3d-printing-vitro-hydrogel-microcarriers-alternating-viscous-inertial

3D Printing of <em>In Vitro</em> Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Tsinghua University

Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing

A new three-dimensional (3D) cell printing system was developed and investigated to organize multiple cells/biomaterials with a control precision within 100 μm. This system can be used for the in vitro construction of heterogeneous tissue models. The proposed printing system was achieved by the integration of extrusion printing and alternating viscous and inertial force jetting (AVIFJ) techniques using dual-nozzle switching. In this technique, hydrogels containing high cell densities were extruded using extrusion printing, while droplets containing single cells were precisely manipulated using AVIFJ. The droplets that contained single cells were at the scale of pico-liters and could be accurately positioned at the micron scale. Stable hydrogel structures with adjustable diameters were also printed, with cell viabilities exceeding 90% after printing. A heterogeneous tumor model that contained spheroids and human umbilical vein endothelial cells (HUVECs) was then constructed using the established integrated cell printing system in a stepwise or simultaneous fashion. HUVEC-loaded droplets were observed to locate around the preformed tumor spheroids as designed. Cells and spheroids in the model maintained high cell viability and sustained growth throughout the culture period. The ELISA results of albumin production also proved that the spheroids maintained increased cellular function during the culture. These results demonstrated the feasibility of this integrated 3D printing system for the engineering of in vitro heterogeneous tissue models for future biological and pathological studies. STATEMENT OF SIGNIFICANCE: Addressing the challenge of multi-scale printing in the construction of heterogeneous tissue models, a new 3D cell printing system was developed to organize cells/biomaterials of a control precision within 100 μm. AVIFJ was integrated with extrusion printing, thereby achieving the construction of cell interactions between single cells and spheroids, the manipulation of single cells in a 3D microenvironment with high accuracy, and the real-time on-demand printing. The printed heterogeneous tumor model maintained cell viability, sustained cell growth, and increased cell function during 7 days of culture. We believed that this work would benefit the production of functional artificial tissues, enabling the construction of more biomimetic cell arrangements and microenvironment to support cell functions.

An integrated cell printing system for the construction of heterogeneous tissue models.

Constructing in vitro drug models with a higher degree of bionics is considered to be an important approach to improve the effectiveness of existing drug screening models. 3D printing, co-culture and microfluidics are important methods that commonly used. This study combined the advantages of the three methods. A 3D co-culture microfluidic model with controllable hepatoma cluster size was constructed, and applied to pharmacodynamic tests of a new anti-CD147 monoclonal antibody, Metuzumab. In the phase of cell preparation, hepatoma cells in the new model were found to proliferate faster than common in vitro 3D models fabricated by cell printing only. The hepatoma cells were also found less affected by the increase of drug dosage in migration performance, proliferation performance and protein expression level in the new model compared with 2D models. These results are in line with those obtained from animal experiments and clinical trials of similar anti-CD147 antibody drugs. In view of the special mechanism of antibody drugs, a modified model was built by adding peripheral blood mononuclear cells into the new model as effector cells, porting traditional antibody-dependent cell-mediated cytotoxicity (ADCC) test to a co-culture 3D microfluidic microenvironment. The results showed that ADCC exhibited higher effectiveness in the microfluidic model than in 3D printing models under the same dose of drug treatment. The new model was proved constructive and it provided a valuable reference for complex in vitro hepatoma model studies and antibody drug screening researches.

3D bioprinting of hepatoma cells and application with microfluidics for pharmacodynamic test of Metuzumab.

Resolving the origin and development of tumor heterogeneity has proven to be a crucial challenge in cancer research. In vitro tumor models have been widely used for both scientific and clinical research. Currently, tumor models based on 2D cell culture, animal models, and 3D cell-laden constructs are widely used. Heterogeneous tumor models, which consist of more than one cell type and mimic cell-cell as well as cell-matrix interactions, are attracting increasing attention. Heterogeneous tumor models can serve as pathological models to study the microenvironment and tumor development such as tumorigenesis, invasiveness, and malignancy. They also provide disease models for drug screening and personalized therapy. In this review, the current techniques, models, and oncological applications regarding 3D heterogeneous tumor models are summarized and discussed.

Review on biofabrication and applications of heterogeneous tumor models.

