The Optimization of a Novel Hydrogel-Egg White-Alginate for 2.5D Tissue Engineering of Salivary Spheroid-Like Structure.

Hydrogels have been used for a variety of biomedical applications; in tissue engineering, they are commonly used as scaffolds to cultivate cells in a three-dimensional (3D) environment allowing the formation of organoids or cellular spheroids. Egg white-alginate (EWA) is a novel hydrogel which combines the advantages of both egg white and alginate; the egg white material provides extracellular matrix (ECM)-like proteins that can mimic the ECM microenvironment, while alginate can be tuned mechanically through its ionic crosslinking property to modify the scaffold's porosity, strength, and stiffness. In this study, a frozen calcium chloride (CaCl2) disk technique to homogenously crosslink alginate and egg white hydrogel is presented for 2.5D culture of human salivary cells. Different EWA formulations were prepared and biologically evaluated as a spheroid-like structure platform. Although all five EWA hydrogels showed biocompatibility, the EWA with 1.5% alginate presented the highest cell viability, while EWA with 3% alginate promoted the formation of larger size salivary spheroid-like structures. Our EWA hydrogel has the potential to be an alternative 3D culture scaffold that can be used for studies on drug-screening, cell migration, or as an in vitro disease model. In addition, EWA can be used as a potential source for cell transplantation (i.e., using this platform as an ex vivo environment for cell expansion). The low cost of producing EWA is an added advantage.

99m Tc-labeled rHuEpo for imaging of the erythropoietin receptor in tumors.

To analyze erythropoietin receptor (EpoR) status in tumors, recombinant human erythropoietin (rHuEpo) was labeled with 99m Tc by 99m Tc-centered 1-pot synthesis, resulting in high radiochemical purity, stability, and biological activity. Both in vitro cell culture experiments and biodistribution studies of normal rats demonstrated successful EpoR targeting. The biodistribution of labeled rHuEpo in a NCI-H1975 xenograft model showed tumor accumulation (tumor-to-muscle ratio, 4.27 ± 1.77), confirming the expression of active EpoR in tumors. Thus, as a novel single positron emission computerized tomography tracer for the imaging of EpoR expression in vivo, 99m Tc-rHuEpo is effective for exploring the role of EpoR in cancer growth, metastasis and angiogenesis.

Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization.

Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of "lipid-raft-mediated endocytosis→lysosome→cell nucleic localization" was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application.

Heat-Induced Radiolabeling of Nanoparticles for Monocyte Tracking by PET.

Heat-induced radiolabeling (HIR) yielded (89) Zr-Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes in vitro. Using an IV injection of HIR (89) Zr-FH (rather than in vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.

Internally bridged nanosilica for loadings and release of sparsely soluble compounds.

The engineering of a new monodisperse colloid with a sea urchin-like structure with a large complex internal structure is reported, in which silica surfaces are bridged by an aromatic organic cross-linker to serve as a nanocarrier host for drugs such as doxorubicin (DOX) against breast cancer cells. While dendritic fibrous nanosilica (DFNS) was employed and we do not observe a dendritic structure, these particles are referred to as sea urchin-like nanostructured silica (SNS). Since the structure of SNS consists of many silica fibrils protruding from the core, similar to the hairs of a sea urchin. For the aromatic structured cross-linker, bis(propyliminomethyl)benzene (b(PIM)B-S or silanated terephtaldehyde) were employed, which are prepared with terephtaldehyde and 3-aminopropyltriethoxy-silane (APTES) through a simple Schiff base reaction. b(PIM)B-S bridges were introduced into SNS under open vessel reflux conditions. SPS refers to the product obtained by incorporating the cross-linker b(PIM)B-S in ultra-small colloidal SNS particles. In-situ incorporation of DOX molecules resulted in SPS-DOX. The pH-responsive SPS nanocomposites were tested as biocompatible nanocarriers for controllable doxorubicin (DOX) delivery. We conclude that SPS is a unique colloid which has promising potential for technological applications such as advanced drug delivery systems, wastewater remediation and as a catalyst for green organic reactions in water.

Bioprinted Multicomponent Hydrogel Co-culture Tumor-Immune Model for Assessing and Simulating Tumor-Infiltrated Lymphocyte Migration and Functional Activation.

