To our best knowledge,this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice.The proposal of transplanting fabricated BA microtissues,the microtissue fabrication method,and the demonstration of efficacy in T2DM mice are all new.Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability,storage,purity,safety,dosage,survival,and efficacy.

Therapeutic effects of adipose-derived stem cells-based microtissues on erectile dysfunction in streptozotocin-induced diabetic rats

This study aimed to explore the therapeutic effects of adipose-derived stem cells （ADSCs）-based microtissues （MTs） on erectile dysfunction （ED） in streptozotocin （STZ）-induced diabetic rats. Fifty-six 8-week-old Sprague-Dawley rats received intraperitoneal injection of STZ （60 mg kg-1）, and 8 weeks later, the determined diabetic rats randomly received intracavernous （IC） injection of phosphate buffer solution （PBS）, ADSCs, or MTs. Another eight normal rats equally got IC injection of PBS. MTs were generated with a hanging drop method, and the injected cells were tracked in ADSC- and MT-injected rats. Four weeks after the treatments, intracavernous pressure （ICP）, histopathological changes in corpus cavernosum （CC）, and functional proteins were measured. Rat cytokine antibody array was used to detect ADSCs or MTs lysate. The results showed that MTs expressed vascular endothelial growth factor （VEGF）, nerve growth factor （NGF）, and tumor necrosis factor-stimulated gene-6 （TSG-6）. MTs injection had a higher retention than ADSCs injection and MTs treatment improved ICP, neuronal nitric oxide synthase （nNOS） expression, smooth muscle, and endothelial contents in diabetic rats, ameliorated local inflammation in CC better. Thus, our findings demonstrate that IC injection of MTs improves erectile function and histopathological changes in STZ-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

The new era of cardiovascular research:revolutionizing cardiovascular research with 3D models in a dish

Cardiovascular research has heavily relied on studies using patient samples and animal models.However,patient studies often miss the data from the crucial early stage of cardiovascular diseases,as obtaining primary tissues at this stage is impracticable.Transgenic animal models can offer some insights into disease mechanisms,although they usually do not fully recapitulate the phenotype of cardiovascular diseases and their progression.In recent years,a promising breakthrough has emerged in the form of in vitro three-dimensional(3D)cardiovascular models utilizing human pluripotent stem cells.These innovative models recreate the intricate 3D structure of the human heart and vessels within a controlled environment.This advancement is pivotal as it addresses the existing gaps in cardiovascular research,allowing scientists to study different stages of cardiovascular diseases and specific drug responses using human-origin models.In this review,we first outline various approaches employed to generate these models.We then comprehensively discuss their applications in studying cardiovascular diseases by providing insights into molecular and cellular changes associated with cardiovascular conditions.Moreover,we highlight the potential of these 3D models serving as a platform for drug testing to assess drug efficacy and safety.Despite their immense potential,challenges persist,particularly in maintaining the complex structure of 3D heart and vessel models and ensuring their function is comparable to real organs.However,overcoming these challenges could revolutionize cardiovascular research.It has the potential to offer comprehensive mechanistic insights into human-specific disease processes,ultimately expediting the development of personalized therapies.

Vascular units as advanced living materials for bottom-up engineering of perfusable 3D microvascular networks

The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regen-eration,remaining a central challenge in tissue engineering.In this study,we present a novel(micro)vascular-ization strategy that explores the use of specialized“vascular units”(VUs)as building blocks to initiate blood vessel formation and create perfusable,stroma-embedded 3D microvascular networks from the bottom-up.We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels.This leads to the formation of VUs-derived capillaries,which fuse with adjacent capillaries to form stable microvascular beds within a supportive,extracellular matrix-rich fibroblastic microenvironment.Using a custom-designed biomimetic fibrin-based vessel-on-chip(VoC),we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused.Moreover,VUs can establish capillary bridges between channels,extending the microvascular network throughout the entire device.When VUs and intestinal organoids(IOs)are combined within the VoC,the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs.This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device.These findings un-derscore the remarkable potential of VUs as building blocks for engineering microvascular networks,with ver-satile applications spanning from regenerative medicine to advanced in vitro models.

Encapsulated three-dimensional bioprinted structure seeded with urothelial cells:a new construction technique for tissue-engineered urinary tract patch

Background:Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold.In this study,we combined three-dimensional(3D)bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch,which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction.Methods:Human adipose-derived stem cells(hADSCs)were dropped into smooth muscle differentiation medium to generate induced microtissues(ID-MTs),flow cytometry was utilized to detect the positive percentage for CD44,CD105,CD45,and CD34 of hADSCs.Expression of vascular endothelial growth factor A(VEGFA)and tumor necrosis factor-stimulated gene-6(TSG-6)in hADSCs and MTs were identified by Western blotting.Then the ID-MTs were employed for 3D bioprinting.The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week.After retrieval of the encapsulated structure,hematoxylin and eosin and Masson’s trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers,integral optical density(IOD)and area of interest were calculated for further semiquantitative analysis.Immunofluorescent double staining of CD31 andα-smooth muscle actin(α-SMA)were used to reveal vascularization of the encapsulated structure.Immunohistochemistry was performed to evaluate the expression of interleukin-2(IL-2),α-SMA,and smoothelin of the MTs in the implanted structure.Afterward,the encapsulated structure was seeded with human urothelial cells.Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure.Results:The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355±0.038 in the hADSCs vs.0.649±0.150 in the MTs(t=3.291,P=0.030),while TSG-6 expression was 0.492±0.092 in the hADSCs vs.1.256±0.401 in the MTs(t=3.216,P=0.032).The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67±1.26,while 12.6±4.79 in the hADSCs group,but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups(t=1.724,P=0.16).The semi-quantitative analysis showed that IOD was 71.7±14.2 in non-induced MTs(NI-MTs)vs.35.7±11.4 in ID-MTs for collagen fibers(t=3.428,P=0.027)and 12.8±1.9 in NI-MTs vs.30.6±8.9 in ID-MTs for smooth muscle fibers(t=3.369,P=0.028);furthermore,the mean IOD was 0.0613±0.0172 in ID-MTs vs.0.0017±0.0009 in NI-MTs forα-SMA(t=5.994,P=0.027),while 0.0355±0.0128 in ID-MTs vs.0.0035±0.0022 in NI-MTs for smoothelin(t=4.268,P=0.013),which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract.After encapsulation of the urinary tract patch for additional cell adhesion,urothelial cells were seeded onto the encapsulated structures,and a monolayer urothelial cell was observed.Conclusion:Through 3D bioprinting and tissue engineering methods,we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation.

