3D bioprinting of in vitro porous hepatoma models:establishment,evaluation,and anticancer drug testing

Traditional tumor models do not tend to accurately simulate tumor growth in vitro or enable personalized treatment and are particularly unable to discover more beneficial targeted drugs.To address this,this study describes the use of threedimensional(3D)bioprinting technology to construct a 3D model with human hepatocarcinoma SMMC-7721 cells(3DP-7721)by combining gelatin methacrylate(GelMA)and poly(ethylene oxide)(PEO)as two immiscible aqueous phases to form a bioink and innovatively applying fluorescent carbon quantum dots for long-term tracking of cells.The GelMA(10%,mass fraction)and PEO(1.6%,mass fraction)hydrogel with 3:1 volume ratio offered distinct pore-forming characteristics,satisfactorymechanical properties,and biocompatibility for the creation of the 3DP-7721 model.Immunofluorescence analysis and quantitative real-time fluorescence polymerase chain reaction(PCR)were used to evaluate the biological properties of the model.Compared with the two-dimensional culture cell model(2D-7721)and the 3D mixed culture cell model(3DM-7721),3DP-7721 significantly improved the proliferation of cells and expression of tumor-related proteins and genes.Moreover,we evaluated the differences between the three culture models and the effectiveness of antitumor drugs in the three models and discovered that the efficacy of antitumor drugs varied because of significant differences in resistance proteins and genes between the three models.In addition,the comparison of tumor formation in the three models found that the cells cultured by the 3DP-7721 model had strong tumorigenicity in nude mice.Immunohistochemical evaluation of the levels of biochemical indicators related to the formation of solid tumors showed that the 3DP-7721 model group exhibited pathological characteristics of malignant tumors,the generated solid tumors were similar to actual tumors,and the deterioration was higher.This research therefore acts as a foundation for the application of 3DP-7721 models in drug development research.

Development of 3D bioprinting:From printing methods to biomedical applications

Biomanufacturing of tissues/organs in vitro is our big dream,driven by two needs:organ transplantation and accurate tissue models.Over the last decades,3D bioprinting has been widely applied in the construction of many tissues/organs such as skins,vessels,hearts,etc.,which can not only lay a foundation for the grand goal of organ replacement,but also be served as in vitro models committed to pharmacokinetics,drug screening and so on.As organs are so complicated,many bioprinting methods are exploited to figure out the challenges of different applications.So the question is how to choose the suitable bioprinting method?Herein,we systematically review the evolution,process and classification of 3D bioprinting with an emphasis on the fundamental printing principles and commercialized bioprinters.We summarize and classify extrusion-based,dropletbased,and photocuring-based bioprinting methods and give some advices for applications.Among them,coaxial and multi-material bioprinting are highlighted and basic principles of designing bioinks are also discussed.

Design of a High Precision Multichannel 3D Bioprinter

Three-dimensional(3D)printing technology is expected to solve the organ shortage problem.However,owing to the accuracy limitations,it is difficult for the current bioprinting technology to achieve an accurate control of the spatial position and distribution of a single cell or single component droplet.In this study,to accurately achieve the directional deposition of different cells and biological materials in the spatial position for the construction of large transplantable tissues and organs,a high-precision multichannel 3D bioprinter with submicron-level motion accuracy is designed,and concurrent and synergistic printing methods are proposed.Based on the high-precision motion characteristics of the gantry structure and the requirements of concurrent and synergistic printing,a 3D bioprint-ing system with a set of 6 channels is designed to achieve six-in-one printing.Based on the Visual C++environ-ment,a control system software that integrates the programmable multi-axis controller(PMAC)motion,pneumatic,and temperature control subsystems was developed and designed.Finally,based on measurements and experiments,the 3D bioprinter and its control system was verified to fulfil the requirements of multichannel,concurrent,and syn-ergistic printing with submicron-level motion accuracy,significantly shortening the printing time and improving the printing efficiency.This study not only provides an equipment basis for printing complex heterogeneous tissue structures,but also improves the flexibility and functionality of bioprinting,and ultimately makes the construction of complex multicellular tissues or organs possible.