The immune response against a tumor is characterized by the interplay among components of the immune system and neoplastic cells. Here, we bioprinted a model with two distinct regions containing gastric cancer patient-derived organoids (PDOs) and tumor-infiltrated lymphocytes (TILs). The initial cellular distribution allows for the longitudinal study of TIL migratory patterns concurrently with multiplexed cytokine analysis. The chemical properties of the bioink were designed to present physical barriers that immune T-cells must breech during infiltration and migration toward a tumor with the use of an alginate, gelatin, and basal membrane mix. TIL activity, degranulation, and regulation of proteolytic activity reveal insights into the time-dependent biochemical dynamics. Regulation of the sFas and sFas-ligand present on PDOs and TILs, respectively, and the perforin and granzyme longitudinal secretion confirms TIL activation when encountering PDO formations. TIL migratory profiles were used to create a deterministic reaction-advection diffusion model. The simulation provides insights that decouple passive from active cell migration mechanisms. The mechanisms used by TILs and other adoptive cell therapeutics as they infiltrate the tumor barrier are poorly understood. This study presents a pre-screening strategy for immune cells where motility and activation across ECM environments are crucial indicators of cellular fitness.

Constructing 3D In Vitro Models of Heterocellular Solid Tumors and Stromal Tissues Using Extrusion-Based Bioprinting.

Malignant tumor tissues exhibit inter- and intratumoral heterogeneities, aberrant development, dynamic stromal composition, diverse tissue phenotypes, and cell populations growing within localized mechanical stresses in hypoxic conditions. Experimental tumor models employing engineered systems that isolate and study these complex variables using in vitro techniques are under development as complementary methods to preclinical in vivo models. Here, advances in extrusion bioprinting as an enabling technology to recreate the three-dimensional tumor milieu and its complex heterogeneous characteristics are reviewed. Extrusion bioprinting allows for the deposition of multiple materials, or selected cell types and concentrations, into models based upon physiological features of the tumor. This affords the creation of complex samples with representative extracellular or stromal compositions that replicate the biology of patient tissue. Biomaterial engineering of printable materials that replicate specific features of the tumor microenvironment offer experimental reproducibility, throughput, and physiological relevance compared to animal models. In this review, we describe the potential of extrusion-based bioprinting to recreate the tumor microenvironment within in vitro models.

Cisplatin-loaded porous Si microparticles capped by electroless deposition of platinum.

The loading and release of the anti-cancer drug platinum cis-dichlorodiamine (cisplatin) from mesoporous silicon (pSi) microparticles is studied. The pSi microparticles are modified with 1-dodecene or with 1,12-undecylenic acid by hydrosilylation, and each modified pSi material acts as a reducing agent, forming a deposit of Pt on its surface that nucleates further deposition, capping the mesoporous structure and trapping free (unreduced) cisplatin within. Slow oxidation and hydrolytic dissolution of the Si/SiO(2) matrix in buffer solution or in culture medium leads to the release of drugs from the microparticles. The drug-loaded particles show significantly greater toxicity toward human ovarian cancer cells (in vitro), relative to an equivalent quantity of free cisplatin. This result is consistent with the mechanism of drug release, which generates locally high concentrations of the drug in the vicinity of the degrading particles. Control assays with pSi particles loaded in a similar manner with the therapeutically inactive trans isomer of the platinum drug, and with pSi particles containing no drug, result in low cellular toxicity. A hydrophobic pro-drug, cis,trans,cis-[Pt(NH(3))(2)(O(2)C(CH(2))(8)CH(3))(2)Cl(2)], is loaded into the pSi films from chloroform without concomitant reduction of the pSi carrier.

Biotemplated Hollow Mesoporous Silica Particles as Efficient Carriers for Drug Delivery.

We designed three types of hollow-shaped porous silica materials via a three-step biotemplate-directed method: porous hollow silica nanorods, hollow dendritic fibrous nanostructured silica (DFNS), and ultraporous sponge-like DFNS. The first step was making a biotemplate, for which we used cellulose nanocrystals (CNCs), consisting of rod-shaped nanoparticles synthesized by conventional acid hydrolysis of cellulose fibers. In a second step, core-shell samples were prepared using CNC particles as hard template by two procedures. In the first one, core-shell CNC-silica nanoparticles were synthesized by a polycondensation reaction, which exclusively took place at the surface of the CNCs. In the second procedure, a typical synthesis of DFNS was conducted in a bicontinuous microemulsion with the assistance of additives. DFNS was assembled on the surface of the CNCs, giving rise to core-shell CNC-DFNS structures. Finally, all of the silica-coated CNC composites were calcined, during which the CNC was removed from the core and hollow structures were formed. These materials are very lightweight and highly porous. All three structures were tested as nanocarriers for drug delivery and absorbents for dye removal applications. Dye removal results showed that they can adsorb methylene blue efficiently, with ultraporous sponge-like DFNS showing the highest adsorption capacity, followed by hollow DFNS and hollow silica nanorods. Furthermore, breast cancer cells show a lower cell viability when exposed to doxorubicin-loaded hollow silica nanorods compared with control or doxorubicin cultures, suggesting that the loaded nanorod has a greater anticancer effect than free doxorubicin.

