Fabrication of paper-based devices for in vitro tissue modeling

Paper devices have recently attracted considerable attention as a class of cost-effective cell culture substrates for various biomedical applications.The paper biomaterial can be used to partially mimic the in vivo cell microenvironments mainly due to its natural three-dimensional characteristic.The paper-based devices provide precise control over their structures as well as cell distributions,allowing recapitulation of certain interactions between the cells and the extracellular matrix.These features have shown great potential for the development of normal and diseased human tissue models.In this review,we discuss the fabrication of paper-based devices for in vitro tissue modeling,as well as the applications of these devices toward drug screening and personalized medicine.It is believed that paper as a biomaterial will play an essential role in the field of tissue model engineering due to its unique performances,such as good biocompatibility,eco-friendliness,cost-effectiveness,and amenability to various biodesign and manufacturing needs.

Digital light processing (DLP)-based (bio)printing strategies for tissue modeling and regeneration

Digital light processing(DLP)-based bioprinting technology has recently aroused considerable concerns as a strategy to deliver biomedical materials and/or specific cells to create sophisticated structures for various tissue modeling and regeneration.In this review,we display a concise introduction of DLP bioprinting,and a further discussion on the design and manufacture of DLP(bio)printer with varied bioinks and their biomedical applications toward drug screening,disease modeling,tissue repair,and regenerative medicine.Finally,the advantages,challenges,and perspectives of the DLP printing platforms are detailed.It is believed that DLP bioprinting will play a decisive role in the field of tissue model and regenerative medicine,mainly due to its time-efficient,higher resolution,and amenability to automation for various tissue needs.

A novel progress of leg tissue properties modeling based on biomechanics

To describe strategies for addressing technical aspects of computational modeling of leg tissue with the finite element （FE） method, a patient＇s leg sample was selected and scanned by CT at the direction parallel to the Frankfort Horizontal plane. A three-dimensional （3D） finite element model of the human leg was developed using the actual geometry of the leg skeleton and soft tissues, which were obtained from 3D reconstruction of CT images. All joints were defined as contact surfaces, which allow relative articulating movement. The major ligaments were simulated using tension-only truss elements by connecting the corresponding attachment points on the bone surfaces. The bony and ligamentous structures were embedded in a volume of soft tissues. The muscles were defined as non-linear viscoelastic material, and the skin, ligaments and tendons were defined as hyperelastic, while the bony structures were assumed to be linearly elastic. The muhilayer FEM model containing thighbone, tibia, fibula, kneecap, soft tissue was formed after meshing. Diverse forces were imposed on the FEM model. The results show that the multilayer FEM model can represent tissue deformation more accurately.

Reconstructing in vivo spatially offset Raman spectroscopy of human skin tissue using a GPU-accelerated Monte Carlo platform

As one type of spatially offset Raman spectroscopy(SORS), inverse SORS is particularly suited to in vivo biomedical measurements due to its ring-shaped illumination scheme. To explain inhomogeneous Raman scattering during in vivo inverse SORS measurements, the light–tissue interactions when excitation and regenerated Raman photons propagate in skin tissue were studied using Monte Carlo simulation. An eight-layered skin model was first built based on the latest transmission parameters. Then, an open-source platform, Monte Carlo e Xtreme(MCX), was adapted to study the distribution of 785 nm excitation photons inside the model with an inverse spatially shifted annular beam. The excitation photons were converted to emission photons by an inverse distribution method based on excitation flux with spatial offsets Δs of 1 mm, 2 mm, 3 mm and 5 mm. The intrinsic Raman spectra from separated skin layers were measured by continuous linear scanning to improve the simulation accuracy. The obtained results explain why the spectral detection depth gradually increases with increasing spatial offset, and address how the intrinsic Raman spectrum from deep skin layers is distorted by the reabsorption and scattering of the superficial tissue constituents. Meanwhile, it is demonstrated that the spectral contribution from subcutaneous fat will be improved when the offset increases to 5 mm, and the highest detection efficiency for dermal layer spectral detection could be achieved when Δs = 2 mm. Reasonably good matching between the calculated spectrum and the measured in vivo inverse SORS was achieved, thus demonstrating great utility of our modeling method and an approach to help understand the clinical measurements.