In order to address the remaining issues of fragile structure and insufficient mass transfer faced in modular assembly-based liver tissue engineering, a Raschig ring-like hollowed micro-scaffold was proposed and fabricated using poly-ε-caprolactone with 60% porosity and 11.4 mm effective surface area for cell immobilization. The method of cell inoculation, the types of cells for co-culture and the scalability of the proposed hollowed micro-scaffold in perfusion were all investigated to obtain an optimized organoid made of tissue modules. Extracellular matrix was found necessary to establish a hierarchical co-culture, and the triple co-culture of Human Hepatoma Hep G2 cells, liver sinusoid cell line TMNK-1 cells and fibroblasts (Swiss 3T3 cells) was recognized to be the most efficient to obtain higher cell attachment, proliferation and hepatic function. The equipped intersecting hollow channels provided in the micro-scaffold functioned as flow paths to promote mass transfer to the immobilized cells after the modules have been randomly packed into a bioreactor for perfusion culture, and resulted in enhanced albumin production and high cellular viability. Cell density comparable to those found in vivo were obtained in the perfused construct, which also maintained its rigid structure. Those results suggest that modular tissues made with hollowed micro-scaffold-based organoids hold great potential for scaling up tissue engineered constructs towards implantation.

Organization of liver organoids using Raschig ring-like micro-scaffolds and triple co-culture: Toward modular assembly-based scalable liver tissue engineering.

Cervical cancer induced by human papillomavirus (HPV) causes severe morbidity worldwide. Although cervical conization has been widely accepted as the most conventional surgery against cervical cancer, tissue defects and high recurrence rates have a significant negative impact on women's mental and physical health. Herein we developed an implantable, personalized cervical implant with drug release function using 3D printing technology. The cervical implant was designed in cone-shape with hieratical porous structures according to the clinical data, 3D-printed using polyurethane by low-temperature deposition manufacturing (LDM), and finished by lyophilization. Anti-HPV protein was loaded into the porous structure under negative pressure afterwards. Elastic biomedical polyurethane and the porous structure ensured that these cervical implants were equipped with tailored mechanical properties comparable to physiological cervix tissue. Cytotoxicity and cytocompatibility tests indicated that these 3D-printed cervical implants supported cell adhesion and growth. More importantly, the cervical implants with regulated pores could help to quantitatively control the loading and release of anti-HPV protein to inhibit dissociative viruses near the cervix validly. As a result, the 3D-printed cervical implants in the present study showed considerable potential for use as functional tissue implants against HPV infection after cervical conization.

Design, modeling and 3D printing of a personalized cervix tissue implant with protein release function.

Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo.

Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure.

Studying biological characteristics of tumors and evaluating the treatment effects require appropriate in vitro tumor models. However, the occurrence, progression, and migration of tumors involve spatiotemporal changes, cell-microenvironment and cell-cell interactions, and signal transmission in cells, which makes the construction of in vitro tumor models extremely challenging. In the past few years, advances in biomaterials and tissue engineering methods, especially development of the bioprinting technology, have paved the way for innovative platform technologies for in vitro cancer research. Bioprinting can accurately control the distribution of cells, active molecules, and biomaterials. Furthermore, this technology recapitulates the key characteristics of the tumor microenvironment and constructs in vitro tumor models with bionic structures and physiological systems. These models can be used as robust platforms to study tumor initiation, interaction with the microenvironment, angiogenesis, motility and invasion, as well as intra- and extravasation. Bioprinted tumor models can also be used for high-throughput drug screening and validation and provide the possibility for personalized cancer treatment research. This review describes the basic characteristics of the tumor and its microenvironment and focuses on the importance and relevance of bioprinting technology in the construction of tumor models. Research progress in the bioprinting of monocellular, multicellular, and personalized tumor models is discussed, and comprehensive application of bioprinting in preclinical drug screening and innovative therapy is reviewed. Finally, we offer our perspective on the shortcomings of the existing models and explore new technologies to outline the direction of future development and application prospects of next-generation tumor models.

Bioprinting of in vitro tumor models for personalized cancer treatment: a review.