Alginate-gelatin-Matrigel hydrogels enable the development and multigenerational passaging of patient-derived 3D bioprinted cancer spheroid models.

Hydrogels consisting of controlled fractions of alginate, gelatin, and Matrigel enable the development of patient-derived bioprinted tissue models that support cancer spheroid growth and expansion. These engineered models can be dissociated to be then reintroduced to new hydrogel solutions and subsequently reprinted to generate multigenerational models. The process of harvesting cells from 3D bioprinted models is possible by chelating the ions that crosslink alginate, causing the gel to weaken. Inclusion of the gelatin and Matrigel fractions to the hydrogel increases the bioactivity by providing cell-matrix binding sites and promoting cross-talk between cancer cells and their microenvironment. Here we show that immortalized triple-negative breast cancer cells (MDA-MB-231) and patient-derived gastric adenocarcinoma cells can be reprinted for at least three 21 d culture cycles following bioprinting in the alginate/gelatin/Matrigel hydrogels. Our drug testing results suggest that our 3D bioprinted model can also be used to recapitulatein vivopatient drug response. Furthermore, our results show that iterative bioprinting techniques coupled with alginate biomaterials can be used to maintain and expand patient-derived cancer spheroid cultures for extended periods without compromising cell viability, altering division rates, or disrupting cancer spheroid formation.

Decellularized Extracellular Matrix Composite Hydrogel Bioinks for the Development of 3D Bioprinted Head and Neck in Vitro Tumor Models.

Reinforced extracellular matrix (ECM)-based hydrogels recapitulate several mechanical and biochemical features found in the tumor microenvironment (TME) in vivo. While these gels retain several critical structural and bioactive molecules that promote cell-matrix interactivity, their mechanical properties tend toward the viscous regime limiting their ability to retain ordered structural characteristics when considered as architectured scaffolds. To overcome this limitation characteristic of pure ECM hydrogels, we present a composite material containing alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, as rheological modifiers which impart mechanical integrity to the biologically active decellularized ECM (dECM). After an optimization process, the reinforced gel proposed is mechanically stable and bioprintable and has a stiffness within the expected physiological values. Our hydrogel's elastic modulus has no significant difference when compared to tumors induced in preclinical xenograft head and neck squamous cell carcinoma (HNSCC) mouse models. The bioprinted cell-laden model is highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks. Cells encapsulated in our bioink produce spheroids of at least 3000 µm2 of cross-sectional area by day 15 of culture and are positive for cytokeratin in immunofluorescence quantification, a common marker of HNSCC model validation in 2D and 3D models. We use this in vitro model system to evaluate the standard-of-care small molecule therapeutics used to treat HNSCC clinically and report a 4-fold increase in the IC50 of cisplatin and an 80-fold increase for 5-fluorouracil compared to monolayer cultures. Our work suggests that fabricating in vitro models using reinforced dECM provides a physiologically relevant system to evaluate malignant neoplastic phenomena in vitro due to the physical and biological features replicated from the source tissue microenvironment.

Triggered micropore-forming bioprinting of porous viscoelastic hydrogels.

Cell-laden scaffolds of architecture and mechanics that mimic those of the host tissues are important for a wide range of biomedical applications but remain challenging to bioprint. To address these challenges, we report a new method called triggered micropore-forming bioprinting. The approach can yield cell-laden scaffolds of defined architecture and interconnected pores over a range of sizes, encompassing that of many cell types. The viscoelasticity of the bioprinted scaffold can match that of biological tissues and be tuned independently of porosity and stiffness. The bioprinted scaffold also exhibits superior mechanical robustness despite high porosity. The bioprinting method and the resulting scaffolds support cell spreading, migration, and proliferation. The potential of the 3D bioprinting system is demonstrated for vocal fold tissue engineering and as an in vitro cancer model. Other possible applications are foreseen for tissue repair, regenerative medicine, organ-on-chip, drug screening, organ transplantation, and disease modeling.

