Rapid spheroid assays in a 3-dimensional cell culture chip
Jia Lin TehSiti Fairus Abdul RahmanGregory DomnicLengishwarra SatiyasilanNelson Jeng‐Yeou ChearDarshan SinghNethia Mohana‐Kumaran
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Abstract Objective The spheroid model provides a physiological platform to study cancer cell biology and drug sensitivity. Usage of bovine collagen I for spheroid assays is costly especially when experiments are conducted in 24-well plates, as high volume of bovine collagen I is needed. The aim of the study was to downsize spheroid assays to a microfluidic 3D cell culture chip and compare the growth, invasion and response to drug/compound of spheroids embedded in the 3D chip to spheroids embedded in 24-well plates. Results Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.Keywords:
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Abstract It is known that cells grown in 3D are more tolerant to drug treatment than those grown in dispersion, but the mechanism for this is still not clear; cells grown in 3D have opportunities to develop inter-cell communication, but are also closely packed which may impede diffusion. In this study we examine methods for dielectrophoresis-based cell aggregation of both suspension and adherent cell lines, and compare the effect of various drugs on cells grown in 3D and 2D. Comparing viability of pharmacological interventions on 3D cell clusters against both suspension cells and adherent cells grown in monolayer, as well as against a unicellular organism with no propensity for intracellular communication, we suggest that 3D aggregates of adherent cells, compared to suspension cells, show a substantially different drug response to cells grown in monolayer, which increases as the IC 50 is approached. Further, a mathematical model of the system for each agent demonstrates that changes to drug response are due to inherent changes in the system of adherent cells from the 2D to 3D state. Finally, differences in the electrophysiological membrane properties of the adherent cell type suggest this parameter plays an important role in the differences found in the 3D drug response.
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A three-dimensional (3D) cell culture system has been fabricated using a magnetic force based cell patterning method, demonstrating a facile approach for the analysis of invasive capacity of BALB/3T3/v-src using an magnetic force and magnetite nanoparticles. The 3D cell patterning was performed using an external magnetic force and a pin holder, which enables the assembly of the magnetically labeled cells on the collagen gel-coated surface as array-like cell patterns, resulting in the development of a 3D in vitro culture model. The cells embedded in type I collagen showed a compacted, spheroid like configuration at each spot, and distinct, accelerated cell growth was observed in cancer model cells compared with the control cells. The developed 3D cell culture array was applied to the susceptibility assay of the GM6001 matrix metalloproteinase (MMP) inhibitor, a collagenase inhibitor; a distinct suppression of cell proliferation was observed, while little change was observed in 2D. The developed 3D cell culture array system is useful to assess the effects of pharmacologic and/or microenvironmental influences on tumor cell invasion.
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Cell micropatterning, a method to place cells at arbitrary regions, is becoming an essential tool to conduct cell biology and tissue engineering. Conventional cell patterning techniques usually allow only single patterning with single cell type on the same culture surface. However, biomedical research today requires even sophisticated fabrication methods that require spatiotemporal control of multiple cell arrangements. Here we introduce in situ cell micropatterning system which enables stepwise cell patterning using a photoresponsive cell culture surface (PRCS) whose cell adhesiveness could be altered by the UV irradiation. To demonstrate an application to tissue engineering, a liver-mimic tissue array was fabricated and liver-specific gene expressions were quantified with real time PCR. Patterned co-culture systems composed of HepG2 spheroids with Balb/3T3 were fabricated, and the optimum spheroid diameter, which yielded the highest cellular functions, was determined to be 150 microm. After 20 days of patterned co-culture of HepG2 spheroids and Balb/3T3, CYP3A4 expression increased 50-fold higher than conventionally cultured HepG2; CYP3A4 expression was 20% higher than randomly co-cultured HepG2 and Balb/3T3. Thus the combination of PRCS and the photomask-free irradiation apparatus showed the versatility of experimental setups and proved to be a powerful tool for biomedical studies.
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Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.
