Abstract There is a need for new in vitro systems that enable pharmaceutical companies to collect more physiologically-relevant information on drug response in a low-cost and high-throughput manner. For this purpose, three-dimensional (3D) spheroidal models have been established as more effective than two-dimensional models. Current commercial techniques, however, rely heavily on self-aggregation of dissociated cells and are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of control over extracellular matrix components and heterogeneity in shape, size, and aggregate forming tendencies. In this study, we overcome these challenges by coupling tissue engineering toolsets with microfluidics technologies to create engineered cancer microspheres. Specifically, we employ biosynthetic hydrogels composed of conjugated poly(ethylene glycol) (PEG) and fibrinogen protein (PEG-Fb) to create engineered breast and colorectal cancer tissue microspheres for 3D culture, tumorigenic characterization, and examination of potential for high-throughput screening (HTS). MCF7 and MDA-MB-231 cell lines were used to create breast cancer microspheres and the HT29 cell line and cells from a stage II patient-derived xenograft (PDX) were encapsulated to produce colorectal cancer (CRC) microspheres. Using our previously developed microfluidic system, highly uniform cancer microspheres (intra-batch coefficient of variation (CV) ≤ 5%, inter-batch CV < 2%) with high cell densities (>20×106 cells/ml) were produced rapidly, which is critical for use in drug testing. Encapsulated cells maintained high viability and displayed cell type-specific differences in morphology, proliferation, metabolic activity, ultrastructure, and overall microsphere size distribution and bulk stiffness. For PDX CRC microspheres, the percentage of human (70%) and CRC (30%) cells was maintained over time and similar to the original PDX tumor, and the mechanical stiffness also exhibited a similar order of magnitude (103 Pa) to the original tumor. The cancer microsphere system was shown to be compatible with an automated liquid handling system for administration of drug compounds; MDA-MB-231 microspheres were distributed in 384 well plates and treated with staurosporine (1 μM) and doxorubicin (10 μM). Expected responses were quantified using CellTiter-Glo® 3D, demonstrating initial applicability to HTS drug discovery. PDX CRC microspheres were treated with Fluorouracil (5FU) (10 to 500 μM) and displayed a decreasing trend in metabolic activity with increasing drug concentration. Providing a more physiologically relevant tumor microenvironment in a high-throughput and low-cost manner, the PF hydrogel-based cancer microspheres could potentially improve the translational success of drug candidates by providing more accurate in vitro prediction of in vivo drug efficacy. Citation Format: Elizabeth A. Lipke, Wen J. Seeto, Yuan Tian, Mohammadjafar Hashemi, Iman Hassani, Benjamin Anbiah, Nicole L. Habbit, Michael W. Greene, Dmitriy Minond, Shantanu Pradhan. Production of cancer tissue-engineered microspheres for high-throughput screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 175.
more »
« less
Characterization and printability of Sodium alginate -Gelatin hydrogel for bioprinting NSCLC co-culture
3D bioprinting improves orientation of
- Award ID(s):
- 1735968
- NSF-PAR ID:
- 10153976
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bioprinting allows the deposition of cell-laden bioink, ensuring predefined scaffold architecture that may offer living tissue regeneration. With a combination of unique characteristics such as biocompatibility, less cell toxicity, and high water content, natural hydrogels are a great candidate for bioink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural integrity. To tackle this challenge, the rheological properties, specifically the shear thinning behavior (reduction of viscosity with increasing the applied load/shear rate on hydrogels) of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TO-NFC), carboxymethyl cellulose (CMC), and commonly used alginate, were explored. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TO-NFC, 1–4% of CMC, and 1–4% of alginate to analyze the shear thinning factors such as the values of n and K, which were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to tune shear thinning factors with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture.more » « less
-
more » « less
Abstract Among various available 3D bioprinting techniques, extrusion-based three-dimensional (3D) bio-printing allows the deposition of cell-laden bio-ink, ensuring predefined scaffold architecture that may offer living tissue regeneration. With a combination of unique characteristics such as biocompatibility, less cell toxicity, and high-water content, natural hydrogels are a great candidate for bio-ink formulation for the extrusion-based 3D bioprinting process. However, due to its low mechanical integrity, hydrogel faces a common challenge in maintaining structural ty. To tackle this challenge, we characterized the rheological properties of a set of hybrid hydrogels composed of cellulose-derived nanofiber (TEMPO-mediated nano-fibrillated cellulose, TONFC), carboxymethyl cellulose (CMC) and commonly used alginate. A total of 46 compositions were prepared using higher (0.5% and 1.0%) and lower percentages (0.005% and 0.01%) of TONFC, 1%–4% of CMC, and 1%–4% of alginate to analyze the rheological properties. The shear thinning coefficients of n and K were determined for each composition from the flow diagram and co-related with the 3D printability. The ability to control rheological properties with various ratios of a nanofiber can help achieve a 3D bio-printed scaffold with defined scaffold architecture.