Engineering biomimetic intestinal topological features in 3D tissue models: retrospects and prospects

Conventional 2D intestinal models cannot precisely recapitulate biomimetic features in vitro and thus are unsuitable for various pharmacokinetic applications,development of disease models,and understanding the host-microbiome interactions.Thus,recently,efforts have been directed toward recreating in vitro models with intestine-associated unique 3D crypt-villus(for small intestine)or crypt-lumen(for large intestine)architectures.This review comprehensively delineates the current advancements in this research area in terms of the different microfabrication technologies(photolithography,laser ablation,and 3D bioprinting)employed and the physiological relevance of the obtained models in mimicking the features of native intestinal tissue.A major thrust of the manuscript is also on highlighting the dynamic interplay between intestinal cells(both the stem cells and differentiated ones)and different biophysical,biochemical,and mechanobiological cues along with interaction with other cell types and intestinal microbiome,providing goals for the future developments in this sphere.The article also manifests an outlook toward the application of induced pluripotent stem cells in the context of intestinal tissue models.On a concluding note,challenges and prospects for clinical translation of 3D patterned intestinal tissue models have been discussed.

Angiogenesis in a 3D model containing adipose tissue stem cells and endothelial cells is mediated by canonical Wnt signaling

Adipose-derived stromal cells （ASCs） have gained great attention in regenerative medicine. Progress in our understanding of adult neovascularization further suggests the potential of ASCs in promoting vascular regeneration, although the specific cues that stimulate their angiogenic behavior remain controversial In this study, we established a three-dimensional （3D） angiogenesis model by co-culturing ASCs and endothelial cells （ECs） in collagen gel and found that ASC-EC-instructed angiogenesis was regulated by the canonical Wnt pathway. Furthermore, the angiogenesis that occurred in implants collected after injections of our collagen gel- based 3D angiogenesis model into nude mice was confirmed to be functional and also regulated by the canonical Wnt pathway. Wnt regulation of angiogenesis involving changes in vessel length, vessel density, vessel sprout, and connection numbers occurred in our system. Wnt signaling was then shown to regulate ASC- mediated paracrine signaling during angiogenesis through the nuclear translocation of β-catenin after its cytoplasmic accumulation in both ASCs and ECs. This translocation enhanced the expression of nuclear cofactor Lef-1 and cyclin D1 and activated the angiogenic transcription of vascular endothelial growth factor A （VEGFA）, basic fibroblast growth factor （bFGF）, and insulin-like growth factor 1 （IGF-1）. The angiogenesis process in the 3D collagen model appeared to follow canonical Wnt signaling, and this model can help us understand the importance of the canonical Wnt pathway in the use of ASCs in vascular regeneration.

Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

Local icariin application enhanced periodontal tissue regeneration and relieved local inflammation in a minipig model of periodontitis

Periodontitis is an inflammatory autoimmune disease. Treatment should alleviate inflammation, regulate the immune reaction and promote periodontal tissue regeneration. Icariin is the main active ingredient of Epimedii Folium, and it is a promising compound for the enhancement of mesenchymal stem cell function, promotion of bone formation, inhibition of bone resorption, alleviation of inflammation and regulation of immunity. The study investigated the effect of icariin on periodontal tissue regeneration in a minipig model of periodontitis. The minipig model of periodontitis was established. Icariin was injected locally. The periodontal clinical assessment index, a computed tomography(CT) scan, histopathology and enzyme-linked immune sorbent assay(ELISA)were used to evaluate the effects of icariin. Quantitative analysis results 12 weeks post-injection demonstrated that probing depth,gingival recession, attachment loss and alveolar bone regeneration values were(3.72 ± 1.18) mm vs.(6.56 ± 1.47) mm,(1.67 ± 0.59)mm vs.(2.38 ± 0.61) mm,(5.56 ± 1.29) mm vs.(8.61 ± 1.72) mm, and(25.65 ± 5.13) mm3 vs.(9.48 ± 1.78) mm3 in the icariin group and0.9% NaCl group, respectively. The clinical assessment, CT scan, and histopathology results demonstrated significant enhancement of periodontal tissue regeneration in the icariin group compared to the 0.9% NaCl group. The ELISA results suggested that the concentration of interleukin-1 beta(IL-1β) in the icariin group was downregulated compared to the 0.9% NaCl group, which indicates that local injection of icariin relieved local inflammation in a minipig model of periodontitis. Local injection of icariin promoted periodontal tissue regeneration and exerted anti-inflammatory and immunomodulatory function. These results support the application of icariin for the clinical treatment of periodontitis.