The use of machine learning to predict the effects of cryoprotective agents on the GelMA-based bioinks used in extrusion cryobioprinting

Cryobioprinting has tremendous potential to solve problems to do with lack of shelf availability in traditional bioprinting by combining extrusion bioprinting and cryopreservation.In order to ensure the viability of cells in the frozen state and avoid the possible toxicity of dimethyl sulfoxide(DMSO),DMSO-free bioink design is critical for achieving successful cryobioprinting.A nontoxic gelatin methacryloyl-based bioink used in cryobioprinting is composed of cryoprotective agents(CPAs)and a buffer solution.The selection and ratio of CPAs in the bioink directly affect the survival of cells in the frozen state.However,the development of universal and efficient cryoprotective bioinks requires extensive experimentation.We first compared two commonly used CPA formulations via experiments in this study.Results show that the effect of using ethylene glycol as the permeable CPA was 6.07%better than that of glycerol.Two datasets were obtained and four machinelearning models were established to predict experimental outcomes.The predictive powers of multiple linear regression(MLR),decision tree(DT),random forest(RF),and artificial neural network(ANN)approaches were compared,suggesting an order of ANN>RF>DT>MLR.The final selected ANN model was then applied to another dataset.Results reveal that this machine-learning method can accurately predict the effects of cryoprotective bioinks composed of different CPAs.Outcomes also suggest that the formulations presented here have universality.Our findings are likely to greatly accelerate research and development on the use of bioinks for cryobioprinting.

Polyvinylpyrrolidone-based bioink:influence of bioink properties on printing performance and cell proliferation during inkjet-based bioprinting

Among the different bioprinting techniques,the drop-on-demand(DOD)jetting-based bioprinting approach facilitates contactless deposition of pico/nanoliter droplets ofmaterials and cells for optimal cell–matrix and cell–cell interactions.Although bioinks play a critical role in the bioprinting process,there is a poor understanding of the influence of bioink properties on printing performance(such as filament elongation,formation of satellite droplets,and droplet splashing)and cell health(cell viability and proliferation)during the DOD jetting-based bioprinting process.An inert polyvinylpyrrolidone(PVP360,molecular weight=360 kDa)polymerwas used in this study to manipulate the physical properties of the bioinks and investigate the influence of bioink properties on printing performance and cell health.Our experimental results showed that a higher bioink viscoelasticity helps to stabilize droplet filaments before rupturing from the nozzle orifice.The highly stretched droplet filament resulted in the formation of highly aligned“satellite droplets,”which minimized the displacement of the satellite droplets away from the predefined positions.Next,a significant increase in the bioink viscosity facilitated droplet deposition on the wetted substrate surface in the absence of splashing and significantly improved the accuracy of the deposited main droplet.Further analysis showed that cell-laden bioinks with higher viscosity exhibited higher measured average cell viability(%),as the presence of polymer within the printed droplets provides an additional cushioning effect(higher energy dissipation)for the encapsulated cells during droplet impact on the substrate surface,improves the measured average cell viability even at higher droplet impact velocity and retains the proliferation capability of the printed cells.Understanding the influence of bioink properties(e.g.,bioink viscoelasticity and viscosity)on printing performance and cell proliferation is important for the formulation of new bioinks,and we have demonstrated precise DOD deposition of living cells and fabrication of tunable cell spheroids(nL–μL range)using multiple types of cells in a facile manner.