Highly Absorbent Antibacterial and Biofilm-Disrupting Hydrogels from Cellulose for Wound Dressing Applications.

In this study, a carboxyl-modified cellulosic hydrogel was developed as the base material for wound dressings. ε-poly-l-lysine, a natural polyamide, was then covalently linked to the hydrogel through a bioconjugation reaction, which was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR). The antibacterial efficacy of the hydrogel was tested against two model bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, two of the most commonly found bacteria in wound infections. Bacterial viability and biofilm formation after exposure of bacteria to the hydrogels were used as efficacy indicators. Live/Dead assay was used to measure the number of compromised bacteria using a confocal laser scanning microscope. The results show that the antibacterial hydrogel was able to kill approximately 99% of the exposed bacteria after 3 h of exposure. In addition, NIH/3T3 fibroblasts were used to study the biocompatibility of the developed hydrogels. Water-soluble tetrazolium salt (WST)-1 assay was used to measure the metabolic activity of the cells and Live/Dead assay was used to measure the viability of the cells after 24, 48, and 72 h. The developed antibacterial hydrogels are light weight, have a high water-uptake capacity, and show high biocompatibility with the model mammalian cells, which make them a promising candidate to be used for wound dressing applications.

3D Cultures of Salivary Gland Cells in Native or Gelled Egg Yolk Plasma, Combined with Egg White and 3D-Printing of Gelled Egg Yolk Plasma.

For salivary gland (SG) tissue engineering, we cultured acinar NS-SV-AC cell line or primary SG fibroblasts for 14 days in avian egg yolk plasma (EYP). Media or egg white (EW) supplemented the cultures as they grew in 3D-Cryo histology well inserts. In the second half of this manuscript, we measured EYP's freeze-thaw gelation and freeze-thaw induced gelled EYP (GEYP), and designed and tested further GEYP tissue engineering applications. With a 3D-Cryo well insert, we tested GEYP as a structural support for 3D cell culture or as a bio-ink for 3D-Bioprinting fluorescent cells. In non-printed EYP + EW or GEYP + EW cultures, sagittal sections of the cultures showed cells remaining above the well's base. Ki-67 expression was lacking for fibroblasts, contrasting NS-SV-AC's constant expression. Rheological viscoelastic measurements of GEYP at 37 °C on seven different freezing periods showed constant increase from 0 in mean storage and loss moduli, to 320 Pa and 120 Pa, respectively, after 30 days. We successfully 3D-printed GEYP with controlled geometries. We manually extruded GEYP bio-ink with fluorescence cells into a 3D-Cryo well insert and showed cell positioning. The 3D-Cryo well inserts reveal information on cells in EYP and we demonstrated GEYP cell culture and 3D-printing applications.

The Applications of 3D Printing for Craniofacial Tissue Engineering.

Three-dimensional (3D) printing is an emerging technology in the field of dentistry. It uses a layer-by-layer manufacturing technique to create scaffolds that can be used for dental tissue engineering applications. While several 3D printing methodologies exist, such as selective laser sintering or fused deposition modeling, this paper will review the applications of 3D printing for craniofacial tissue engineering; in particular for the periodontal complex, dental pulp, alveolar bone, and cartilage. For the periodontal complex, a 3D printed scaffold was attempted to treat a periodontal defect; for dental pulp, hydrogels were created that can support an odontoblastic cell line; for bone and cartilage, a polycaprolactone scaffold with microspheres induced the formation of multiphase fibrocartilaginous tissues. While the current research highlights the development and potential of 3D printing, more research is required to fully understand this technology and for its incorporation into the dental field.

Engineering bioprintable alginate/gelatin composite hydrogels with tunable mechanical and cell adhesive properties to modulate tumor spheroid growth kinetics.

Tunable bioprinting materials are capable of creating a broad spectrum of physiological mimicking 3D models enabling in vitro studies that more accurately resemble in vivo conditions. Tailoring the material properties of the bioink such that it achieves both bioprintability and biomimicry remains a key challenge. Here we report the development of engineered composite hydrogels consisting of gelatin and alginate components. The composite gels are demonstrated as a cell-laden bioink to build 3D bioprinted in vitro breast tumor models. The initial mechanical characteristics of each composite hydrogel are correlated to cell proliferation rates and cell spheroid morphology spanning month long culture conditions. MDA-MB-231 breast cancer cells show gel formulation-dependency on the rates and frequency of self-assembly into multicellular tumor spheroids (MCTS). Hydrogel compositions comprised of decreasing alginate concentrations, and increasing gelatin concentrations, result in gels that are mechanically soft and contain a greater number of cell-adhesion moieties driving the development of large MCTS; conversely gels containing increasing alginate, and decreasing gelatin concentrations are mechanically stiffer, with fewer cell-adhesion moieties present in the composite gels yielding smaller and less viable MCTS. These composite hydrogels can be used in the biofabrication of tunable in vitro systems that mimic both the mechanical and biochemical properties of the native tumor stroma.