3D printing of functional bioengineered constructs for neural regeneration: a review

Three-dimensional(3D)printing technology has opened a new paradigm to controllably and reproducibly fabricate bioengineered neural constructs for potential applications in repairing injured nervous tissues or producing in vitro nervous tissue models.However,the complexity of nervous tissues poses great challenges to 3D-printed bioengineered analogues,which should possess diverse architectural/chemical/electrical functionalities to resemble the native growth microenvironments for functional neural regeneration.In this work,we provide a state-of-the-art review of the latest development of 3D printing for bioengineered neural constructs.Various 3D printing techniques for neural tissue-engineered scaffolds or living cell-laden constructs are summarized and compared in terms of their unique advantages.We highlight the advanced strategies by integrating topographical,biochemical and electroactive cues inside 3D-printed neural constructs to replicate in vivo-like microenvironment for functional neural regeneration.The typical applications of 3D-printed bioengineered constructs for in vivo repair of injured nervous tissues,bio-electronics interfacing with native nervous system,neural-on-chips as well as brain-like tissue models are demonstrated.The challenges and future outlook associated with 3D printing for functional neural constructs in various categories are discussed.

A high‑throughput three‑dimensional cell culture platform for drug screening

Traditional two-dimensional(2D)cell cultures lack the extracellular matrix(ECM)-like structure or dynamic fluidic microenvironment for cells to maintain in vivo functionality.Three-dimensional(3D)tissue scaffolds,on the other hand,could provide the ECM-like microenvironment for cells to reformulate into tissue or organoids that are highly useful for in vitro drug screening.In this study,a high-throughput two-chamber 3D microscale tissue model platform is developed.Porous scaffolds are selectively foamed on a commercially available compact disk using laser.Perfusion of cell culture medium is achieved with centrifugal force-driven diffusion by disk rotation.Experimental studies were conducted on the fabrication process under various gas saturation and laser power conditions.Cell cultures were performed with two types of human cell lines:M059K and C3A-sub28.It is shown that the structure of microscale porous scaffolds can be controlled with laser foaming parameters and that coating with polydopamine these scaffolds are inducive for cell attachment and aggregation,forming a 3D network.With many such two-chamber models fabricated on a single CD and perfusion driven by the centrifugal force from rotation,the proposed platform provides a simple solution to the high-cost and lengthy drug development process with a high-throughput and physiologically more relevant tissue model system.

A Systematic Review of Animal and Clinical Studies on the Use of Scaffolds for Urethral Repair

Replacing urethral tissue with functional scaffolds has been one of the challenging problems in the field of urethra reconstruction or repair over the last several decades. Various scaffold materials have been used in animal studies, but clinical studies on use of scaffolds for urethral repair are scarce. The aim of this study was to review recent animal and clinical studies on the use of different scaffolds for urethral repair, and to evaluate these scaffolds based on the evidence from these studies. Pub Med and OVID databases were searched to identify relevant studies, in conjunction with further manual search. Studies that met the inclusion criteria were systematically evaluated. Of 555 identified studies, 38 were included for analysis. It was found that in both animal and clinical studies, scaffolds seeded with cells were used for repair of large segmental defects of the urethra, such as in tubular urethroplasty. When the defect area was small, cell-free scaffolds were more likely to be applied. A lot of pre-clinical and limited clinical evidence showed that natural or artificial materials could be used as scaffolds for urethral repair. Urinary tissue engineering is still in the immature stage, and the safety, efficacy, cost-effectiveness of the scaffolds are needed for further study.

Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

Vascularization of acellular nerves has been shown to contribute to nerve bridging.In this study,we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves.The rat nerve defects were treated with acellular nerve grafting（control group） alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein（experimental group）.As shown through two-dimensional imaging,the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation,and gradually covered the entire graft at day 21.The vascular density,vascular area,and the velocity of revascularization in the experimental group were all higher than those in the control group.These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves.

Training gastroenterology fellows to perform gastric polypectomy using a novel ex vivo model

AIM:To evaluate the effect of hands-on training of gastroenterology fellows in gastric polypectomy using an ex vivo simulator.METHODS:Eight gastroenterology fellows at Mackay Memorial Hospital,Taipei were evaluated in gastricpolypectomy techniques using a pig stomach with artificial polyps created by a rubber band ligation device.The performance of four second year(year-2)fellows who had undergone one year of clinical training was compared with that of four f irst year(year-1)fellows both before and after a 4-h workshop using the ex vivo simulator.The workshop allowed for hands-on train-ing in the removal of multiple artif icial polyps and the placement of hemoclips at the excision site.Evaluation included observation of technical skills,procedure time,and the fellows' conf idence scale.RESULTS:One week after the workshop,the year-1 fellows were re-evaluated and had significantly im-proved mean performance scores(from 17.9 ± 1.8 to 22.5 ± 0.7),conf idence scale(from 4.5 ± 1.0 to 7.8 ± 0.5)and procedure time(from 615.0 ± 57.4 s to 357.5 ± 85.0 s)compared with their baseline performance.After 4 h of training using the ex vivo simulator,the skills of the year-1 fellows were statistically similar to those of the year-2 fellows.CONCLUSION:Use of this ex vivo simulator significantly improved the endoscopic gastric polypectomy skills of gastroenterology fellows who had not had previous clinical training in gastric polypectomy.

Synthesis of easily-processable collagen bio-inks using ionic liquid for 3D bioprinted liver tissue models with branched vascular networks

Bioprinting has been a flouring way to fabricate complex tissue and organ mimics via precisely depositing printable cell-laden biomaterials.However,there is a limited number of biomaterials that fulfill the mechanical property of printing while meeting the responsive environment desired for the cells.Despite excellent cell compatibility and bioactivity,collagen suffers from difficulties in processing and printability which inhibited its utilization in three-dimensional(3D)bioprinting.Herein,we address this limitation by using ionic liquid as the solvent in the modification process,enabling collagens modified with quantified norbornene for chemical crosslink and extrusion-based 3D printing.With improved solubility and rheological properties,norbornene-functionalized collagen(Col-Nor)exhibited better shape fidelity in extrusion-based 3D printing compared with the one before modification.Photo-crosslinked Col-Nor hydrogel provided structural support and promoted the adhesion,proliferation,and differentiation of various types of cells,which afforded a centimeter-scale liver tissue model.This highly generalizable methodology expands printable,versatile,and tunable hydrogels developed from the natural extracellular matrix,allowing the biofabrication of 3D liver tissue model with branched vascular networks.

3D engineered tissue models for studying human-specific infectious viral diseases

Viral infections cause damage to various organ systems by inducing organ-specific symptoms or systemic multi-organ damage.Depending on the infection route and virus type,infectious diseases are classified as respiratory,nervous,immune,digestive,or skin infections.Since these infectious diseases can widely spread in the com-munity and their catastrophic effects are severe,identification of their causative agent and mechanisms un-derlying their pathogenesis is an urgent necessity.Although infection-associated mechanisms have been studied in two-dimensional(2D)cell culture models and animal models,they have shown limitations in organ-specific or human-associated pathogenesis,and the development of a human-organ-mimetic system is required.Recently,three-dimensional(3D)engineered tissue models,which can present human organ-like physiology in terms of the 3D structure,utilization of human-originated cells,recapitulation of physiological stimuli,and tight cell–cell interactions,were developed.Furthermore,recent studies have shown that these models can recapitulate infection-associated pathologies.In this review,we summarized the recent advances in 3D engineered tissue models that mimic organ-specific viral infections.First,we briefly described the limitations of the current 2D and animal models in recapitulating human-specific viral infection pathology.Next,we provided an overview of recently reported viral infection models,focusing particularly on organ-specific infection pathologies.Finally,a future perspective that must be pursued to reconstitute more human-specific infectious diseases is presented.