3D-bioprinted cholangiocarcinoma-on-a-chip model for evaluating drug responses

Cholangiocarcinoma(CCA)is characterized by heterogeneous mutations and a refractory nature.Thus,the development of a model for effective drug screening is urgently needed.As the established therapeutic testing models for CCA are often ineffective,we fabricated an enabling three-dimensional(3D)-bioprinted CCA-on-a-chip model that to a good extent resembled the multicellular microenvironment and the anatomical microstructure of the hepato-vascular-biliary system to perform high-content antitumor drug screening.Specifically,cholangiocytes,hepatocytes,and vascular endotheliocytes were employed for 3D bioprinting of the models,allowing for a high degree of spatial and tube-like microstructural control.Interestingly,it was possible to observe CCA cells attached to the surfaces of the gelatin methacryloyl(GelMA)hydrogelembedded microchannels and overgrown in a thickening manner,generating bile duct stenosis,which was expected to be analogous to the in vivo configuration.Over 4000 differentially expressed genes were detected in the CCA cells in our 3D coculture model compared to the traditional two-dimensional(2D)monoculture.Further screening revealed that the CCA cells grown in the 3D traditional model were more sensitive to the antitumoral prodrug than those in the 2D monoculture due to drug biotransformation by the neighboring functional hepatocytes.This study provides proof-of-concept validation of our bioprinted CCA-on-a-chip as a promising drug screening model for CCA treatment and paves the way for potential personalized medicine strategies for CCA patients in the future.

Visible Light-Induced 3D Bioprinting Technologies and Corresponding Bioink Materials for Tissue Engineering: A Review

Three-dimensional(3D)bioprinting based on traditional 3D printing is an emerging technology that is used to precisely assemble biocompatible materials and cells or bioactive factors into advanced tissue engineering solutions.Similar technology,particularly photo-cured bioprinting strategies,plays an important role in the field of tissue engineering research.The successful implementation of 3D bioprinting is based on the properties of photopolymerized materials.Photocrosslinkable hydrogel is an attractive biomaterial that is polymerized rapidly and enables process control in space and time.Photopolymerization is frequently initiated by ultraviolet(UV)or visible light.However,UV light may cause cell damage and thereby,affect cell viability.Thus,visible light is considered to be more biocompatible than UV light for bioprinting.In this review,we provide an overview of photo curing-based bioprinting technologies,and describe a visible light crosslinkable bioink,including its crosslinking mechanisms,types of visible light initiator,and biomedical applications.We also discuss existing challenges and prospects of visible light-induced 3D bioprinting devices and hydrogels in biomedical areas.

Rapid printing of 3D porous scaffolds for breast reconstruction

Prosthesis implantation and fat transplantation are common breast reconstructionmethods.In general,prosthesis implantation alone does not achieve a realistic enough appearance,and fat transplantation alone is difficult to achieve in the correct capacity.To date,no reports have focused on methods of combining fat with implanted prostheses for breast reconstruction.Using a newly designed bionic ink(i.e.,polyether F127 diacrylate(F127DA)&poly(ethylene glycol)diacrylate(PEGDA))and projection-based three-dimensional bioprinting(PBP),we report the development of a new method for printing porous prostheses.PEGDA was used to improve the printing precision of the prosthesis by increasing the gel point of F127DA and reducing the impact of external temperature.The compression modulus of the printed prosthesis was very close to that of prostheses currently used in clinical practice and to that of natural breasts.Finally,stromal vascular fraction gel(SVF-gel),a human fat extract,was injected into the pores of the synthesized prostheses to prepare a prosthesis mixed with adipose tissue.These were implanted subcutaneously in nude mice to observe their biological performance.After 14 and 28 days of observation,the prosthesis showed good biocompatibility,and adipose tissues grew well in and around the prosthesis.This result shows that a porous prosthesis containing pre-placed adipose tissues is a promising breast reconstruction material.

Three-dimensional bioprinting in ophthalmic care

Three-dimensional(3D)bioprinting is widely used in ophthalmic clinic,including in diagnosis,surgery,prosthetics,medications,drug development and delivery,and medical education.Articles published in 2011–2022 into bioinks,printing technologies,and bioprinting applications in ophthalmology were reviewed and the strengths and limitations of bioprinting in ophthalmology highlighted.The review highlighted the trade-offs of printing technologies and bioinks in respect to,among others,material type cost,throughput,gelation technique,cell density,cell viability,resolution,and printing speed.There is already widespread ophthalmological application of bioprinting outside clinical settings,including in educational modelling,retinal imaging/visualization techniques and drug design/testing.In clinical settings,bioprinting has already found application in pre-operatory planning.Even so,the findings showed that even with its immense promise,actual translation to clinical applications remains distant,but relatively closer for the corneal(except stromal)tissues,epithelium,endothelium,and conjunctiva,than it was for the retina.This review similarly reflected on the critical on the technical,practical,ethical,and cost barrier to rapid progress of bioprinting in ophthalmology,including accessibility to the most sophisticated bioprinting technologies,choice,and suitability of bioinks,tissue viability and storage conditions.The extant research is encouraging,but more work is clearly required for the push towards clinical translation of research.