The existing  models for antitumor drug screening have great limitations. Many compounds that inhibit 2D cultured cells do not exhibit the same pharmacological effects , thereby wasting human and material resources as well as time during drug development. Therefore, developing new models is critical. The 3D bioprinting technology has greater advantages in constructing human tissue compared with sandwich culture and organoid construction. Here, we used 3D bioprinting technology to construct a 3D model with HepG2 cells (3DP-HepG2). The biological activities of the model were evaluated by immunofluorescence, real-time quantitative PCR, and transcriptome sequencing. Compared with the traditional 2D cultured tumor cells (2D-HepG2), 3DP-HepG2 showed significantly improved expression of tumor-related genes, including ALB, AFP, CD133, IL-8, EpCAM, CD24, and β-TGF genes. Transcriptome sequencing analysis revealed large differences in gene expression between 3DP-HepG2 and 2D-HepG2, especially genes related to hepatocyte function and tumor. We also compared the effects of antitumor drugs in 3DP-HepG2 and 2D-HepG2, and found that the large differences in drug resistance genes between the models may cause differences in the drugs' pharmacodynamics.

Application of a 3D Bioprinted Hepatocellular Carcinoma Cell Model in Antitumor Drug Research.

Towards the development of in vivo-mimicking tumor model for extensive study of tumorigenesis and establishment of personalized therapy, patient-derived primary tumor cells were employed in this work for three-dimensional (3D) bioprinting. Intrahepatic cholangiocarcinoma cells isolated from patient were bioprinted using a composite hydrogel system of gelatin-alginate-Matrigel into pre-designed grid architecture. ICC cells were observed to process a colony forming ability with high survival rate and active proliferation. Expression levels of tumor markers, cancer stem cell markers, matrix metalloproteinase protein, index of tumor fibrosis, index of liver function, and epithelial-mesenchymal transition regulatory proteins confirmed the development of the invasive and metastatic phenotype of the intrahepatic cholangiocarcinoma cells in the 3D printed tumor microenvironment. Similar results were obtained in anti-cancer drug resistance of the intrahepatic cholangiocarcinoma cells in the 3D bioprinted construct that demonstrated stem-like properties, which suggested the promising potential of current 3D printed tumor model in the development of personalized therapy, especially for discovery of more conducive targeted drugs.

Bioprinting of patient-derived in vitro intrahepatic cholangiocarcinoma tumor model: establishment, evaluation and anti-cancer drug testing.

Hepatocellular carcinoma (HCC) is one of the most lethal tumors worldwide. This study aims to address the lack of faithful and available in vitro models for patient-specific drug screening for HCC. We recently established a novel modeling system using three-dimensional (3D) bioprinting technology and constructed hepatorganoids with HepaRG cells, which retain the liver function and prolong the survival of mice with liver failure after abdominal transplantation. Here we extend this modeling system to establish individualized model for hepatocellular carcinoma. HCC specimens were obtained from six patients after surgery. Primary HCC cells were isolated and mixed with gelatin and sodium alginate to form the bioink for printing. Patient-derived three-dimensional bio-printed HCC (3DP-HCC) models were successfully established afterward and grew well during long-term culture. These models retained the features of parental HCCs, including stable expression of the biomarker, stable maintenances of the genetic alterations and expression profiles. 3DP-HCC models are capable of displaying the results of drug screening intuitively and quantitatively. In conclusion, 3DP-HCC models are faithful in vitro models that are reliable in long-term culture and able to predict patient-specific drugs for personalized treatment.

Three-dimensional bio-printing of primary human hepatocellular carcinoma for personalized medicine.

As a precursor of graphene, graphene oxide (GO) exhibits excellent mechanical, thermal, and electrical properties, besides appreciable biocompatibility in tissue engineering applications. However, the current GO-3D fabrication technology is still in need of optimization and simplification to ensure fine architecture and reasonable mechanical properties, which would further promote the performance of GO as bio-scaffolds in cell or microorganism attachment and in material transformation. To address this issue, we proposed a GO ink, with appreciable rheological properties and excellent printing performance via high-speed centrifugation and ferric ion-assisted cross-linking. A woodpile structure with controllable micro-pores was produced by micro-extrusion-based 3D printing technology followed by an optimized freeze-drying process. Cellular adhesion and viability were verified by inoculation and culture of HepaRG cells using the fabricated GO 3D structure, thus suggesting ferric ion-assisted cross-linking and controllable pore distribution for improving the performance of the GO construct as a bio-scaffold for in vitro liver tissue models.

Ferric ion crosslinking-based 3D printing of a graphene oxide hydrogel and its evaluation as a bio-scaffold in tissue engineering.

