Alginate hydrogel based 3-dimensional cell culture and chemical screening platform using digital microfluidics

To meet the increasing demand of efficient cooling performance in small scale, this paper presents a digital microfluidics (DMF) microscale liquid cooling system which works on the principle of electrowetting on dielectric (EWOD). In EWOD DMF, fluids are handled drop-wise by external electric field. When the dispensed liquid droplet arrives at the hotspot by EWOD DMF operation, it picks up heat and removes heat when it leaves. This process can be repeated for a series of droplets by using a completely automated LabVIEW controlled system connected to the PCB package. With the help of indium tin oxide (ITO) thin film resistance temperature detectors (RTD) and pre-calibrated temperature coefficient of resistance (TCR) data, the temperatures of the hotspot before and after the residence of liquid droplet (i.e., cooling) can be recorded for different frequencies (dwelling time period of droplet on the hotspot) of the drop motion and varying heater power. Future work will involve RTD resistance data collection to plot the heat flux and the temperature difference (before and after cooling) for different frequencies of drop motion. Although the primary focus is to study single phase cooling, the DI water drop will experience considerable evaporation resulting in higher cooling performance. The single phase cooling studies will help in establishing a robust platform for future two-phase cooling analysis in which evaporation effects will be considered.

The healing process of diabetic foot ulcers (DFUs) presents a slow pattern with an increased risk of infections, ischemia, and thrombosis correlated with high levels of reactive oxygen species production. Vascular injury is one of the factors contributing to the difficulty of wound healing in diabetic patients. Although the understanding of the pathophysiology of DFUs has significantly increased in recent years, associated treatments still have a high level of failure, leading to high morbidity rates, mortality, and amputations. Three-dimensional (3D) cell culture platforms offer a new approach to investigating and treating these wounds, as they can reproduce one or more physiological systems in a relevant microenvironment. This systematic review describes the advancements, challenges, and future implications of advanced 3D culture models in vascularization, encompassing pathophysiological understanding, treatment, and prognostic perspectives. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This work was registered under PROSPERO protocol number CRD42022336473. The eligibility criteria addressed studies related to chronic DFUs that analyzed vascularization during the healing process. The selected study designs involved 3D cultures and organ-on-a- chip (OoC) platforms, utilizing either primary or secondary human cell lines. Studies published more than 10 years ago or using only animal cells in 2D culture environments were excluded. The search was conducted in PubMed, LILACS, Embase, MEDLINE, IEEE, the BSV regional portal, ScienceDirect, Scopus, CINAHL, EBSCO, and Web of Science. A total of 2,539 relevant studies were identified as of June 1, 2025. After screening, only seven met the inclusion criteria. These studies reflect a growing interest in using hydrogel scaffolds and microfluidic systems to replicate diabetic skin environments; however, the field remains in an early stage of development. OoC platforms, in particular, stand out for their ability to recreate dynamic, tissue-like conditions and vascular function. While some promising attempts have been made to combine hydrogels with these technologies, the evidence is still limited and inconclusive for chronic wound modeling. This review underscores both the potential and the urgent need for more robust and translational research in this area—especially toward building personalized, clinically relevant models to support future drug testing and therapeutic innovation.

The demands of tissue engineering have driven a tremendous amount of research effort in 3D tissue culture technology and, more recently, in 3D printing. The need to use 3D tissue culture techniques more broadly in all of cell biology is well-recognized, but the transition to 3D has been impeded by the convenience, effectiveness, and ubiquity of 2D culture materials, assays, and protocols, as well as the lack of 3D counterparts of these tools. Interestingly, progress and discoveries in 3D bioprinting research may provide the technical support needed to grow the practice of 3D culture. Here we investigate an integrated approach for 3D printing multicellular structures while using the same platform for 3D cell culture, experimentation, and assay development. We employ a liquid-like solid (LLS) material made from packed granular-scale microgels, which locally and temporarily fluidizes under the focused application of stress and spontaneously solidifies after the applied stress is removed. These rheological properties enable 3D printing of multicellular structures as well as the growth and expansion of cellular structures or dispersed cells. The transport properties of LLS allow molecular diffusion for the delivery of nutrients or small molecules for fluorescence-based assays. Here, we measure viability of 11 different cell types in the LLS medium, we 3D print numerous structures using several of these cell types, and we explore the transport properties in molecular time-release assays.

IntroductionCholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to in vivo tumor. MethodsHere, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Results and discussionFirstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. ConclusionsThus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes in vivo.

BackgroundThree-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers.ResultsSeven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen.ConclusionHere we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

Liquid-liquid extraction techniques are one of the major tools in chemical engineering, analytical chemistry, and biology, especially in a system where two immiscible liquids have an interface solutes exchange between the two liquid phases along the interface up to a point where the concentration ratios in the two liquids reach their equilibrium values [1]. Solutes including nucleic acids and proteins of interests can be extracted from one liquid phase to the other immiscible liquid phase as a preparation step for many analytical processes. There are several advantages in miniaturizing the liquid-liquid extraction methods to on-chip level extraction. Usual advantages of miniaturization are the reduction in the sample size and portability. In addition, transport phenomena is faster in Micro-systems than in ordinary size systems, and therefore, one may expect that liquid-liquid extraction takes less time to achieve in miniaturized devices. It is due to shorter diffusion time in micro scale as well as high surface to volume ratio of Microsystems. Electrowetting on dielectric (EWOD) digital microfluidics is an efficient platform to process droplet based analytical processes [2]. Nanoliter (nL) or smaller volume of aqueous liquid droplets can be generated and transported on a chip by EWOD process. In addition to the high surface to volume ratio, high chemical potential can be expected in droplet based extraction when the droplets are in motion. In this paper, we propose to use room temperature ionic liquid (RTIL) as a second liquid phase for extraction, which forms immiscible interface with aqueous solutions. Properties of RTIL can be tailored by choice of cation, anion and substituents. RTIL has been investigated as replacements for the organic solvents and various “task-specific” ionic liquid are being developed which exhibit many attractive properties such as very low vapor pressure, high thermal stability [3]. We recently published EWOD properties of various RTILs toward microfluidic applications [4]. To demonstrate liquid-liquid micro extraction on chip, we fabricated and tested EWOD digital microfluidic devices. Fig. 1 shows (a) top and (b) cross sectional views of EWOD device. Two model extraction systems were tested. One is organic dye extracted from RTIL (1-butyl-3-methylimidazolium bis(trifluoromethanesulfonylimide or BMIMNTf2) to water and the other is iodine (I2) extracted from water to BMIMNTf2. The later model experiment is demonstrated in Fig. 2. Droplets of aqueous solution and BMIMNTf2 solution were generated on chip reservoir then transported for extraction and separated by EWOD actuation. When an aqueous solution and BMIMNTf2 solution join together, they created an interface, since water and BMIMNTf2 are immiscible. Extraction of I2 was done along the interface. After successful extraction, two immiscible liquid phases were separated by EWOD actuation and formed two separate droplets. From the result shown in Fig 2 (g), it is expected that extraction performance at the interface of moving droplet would be enhanced compared to the stationary droplet, because a moving interface prevent the chemical equilibrium, thus more chemical extraction potential can be provided with a moving interface than at a stationary interface. This demonstration is the first step toward total analysis system. The presented result opens the way to on-chip micro extraction, which will be readily integrated with other sample preparation microfluidic components and detection components. Currently, micro extraction systems for larger molecules such as nucleic acids, proteins and biological cells are being developed for further analytical applications.