Using 3D bioprinting to produce mini-brain

Recent progresses in three-dimensional （3D） bioprinting technology accelerate the coming of the era of personalized medicine. With vari- ous printing approaches and materials developed, 3D bioprinting may have a broad range of medical applications, including the fabrication of delicate tissues/organs/or the clinical use in the future or for the es- tablishment of tissues in disease models. The principal advantages of 3D bioprinting are personalized design and precise fabrication, which are of critical importance for tissue engineering. To date, several types of biomimetic tissues, such as cartilage, skin, and vascular tissues have been fabricated by 3D bioprinting （Liaw and Guvendiren, 2017）.

Sacrificial microgel‑laden bioink‑enabled 3D bioprinting of mesoscale pore networks

Three-dimensional(3D)bioprinting is a powerful approach that enables the fabrication of 3D tissue constructs that retain complex biological functions.However,the dense hydrogel networks that form after the gelation of bioinks often restrict the migration and proliferation of encapsulated cells.Herein,a sacrificial microgel-laden bioink strategy was designed for directly bioprinting constructs with mesoscale pore networks(MPNs)for enhancing nutrient delivery and cell growth.The sacrificial microgel-laden bioink,which contains cell/gelatin methacryloyl(GelMA)mixture and gelled gelatin microgel,is first thermo-crosslinked to fabricate temporary predesigned cell-laden constructs by extrusion bioprinting onto a cold platform.Then,the construct is permanently stabilized through photo-crosslinking of GelMA.The MPNs inside the printed constructs are formed after subsequent dissolution of the gelatin microgel.These MPNs allowed for effective oxygen/nutrient diffusion,facilitating the generation of bioactive tissues.Specifically,osteoblast and human umbilical vein endothelial cells encapsulated in the bioprinted large-scale constructs(≥1 cm)with MPNs showed enhanced bioactivity during culture.The 3D bioprinting strategy based on the sacrificial microgel-laden bioink provided a facile method to facilitate formation of complex tissue constructs with MPNs and set a foundation for future optimization of MPN-based tissue constructs with applications in diverse areas of tissue engineering.

Liquid-phase 3D bioprinting of gelatin alginate hydrogels:influence of printing parameters on hydrogel line width and layer height

Extrusion-based 3D bioprinting is a direct deposition approach used to create three-dimensional(3D)tissue scaffolds typically comprising hydrogels.Hydrogels are hydrated polymer networks that are chemically or physically cross-linked.Often,3D bioprinting is performed in air,despite the hydrated nature of hydrogels and the potential advantage of using a liquid phase to provide cross-linking and otherwise functionalize the hydrogel.In this work,we print gelatin alginate hydrogels directly into a cross-linking solution of calcium chloride and investigate the influence of nozzle diameter,distance between nozzle and surface,calcium chloride concentration,and extrusion rate on the dimensions of the printed hydrogel.The hydrogel layer height was generally found to increase with increasing extrusion rate and nozzle distance,according to the increased volume extruded and the available space,respectively.In addition,the hydrogel width was generally found to increase with decreasing nozzle distance and cross-linking concentration corresponding to confinement-induced spreading and low crosslinking regimes,respectively.Width/height ratios of^1 were generally achieved when the nozzle diameter and distance were comparable above a certain cross-linking concentration.Using these relationships,biocompatible 3D multilayer structures were successfully printed directly into calcium chloride cross-linking solution.