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Abstract Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures. Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiterTM-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent. Results: In 3D spheroids/aggregates, HER2 IHC staining and GPA assay showed for SK-BR-3 and BT-474 HER2 3+ expression and HER2/CEP17 of ≥ 2; MDA-MB-361 cells with HER2 2+ expression and HER2/CEP17 of ≥ 2.0; MDA-MB-175 cells with HER2 1+ expression and HER2/CEP17 < 2.0 and MCF-7 cells with HER2 0+(IHC staining only without HER2 GPA data). Some of the 3D spheroids/aggregates in MDA-MB-361 cells showed heterogeneous expression of HER2 protein. The IC50 values of 3D spheroids/aggregates for some cell lines were significantly higher than were demonstrated for cell lines grown in 2D cell cultures. The fold changes between 3D spheroids and 2D cells (72h T-DM1 treatment time) are: 4.2 for SK-BR-3; ≳ 10 for BT-474 and 22 for MDA-MB-361. Additionally, the fluorescent images showed that a longer incubation time was required for the T-DM1 drug (3 µg/ml) to be internalized effectively into BT-474 3D spheroids; for example, about 120h for 3D spheroids in comparison to about 36h in 2D cells. Interestingly, the 3D spheroids incubated for 120h with T-DM1 (470 µm) are smaller in size than 3D spheroids in the control group (600 µm) incubated for 120h without T-DM1 treatment. Conclusions: Drug efficacy studies performed on 3D cultured spheroids/aggregates are expected to be very important and biologically relevant for determining drug activity in tumor tissue. Our drug efficacy study using 3D cell culture demonstrated greater concentrations of T-DM1 and longer incubation times were required than for cells grown as 2D in some cell lines, likely due to less efficient internalization. Citation Format: Jean Z. Boyer, Gail Lewis Phillips, Hiro Nitta, Karl Garsha, Eric May, Brittany Admire, Robert Kraft, Megan Peccarelli, Andre Zamorano, Scott Gill, Eslie Dennis, Liz Vela, Penny Towne. Activity of trastuzumab emtansine (T-DM1) in 3D cell culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 29.
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Cell culture is an important life science technology. Compared with the traditional two-dimensional cell culture, three-dimensional cell culture can simulate the natural environment and structure specificity of cell growth in vivo. As such, it has become a research hotspot. The existing three-dimensional cell culture techniques include the hanging drop method, spinner flask method, etc., making it difficult to ensure uniform morphology of the obtained cell spheroids while performing high-throughput. Here, we report a method for amplifying cell spheroids with the advantages of quickly enlarging the culture scale and obtaining cell spheroids with uniform morphology and a survival rate of over 95%. Technically, it is easy to operate and convenient to change substances. These results indicate that this method has the potential to become a promising approach for cell-cell, cell-stroma, cell-organ mutual interaction research, tissue engineering, and anti-cancer drug screening.
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Traditional studies using cancer cell lines are often performed on a two-dimensional (2D) cell culture model with a low success rate of translating to Phase I or Phase II clinical studies. In comparison, with the advent of developments three-dimensional (3D) cell culture has been championed as the latest cellular model system that better mimics in vivo conditions and pathological conditions such as cancer. In comparison to biospecimens taken from in vivo tissue, the details of gene expression of 3D culture models are largely undefined, especially in mesothelioma - an aggressive cancer with very limited effective treatment options. In this study, we examined the veracity of the 3D mesothelioma cell culture model to study cell-to-cell interaction, gene expression and drug response from 3D cell culture, and compared them to 2D cell and tumor samples. We confirmed via SEM analysis that 3D cells grown using the spheroid methods expressed highly interconnected cell-to-cell junctions. The 3D spheroids were revealed to be an improved mini-tumor model as indicated by the TEM visualization of cell junctions and microvilli, features not seen in the 2D models. Growing 3D cell models using decellularized lung scaffold provided a platform for cell growth and infiltration for all cell types including primary cell lines. The most time-effective method was growing cells in spheroids using low-adhesive U-bottom plates. However, not every cell type grew into a 3D model using the the other methods of hanging drop or poly-HEMA. Cells grown in 3D showed more resistance to chemotherapeutic drugs, exhibiting reduced apoptosis. 3D cells stained with H&E showed cell-to-cell interactions and internal architecture that better represent that of in vivo patient tumors when compared to 2D cells. IHC staining revealed increased protein expression in 3D spheroids compared to 2D culture. Lastly, cells grown in 3D showed very different microRNA expression when compared to that of 2D counterparts. In conclusion, 3D cell models, regardless of which method is used. Showed a more realistic tumor microenvironment for architecture, gene expression and drug response, when compared to 2D cell models, and thus are superior preclinical cancer models.
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