Pancreatic cancer is one of the most deadly forms of cancer, with overall 5-year survival rates under 20%. The only cure for this type of cancer is surgical, however this option is rarely available due in part to detection that is typically late in the progression of the disease. The aim of this study was to understand the differences in progression between L3.6, PANC-1, and M1A-PaCa-2 cells as representatives of varied levels of metastatic character through aggregation analysis in 3D cell culture. It is well established that 3D culture more closely mimics the in vivo condition, as the cells adhere to each other forming “microtumors” as opposed to being attached to a plastic substratum as is the case with standard 2D cell culture. We also examined the effectiveness of a novel target for chemotherapeutic intervention in these cell lines in 3D culture. The sigma-2 receptor is highly upregulated in rapidly proliferating cancer tissues as compared to non-cancerous tissue, and induces apoptosis when activated. We previously reported the ability of the sigma-2 receptor partial agonist CM572 (sigma-2 Ki = 14.7 nM) to induce apoptosis in PANC-1 pancreatic carcinoma cells in standard 2D culture (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 74: #3237, 2014). Here, we measured the aggregation rate and sigma-2 agonist sensitivity of L3.6, a highly metastatic cell line, in comparison to M1A-PaCa-2 and PANC-1 cells, which are both significantly less aggressive. The sigma-2 receptor agonist siramesine (Ki = 0.12nM) was found to induce cell death in 3D tumor cell culture in all three pancreatic cancer cell lines. However, the potency was 30-50% of the potency observed in traditional 2D cultured cells. Interestingly, in 3D culture, L3.6 and PANC-1 cells both exhibited ∼45% of the maximal cell death that was achieved in 2D culture using the same dose of siramesine, and M1A-PaCa-2 cells exhibited ∼25% of the maximal 2D toxicity at this dose. Additionally, both siramesine and CM572 were able to slow aggregation of the pancreatic cancer cells. This data indicates that the sigma-2 receptor may provide a novel target for therapeutic intervention in pancreatic cancer, both for slowing progression of the disease and as a chemotherapeutic target. The data also indicate that cell-cell contact in 3D culture either modifies the sigma-2 receptor signaling process or that cells in 3D culture present more of a barrier for drug access to the tumor cells. Further studies are needed to examine these possibilities. Citation Format: Timothy Chou, Michaela Jacobs, Hilary Nicholson, Wayne D. Bowen. Differential aggregation rates and therapeutic response of pancreatic cancer cell lines to sigma-2 receptor activation in 3D culture. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2015-1699

Combining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. Combining imaging with precise cell retrieval is of particular interest when studying highly dynamic or transient, asynchronous, or heterogeneous cell biological and developmental processes. Here, we present a method to encapsulate live cells in a 3D hydrogel matrix, via hydrogel bead compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we can long-term culture and subsequently extract individual cells-in-beads by media flow only, obviating the need for enzymatic cell removal from the platform. Specific beads may be extracted from the platform in isolation, without disrupting the adjacent beads. We show that we can differentiate mouse embryonic stem cells, monitor reporter expression by live imaging, and retrieve individual beads for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

ABSTRACTExtracellular matrix (ECM) materials have had remarkable success as scaffolds in tissue engineering (TE) and as therapies for tissue injury whereby the ECM microenvironment promotes constructive remodeling and tissue regeneration. ECM powder and solubilized derivatives thereof have novel applications in TE and RM afforded by the capacity of these constructs to be dynamically modulated. The powder form allows for effective incorporation and penetration of reagents; hence, ECM powder is an efficacious platform for 3D cell culture and vehicle for small molecule delivery. ECM powder offers minimally invasive therapy for tissue injury and successfully treatment for wounds refractory to first-line therapies. Comminution of ECM and fabrication of powder-derived constructs, however, may compromise the biological integrity of the ECM. The current lack of optimized fabrication protocols prevents a more extensive and effective clinical application of ECM powders. Further study on methods of ECM powder fabrication and modification is needed.

Three-dimensional (3D) cell culture has developed rapidly over the past 5-10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.

Artonin E is a naturally isolated compound from Artocarpus elasticus and was evaluated for their anticancer property in the present study. This study aimed to develop a 3D ovarian cell culture model and to examine the cytotoxicity of artonin E in 2D and 3D cell culture models. The 3D cell culture was performed using BD™ Puramatrix™ Peptide Hydrogel. Alamar blue assay, selectivity index analysis, morphological apoptotic double staining and immunofluorescence study were conducted to study the antiproliferative and apoptotic induction mechanism of artonin E in 3D SKOV-3 spheroid culture. SKOV-3 cells encapsulated in BD™ Puramatrix™ Peptide Hydrogel clearly demonstrated 3D-spheroid formation. The Alamar blue assay result showed that artonin E inhibited the growth of SKOV-3 cells in 2D and 3D culture with the IC50 values at 72 h treatment of 6.0 ± 0.8 µg/mL and 25.0 ± 0.8 µg/mL, respectively. This compound was found to be less toxic to the normal human ovarian cell lines, T1074, with IC50 values at 72 h of 28.0 ± 0.8 µg/mL in 2D culture and 85.0 ± 0.5 µg/mL in 3D culture, respectively. The selectivity index analysis more than 2 indicated that artonin E was selective against cancer cells compared to normal cells. Artonin E treatment caused apoptotic morphological changes in 3D SKOV-3 spheroids. In a 3D immunofluorescence study, elevated levels of cleaved caspase-3, cleaved caspase-9, apoptotic proteins bax, and decreased levels of antiapoptotic proteins bcl-2, Hsp70, and survivin were observed. In conclusion, artonin E increases chemoresistance and induces cell death in a 3D SKOV-3 spheroids culture via a pro- and anti-apoptotic protein pathway. These findings demonstrate that 3D spheroid culture is an effective platform for testing artonin E therapeutic candidates in an in vivo mimic microenvironment.