A rapidly magnetically assembled stem cell microtissue with“hamburger”architecture and enhanced vascularization capacity

With the development of magnetic manipulation technology based on magnetic nanoparticles(MNPs),scaffold-free microtissues can be constructed utilizing the magnetic attraction of MNP-labeled cells.The rapid in vitro construction and in vivo vascularization of microtissues with complex hierarchical architectures are of great importance to the viability and function of stem cell microtissues.Endothelial cells are indispensable for the formation of blood vessels and can be used in the prevascularization of engineered tissue constructs.Herein,safe and rapid magnetic labeling of cells was achieved by incubation with MNPs for 1 h,and ultrathick scaffold-free microtissues with different sophisticated architectures were rapidly assembled,layer by layer,in 5 min intervals.The in vivo transplantation results showed that in a stem cell microtissue with trisection architecture,the two separated human umbilical vein endothelial cell(HUVEC)layers would spontaneously extend to the stem cell layers and connect with each other to form a spatial network of functional blood vessels,which anastomosed with the host vasculature.The“hamburger”architecture of stem cell microtissues with separated HUVEC layers could promote vascularization and stem cell survival.This study will contribute to the construction and application of structural and functional tissues or organs in the future.

Engineered neuronal microtissue provides exogenous axons for delayed nerve fusion and rapid neuromuscular recovery in rats

Nerve injury requiring surgical repair often results in poor functional recovery due to the inability of host axons to re-grow long distances and reform meaningful connections with the target muscle. While surgeons can re-route local axon fascicles to the target muscle, there are no technologies to provide an exogenous source of axons without sacrificing healthy nerves. Accordingly, we have developed tissue engineered neuromuscular interfaces (TE-NMIs) as the first injectable microtissue containing motor and sensory neurons in an anatomically-inspired architecture. TE-NMIs provide axon tracts that are intended to integrate with denervated distal structures and preserve regenerative capacity during prolonged periods without host innervation. Following implant, we found that TE-NMI axons promoted Schwann cell maintenance, integrated with distal muscle, and preserved an evoked muscle response out to 20-weeks post nerve transection in absence of innervation from host axons. By repopulating the distal sheath with exogenous axons, TE-NMIs also enabled putative delayed fusion with proximal host axons, a phenomenon previously not achievable in delayed repair scenarios due to distal axon degeneration. Here, we found immediate electrophysiological recovery after fusion with proximal host axons and improved axon maturation and muscle reinnervation at 24-weeks post-transection (4-weeks following delayed nerve fusion). These findings show that TE-NMIs provide the potential to improve functional recovery following delayed nerve repair.

Development of a multicellular 3D-bioprinted microtissue model of human periodontal ligament-alveolar bone biointerface:Towards a pre-clinical model of periodontal diseases and personalized periodontal tissue engineering

While periodontal (PD) disease is among principal causes of tooth loss worldwide, regulation of concomitant soft and mineralized PD tissues, and PD pathogenesis have not been completely clarified yet. Besides, relevant pre-clinical models and in vitro platforms have limitations in simulating human physiology. Here, we have harnessed three-dimensional bioprinting (3DBP) technology for developing a multi-cellular microtissue model resembling PD ligament-alveolar bone (PDL-AB) biointerface for the first time. 3DBP parameters were optimized;the physical, chemical, rheological, mechanical, and thermal properties of the constructs were assessed. Constructs containing gelatin methacryloyl (Gel-MA) and hydroxyapatite-magnetic iron oxide nanoparticles showed higher level of compressive strength when compared with that of Gel-MA constructs. Bioprinted self-supporting microtissue was cultured under flow in a microfluidic platform for >10 days without significant loss of shape fidelity. Confocal microscopy analysis indicated that encapsulated cells were homogenously distributed inside the matrix and preserved their viability for >7 days under microfluidic conditions. Immunofluorescence analysis showed the cohesion of stromal cell surface marker-1+ human PDL fibroblasts containing PDL layer with the osteocalcin+ human osteoblasts containing mineralized layer in time, demonstrating some permeability of the printed constructs to cell migration. Preliminary tetracycline interaction study indicated the uptake of model drug by the cells inside the 3D-microtissue. Also, the non-toxic levels of tetracycline were determined for the encapsulated cells. Thus, the effects of tetracyclines on PDL-AB have clinical significance for treating PD diseases. This 3D-bioprinted multi-cellular periodontal/osteoblastic microtissue model has potential as an in vitro platform for studying processes of the human PDL.