Advances in hydrogel-based vascularized tissues for tissue repair and drug screening

The construction of biomimetic vasculatures within the artificial tissue models or organs is highly required for conveying nutrients,oxygen,and waste products,for improving the survival of engineered tissues in vitro.In recent times,the remarkable progress in utilizing hydrogels and understanding vascular biology have enabled the creation of three-dimensional(3D)tissues and organs composed of highly complex vascular systems.In this review,we give an emphasis on the utilization of hydrogels and their advantages in the vascularization of tissues.Initially,the significance of vascular elements and the regeneration mechanisms of vascularization,including angiogenesis and vasculogenesis,are briefly introduced.Further,we highlight the importance and advantages of hydrogels as artificial microenvironments in fabricating vascularized tissues or organs,in terms of tunable physical properties,high similarity in physiological environments,and alternative shaping mechanisms,among others.Furthermore,we discuss the utilization of such hydrogels-based vascularized tissues in various applications,including tissue regeneration,drug screening,and organ-on-chips.Finally,we put forward the key challenges,including multifunctionalities of hydrogels,selection of suitable cell phenotype,sophisticated engineering techniques,and clinical translation behind the development of the tissues with complex vasculatures towards their future development.

Studies of nanoparticle delivery with in vitro bio-engineered microtissues

A variety of engineered nanoparticles,including lipid nanoparticles,polymer nanoparticles,gold nanoparticles,and biomimetic nanoparticles,have been studied as delivery vehicles for biomedical applications.When assessing the efficacy of a nanoparticle-based delivery system,in vitro testing with a model delivery system is crucial because it allows for real-time,in situ quantitative transport analysis,which is often difficult with in vivo animal models.The advent of tissue engineering has offered methods to create experimental models that can closely mimic the 3D microenvironment in the human body.This review paper overviews the types of nanoparticle vehicles,their application areas,and the design strategies to improve delivery efficiency,followed by the uses of engineered microtissues and methods of analysis.In particular,this review highlights studies on multicellular spheroids and other 3D tissue engineering approaches for cancer drug development.The use of bio-engineered tissues can potentially provide low-cost,high-throughput,and quantitative experimental platforms for the development of nanoparticle-based delivery systems.

Inkjet 3D bioprinting for tissue engineering and pharmaceutics

3D bioprinting has the capability to create 3D cellular constructs with the desired shape using a layer-by-layer approach.Inkjet 3D bioprinting,as a key component of 3D bioprinting,relies on the deposition of cell-laden droplets to create native-like tissues/organs which are envisioned to be transplantable into human body for replacing damaged ones.Benefiting from its superiorities such as high printing resolution and deposition accuracy,inkjet 3D bioprinting has been widely applied to various areas,including,but not limited to,tissue engineering and drug screening in pharmaceutics.Even though inkjet 3D bioprinting has proved its feasibility and versatility in various fields,the current applications of inkjet 3D bioprinting are still limited by the printing technique and material selection.This review,which specifically focuses on inkjet 3D bioprinting,firstly summarizes the techniques,materials,and applications of inkjet 3D bioprinting in tissue engineering and drug screening,subsequently discusses the major challenges that inkjet 3D bioprinting is facing,and lastly summarizes potential solutions to those challenges.

Study the lipidoid nanoparticle mediated genome editing protein delivery using 3D intestinal tissue model

Lipid nanoparticles are promising carriers for oral drug delivery.For bioactive cargos with intracellular targets,e.g.gene-editing proteins,it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells.However,limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier.Herein,we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle(carrier)and protein(cargo)nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells.We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase,penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer.After transporting across the intestinal epithelia,the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination.These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.