In liver tissue engineering, co-culturing hepatocytes with typical non-parenchymal hepatic cells to form cell aggregates is available to mimic the  microenvironment and promote cell biological functions. With a modular assembly approach, endothelialized hepatic cell aggregates can be packed for perfusion culture, which enables the construction of large-scale liver tissues. Since tightly packed aggregates tend to fuse with each other and block perfusion flows, a loosely packed mode was introduced in our study.

Modular assembly-based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering.

To address the remaining issue of poor cell immobilization and insufficient mass transfer in scaffold-based tissue engineering approach for future islet transplantation, we employed a macro-porous poly-l-lactide (PLLA) scaffold immobilizing mouse insulinoma cells and studied its function toward an implantable pancreatic tissue in 7-day perfusion culture. The murine pancreatic β cells could be immobilized in the PLLA scaffold at a high density of 10 cells per cm close to the estimated range in normal pancreas. The perfusion culture promoted the 3D cellular organization as observed with live/dead staining and histological staining. The insulin production was significantly enhanced in comparison with static 2D culture and 3D rotational suspension culture by two and six folds, respectively ( < 0.001). As enhanced insulin response was only observed where both the perfusion and 3D cellular organization were present, this could represent important elements in engineering a functional bioartificial pancreas.

3D perfusion culture of mouse insulinoma in macro-porous scaffolds enhanced insulin production response.

Bioelectrochemical systems (BESs) can fulfill the demand for renewable energy and wastewater treatment but still face significant challenges to improve their overall performance. Core efforts have been made to enhance the bioelectrode performance, yet, previous approaches are fragmented and have limited applicability, unable to flexibly adjust physicochemical and structural properties of electrodes for specific requirements in various applications. Here, we propose a facile electrode design strategy that integrates three-dimensional printing technology and functionalized modular electrode materials. A customized graphene-based electrode with hierarchical pores and functionalized components (i.e., ferric ions and magnetite nanoparticles) was fabricated. Owing to efficient mass and electron transfer, a high volumetric current density of 10,608 ± 1,036 A/m was achieved, the highest volumetric current density with pure  to date. This strategy can be readily applied to existing BESs (e.g., microbial fuel cells and microbial electrosynthesis) and provide a feasibility for practical application.

Article	Year
An integrated cell printing system for the construction of heterogeneous tissue models. Acta biomaterialia\| PubMed ID: 31128321	2019
3D bioprinting of hepatoma cells and application with microfluidics for pharmacodynamic test of Metuzumab. Biofabrication\| PubMed ID: 31141796	2019
Review on biofabrication and applications of heterogeneous tumor models. Journal of tissue engineering and regenerative medicine\| PubMed ID: 31359625	2019
Organization of liver organoids using Raschig ring-like micro-scaffolds and triple co-culture: Toward modular assembly-based scalable liver tissue engineering. Medical engineering & physics\| PubMed ID: 31883633	2020
Design, modeling and 3D printing of a personalized cervix tissue implant with protein release function. Biomedical materials (Bristol, England)\| PubMed ID: 32109897	2020
Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure. Gut\| PubMed ID: 32434830	2021
Bioprinting of in vitro tumor models for personalized cancer treatment: a review. Biofabrication\| PubMed ID: 32470967	2020
Application of a 3D Bioprinted Hepatocellular Carcinoma Cell Model in Antitumor Drug Research. Frontiers in oncology\| PubMed ID: 32582546	2020
Bioprinting of patient-derived in vitro intrahepatic cholangiocarcinoma tumor model: establishment, evaluation and anti-cancer drug testing. Biofabrication\| PubMed ID: 32599574	2020
Three-dimensional bio-printing of primary human hepatocellular carcinoma for personalized medicine. Biomaterials\| PubMed ID: 33007612	2021
Ferric ion crosslinking-based 3D printing of a graphene oxide hydrogel and its evaluation as a bio-scaffold in tissue engineering. Biotechnology and bioengineering\| PubMed ID: 33022744	2021
Modular assembly-based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering. Annals of translational medicine\| PubMed ID: 33313145	2020
3D perfusion culture of mouse insulinoma in macro-porous scaffolds enhanced insulin production response. The International journal of artificial organs\| PubMed ID: 33380250	2020
Customizable design strategies for high-performance bioanodes in bioelectrochemical systems. iScience\| PubMed ID: 33665579	2021

Yuan Pang

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