The construction of in vitro tumormodels based on 3D bioprinting

Cancer is characterized by a high fatality rate,complex molecular mechanism,and costly therapies.The microenvironment of a tumor consists of multiple biochemical cues and the interaction between tumor cells,stromal cells,and extracellular matrix plays a key role in tumor initiation,development,angiogenesis,invasion and metastasis.To better understand the biological features of tumor and reveal the critical factors of therapeutic treatments against cancer,it is of great significance to build in vitro tumor models that could recapitulate the stages of tumor progression and mimic tumor behaviors in vivo for efficient and patient-specific drug screening and biological studies.Since conventional tissue engineering methods of constructing tumor models always fail to simulate the later stages of tumor development due to the lack of ability to build complex structures and angiogenesis potential,three-dimensional(3D)bioprinting techniques have gradually found its applications in tumor microenvironment modeling with accurate composition and well-organized spatial distribution of tumor-related cells and extracellular components in the past decades.The capabilities of building tumor models with a large range of scale,complex structures,multiple biomaterials and vascular network with high resolution and throughput make 3D bioprinting become a versatile platform in bio-manufacturing aswell as inmedical research.In this review,wewill focus on 3D bioprinting strategies,design of bioinks,current 3D bioprinted tumor models in vitro classified with their structures and propose future perspectives.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellularmatrix for the differentiation of skeletal stem and progenitor cells

Autograft or metal implants are routinely used in skeletal repair.However,they fail to provide long-term clinical resolution,necessitating a functional biomimetic tissue engineering alternative.The use of native human bone tissue for synthesizing a biomimeticmaterial inkfor three-dimensional(3D)bioprintingof skeletal tissueis anattractivestrategyfor tissueregeneration.Thus,human bone extracellular matrix(bone-ECM)offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells(HBMSCs)to proliferate and differentiate along the osteogenic lineage.In this study,we engineered a novel material ink(LAB)by blending human bone-ECM(B)with nanoclay(L,Laponite®)and alginate(A)polymers using extrusion-based deposition.The inclusion of the nanofiller and polymeric material increased the rheology,printability,and drug retention properties and,critically,the preservation of HBMSCs viability upon printing.The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor(VEGF)enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane(CAM)model.The inclusion of bone morphogenetic protein-2(BMP-2)with the HBMSCs further enhanced vascularization and mineralization after only seven days.This study demonstrates the synergistic combination of nanoclay with biomimetic materials(alginate and bone-ECM)to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.

3D bioprinting:current status and trends-a guide to the literature and industrial practice

The multidisciplinary research field of bioprinting combines additive manufacturing,biology and material sciences to cre-ate bioconstructs with three-dimensional architectures mimicking natural living tissues.The high interest in the possibility of reproducing biological tissues and organs is further boosted by the ever-increasing need for personalized medicine,thus allowing bioprinting to establish itself in the field of biomedical research,and attracting extensive research efforts from companies,universities,and research institutes alike.In this context,this paper proposes a scientometric analysis and critical review of the current literature and the industrial landscape of bioprinting to provide a clear overview of its fast-changing and complex position.The scientific literature and patenting results for 2000-2020 are reviewed and critically analyzed by retrieving 9314 scientific papers and 309 international patents in order to draw a picture of the scientific and industrial landscape in terms of top research countries,institutions,journals,authors and topics,and identifying the technology hubs worldwide.This review paper thus offers a guide to researchers interested in this field or to those who simply want to under-stand the emerging trends in additive manufacturing and 3D bioprinting.

Development of a 3D subcutaneous construct containing insulin-producing beta cells using bioprinting

Type 1 diabetes is caused by insulin deficiency due to the loss of beta cells in the islets of Langerhans.In severe cases,islet transplantation into the portal vein is performed.However,due to the loss of transplanted islets and the failure of islet function,the 5-year insulin independence rate of these patients is<50%.In this study,we developed a long-term,insulin-secreting,3 Dbioprinted construct implanted subcutaneously with the aim of preventing islet loss.The bioprinted construct was fabricated by the multi-layer bioprinting of beta-cell(mouse insulinoma-6:MIN-6)-encapsulated alginate bioink and poly(caprolactone)biodegradable polymer.A glucose response assay revealed that the bioprinted constructs proliferated and released insulin normally during the 4-week in vitro period.Bioprinted MIN-6 generated clusters with a diameter of 100-200μm,similar to the original pancreatic islets in the construct.In an in vivo study using type 1 diabetes mice,animals implanted with bioprinted constructs showed three times higher insulin secretion and controlled glucose levels at 8 weeks after implantation.Because the implanted,bioprinted constructs had a positive effect on insulin secretion in the experimental animals,the survival rate of the implanted group(75%)was three times higher than that of the non-implanted group(25%).The results suggest that the proposed,3 D-bioprinted,subcutaneous construct can be a better alternative to portal vein islet transplantation.