Three-dimensional (3D) multicellular models are considered ideal methods for bridging the gap between two-dimensional (2D) cell culture and animal models, which are widely used in organogenesis, disease models, drug development, and regenerative medicine. Cell culture technologies determine the physical and biological properties of multicellular spheroids or organoids that affect the authenticity, stability, assessment, and throughput of the 3D multicellular system. Micro patterns, characterized as a coating of specific adhesion matrices on substrates, can control cell behaviors and fate by limiting the space available for cell spreading. micro patterns are used to culture non-tumor or tumor spheroids and organoids with effective control of size and arrangement, which is suitable for large-scale and standardized culture to generate 3D multicellular models. This comprehensive review summarizes the advantages and applications of 3D multicellular models and discusses the characteristics of general 3D cell culture technologies. We discuss the basic applications of micro pattern technologies and highlight the specific advantages and features of micropattern (as a 3D cell culture platform) in non-tumor research (regenerative medicine, developmental biology, disease modelling, and monoclonal cell culture) and tumor research (tumor microenvironment (TME) and drug screening). Finally, the fabrication of micro patterns (bio inks, fabrication methods for micro patterns, morphology, and quality of micro patterns) is described.

Tumor spheroids are considered a valuable three dimensional (3D) tissue model to study various aspects of tumor physiology for biomedical applications such as tissue engineering and drug screening as well as basic scientific endeavors, as several cell types can efficiently form spheroids by themselves in both suspension and adherent cell cultures. However, it is more desirable to utilize a 3D scaffold with tunable properties to create more physiologically relevant tumor spheroids as well as optimize their formation. In this study, bioactive spherical microgels supporting 3D cell culture are fabricated by a flow-focusing microfluidic device. Uniform-sized aqueous droplets of gel precursor solution dispersed with cells generated by the microfluidic device are photocrosslinked to fabricate cell-laden microgels. Their mechanical properties are controlled by the concentration of gel-forming polymer. Using breast adenocarcinoma cells, MCF-7, the effect of mechanical properties of microgels on their proliferation and the eventual spheroid formation was explored. Furthermore, the tumor cells are co-cultured with macrophages of fibroblasts, which are known to play a prominent role in tumor physiology, within the microgels to explore their role in spheroid formation. Taken together, the results from this study provide the design strategy for creating tumor spheroids utilizing mechanically-tunable microgels as 3D cell culture platform.

The effect of IFNα-2b on the migration, proliferation, and expression of epithelial and mesenchymal markers of MCF-7 tumor adenocarcinoma cells in 2D and 3D cell cultures was examined. A significant cytostatic effect of IFNα-2b on the tumor population was detected. It was found that changes in the expression of epithelial (CKs and EpCAM) and mesenchymal markers were caused by changing the growth type of the tumor population. IFNα-2b inhibited migration of tumor cells to the suspension fraction and promoted an increase in expression of CK and EpCAM in 2D and 3D cell cultures, but only in the 3D culture was expression of vimentin increased. IFNα-2b caused an increase in CK and EpCAM expression by 50.5% and 47.8%, respectively, compared with the control in the 2D cell culture. In the 3D cell culture this increase was 33% and 34%, respectively, compared with the control. IFNα-2b stimulated the differentiation and inhibited the migrational ability of tumor cells in the early stages of breast cancer development.

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.