-
more » « less
Abstract Fibroblasts are an abundant cell type in tumor microenvironments. Activated fibroblasts, known as carcinoma‐associated fibroblasts (CAFs), interact with cancer cells through biochemical signaling and render cancer cells proliferative, invasive, and resistant to therapeutics. Targeting CAFs–cancer cells interactions offers a strategy to block cancer progression. 2D and 3D co‐cultures of human mammary fibroblasts and triple negative breast cancer (TNBC) cells are used to investigate the impact of heterotypic cellular interactions on the proliferation of matrix invasion of TNBC cells. The results show that fibroblasts secreting a chemokine, CXCL12, significantly enhance proliferation of TNBC cells expressing the chemokine receptor, CXCR4. Disrupting this interaction with a receptor antagonist normalizes cancer cell proliferation to that of a co‐culture model lacking this signaling. When co‐culture spheroids are embedded in collagen, fibroblasts producing CXCL12 promote collagen invasion of TNBC cells. Although co‐cultures containing normal fibroblasts also lead to TNBC cell spreading into the matrix, a morphological analysis of cells and inhibition of chemokine‐receptor signaling shows that this spreading is due to the incompatibility of fibroblasts and cancer cells leading to the segregation of the two cell types from the spheroid.
-
Abstract Three-dimensional (3D) bioprinting has emerged as a powerful engineering approach for various tissue engineering applications, particularly for the development of 3D cellular structures with unique mechanical and/or biological properties. For the jammed gelatin microgel-gelatin solution composite bioink, comprising a discrete phase of microgels (enzymatically gelled gelatin microgels) and a cross-linkable continuous gelatin precursor solution-based phase containing transglutaminase (TG), its rheological properties and printability change gradually due to the TG enzyme-induced cross-linking process. The objective of this study is to establish a direct mapping between the printability of the gelatin microgel-gelatin solution based cross-linkable composite bioink and the TG concentration and cross-linking time, respectively. Due to the inclusion of TG in the composite bioink, the bioink starts cross-linking once prepared and is usually prepared right before a printing process. Herein, the bioink printability is evaluated based on the three metrics: injectability, feature formability, and process-induced cell injury. In this study, the rheological properties such as the storage modulus and viscosity have been first systematically investigated and predicted at different TG concentrations and times during the cross-linking process using the first-order cross-linking kinetics model. The storage modulus and viscosity have been satisfactorily modeled as exponential functions of the TG concentration and time with an experimentally calibrated cross-linking kinetic rate constant. Furthermore, the injectability, feature formability, and process-induced cell injury have been successfully correlated to the TG concentration and cross-linking time via the storage modulus, viscosity, and/or process-induced shear stress. By combing the good injectability, good feature formability, and satisfactory cell viability zones, a good printability zone (1.65, 0.61, and 0.31 h for the composite bioinks with 1.00, 2.00, and 4.00% w/v TG, respectively) has been established during the printing of mouse fibroblast-based 2% gelatin B microgel-3% gelatin B solution composite bioink. This printability zone approach can be extended to the use of other cross-linkable bioinks for bioprinting applications.more » « less