Printabilitye-A key issue in extrusion-based bioprinting

Three-dimensional(3D)extrusion-based bioprinting is widely used in tissue engineering and regenerative medicine to create cell-incorporated constructs or scaffolds based on the extrusion technique.One critical issue in 3D extrusion-based bioprinting is printability or the capability to form and maintain reproducible 3D scaffolds from bioink(a mixture of biomaterials and cells).Research shows that printability can be affected by many factors or parameters,including those associated with the bioink,printing process,and scaffold design,but these are far from certain.This review highlights recent developments in the printability assessment of extrusion-based bioprinting with a focus on the definition of printability,printability measurements and characterization,and printability-affecting factors.Key issues and challenges related to printability are also identified and discussed,along with approaches or strategies for improving printability in extrusion-based bioprinting.

3D bioprinted hyaluronic acid‑based cell‑laden scaffold for brain microenvironment simulation

Treatments for lesions in central nervous system(CNS)are always faced with challenges due to the anatomical and physiological particularity of the CNS despite the fact that several achievements have been made in early diagnosis and precision medicine to improve the survival and quality of life of patients with brain tumors in recent years.Understanding the complexity as well as role of the microenvironment of brain tumors may suggest a better revealing of the molecular mechanism of brain tumors and new therapeutic directions,which requires an accurate recapitulation of the complex microenvironment of human brain in vitro.Here,a 3D bioprinted in vitro brain matrix-mimetic microenvironment model with hyaluronic acid(HA)and normal glial cells(HEBs)is developed which simulates both mechanical and biological properties of human brain microenvironment in vivo through the investigation of the formulation of bioinks and optimization of printing process and parameters to study the effects of different concentration of gelatin(GA)within the bioink and different printing structures of the scaffold on the performance of the brain matrix-mimetic microenvironment models.The study provides experimental models for the exploration of the multiple factors in the brain microenvironment and scaffolds for GBM invasion study.

3D biofabrication of vascular networks for tissue regeneration:A report on recent advances

Rapid progress in tissue engineering research in past decades has opened up vast possibilities to tackle the challenges of generating tissues or organs that mimic native structures. The success of tissue engineered constructs largely depends on the incorporation of a stable vascular network that eventually anastomoses with the host vasculature to support the various biological functions of embedded cells. In recent years, significant progress has been achieved with respect to extrusion, laser, micro-molding, and electrospinning-based techniques that allow the fabrication of any geometry in a layer-by-layer fashion. Moreover, decellularized matrix, self-assembled structures, and cell sheets have been explored to replace the biopolymers needed for scaffold fabrication. While the techniques have evolved to create specific tissues or organs with outstanding geometric precision, formation of interconnected, functional, and perfused vascular networks remains a challenge. This article briefly reviews recent progress in 3D fabrication approaches used to fabricate vascular networks with incorporated cells, angiogenic factors, proteins, and/or peptides. The influence of the fabricated network on blood vessel formation, and the various features, merits, and shortcomings of the various fabrication techniques are discussed and summarized.

3D tumor model biofabrication

Animal models have been extensively used in cancer pathology studies and drug discovery.These models,however,fail to reflect the complex human tumor microenvironment and do not allow for high-throughput drug screening in more human-like physiological conditions.Three-dimensional(3D)cancer models present an alternative to automated high-throughput cancer drug discovery and oncology.In this review,we highlight recent technology innovations in building 3D tumor models that simulate the complex human tumor microenvironment and responses of patients to treatment.We discussed various biofabrication technologies,including 3D bioprinting techniques developed for characterizing tumor progression,metastasis,and response to treatment.