Bone extracts immunomodulate and enhance the regenerative performance of dicalcium phosphates bioceramics.

Immunomodulation strategies are believed to improve the integration and clinical performance of synthetic bone substitutes. One potential approach is the modification of biomaterial surface chemistry to mimic bone extracellular matrix (ECM). In this sense, we hypothesized that coating synthetic dicalcium phosphate (DCP) bioceramics with bone ECM proteins would modulate the host immune reactions and improve their regenerative performance. To test this, we evaluated the in vitro proteomic surface interactions and the in vivo performance of ECM-coated bioceramic scaffolds. Our results demonstrated that coating DCP scaffolds with bone extracts, specifically those containing calcium-binding proteins, dramatically modulated their interaction with plasma proteins in vitro, especially those relating to the innate immune response. In vivo, we observed an attenuated inflammatory response against the bioceramic scaffolds and enhanced peri-scaffold new bone formation supported by the increased osteoblastogenesis and reduced osteoclastogenesis. Furthermore, the bone extract rich in calcium-binding proteins can be 3D-printed to produce customized hydrogels with improved regeneration capabilities. In summary, bone extracts containing calcium-binding proteins can enhance the integration of synthetic biomaterials and improve their ability to regenerate bone probably by modulating the host immune reaction. This finding helps understand how bone allografts regenerate bone and opens the door for new advances in tissue engineering and bone regeneration. STATEMENT OF SIGNIFICANCE: Foreign-body reaction is an important determinant of in vivo biomaterial integration, as an undesired host immune response can compromise the performance of an implanted biomaterial. For this reason, applying immunomodulation strategies to enhance biomaterial engraftment is of great interest in the field of regenerative medicine. In this article, we illustrated that coating dicalcium phosphate bioceramic scaffolds with bone-ECM extracts, especially those rich in calcium-binding proteins, is a promising approach to improve their surface proteomic interactions and modulate the immune responses towards such biomaterials in a way that improves their bone regeneration performance. Collectively, the results of this study may provide a conceivable explanation for the mechanisms involved in presenting the excellent regenerative efficacy of natural bone grafts.

Circular dichroism study of the mechanism of formation of DNA templated nanowires.

In order to control the fabrication method, the mechanism used in the formation of DNA templated nanowires is investigated through circular dichroism (CD) spectroscopy. Metallic (Au) and magnetic (Fe(2)O(3) and CoFe(2)O(4)) nanoparticles (NP) are aligned along the DNA strand at various mass ratios. The DNA templated nanowires are compared to the structure of B-form dsDNA through CD experiments. Absorbance and thermal melting tests are performed to verify the structural changes of DNA templated nanowires. Low concentrations of nanoparticles preserve the DNA B-form through electrostatic interactions. Conversely, at higher concentrations of nanoparticles aligned along the DNA strand, the template is denatured. Information on the mode of nanoparticle binding and DNA helix alterations are explored for metallic and magnetic nanowires based upon the results.

Fabrication of ordered metallic and magnetic heterostructured DNA-Nanoparticle hybrids.

Here we provide a method based on enzymatically catalyzed reactions to cleave and ligate DNA molecules coated with nanoparticles to fabricate multi-component structures. This is done by simultaneously digesting two solutions of nanoparticle coated DNA, one with iron oxide particles the other gold particles, which yields short DNA fragments with complementary single stranded overhangs. When added together and re-attached using ligase enzymes multi-component nanoparticle coated structures are formed providing a novel method to fabricate complicated nanoparticle arrangements from the bottom up. We evaluated the fabrication by characterizing the samples with gel electrophoresis and magnetic force microscopy (MFM). The electrophoresis provides proof that the coated DNA molecules were digested with restriction enzymes and ligated by the T4 ligase enzymes. MFM experiments allow us to visualize the multi-component strands and analyze the magnetic versus metallic segments.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (> 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.