Suspension Cell Cultures Research Articles

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi

Phytochemical Profiling and Biological Activities of Extracts from Bioreactor-Grown Suspension Cell Cultures of Schisandra henryi

Whole-pathway stimulation of anthocyanin biosynthesis by UDP-glycose: flavonoid glycosyltransferase gene (UFGT) providing new insights into natural product development based on plant cell factory

Anthocyanins are a type of plant natural products (PNPs) that play a crucial role in various fields, including food, medicine, cosmetics, and dyes. However, their primary source is plant extraction, leading to significant ecological damage and resource wastage issues. To address the challenge of achieving ecologically sustainable high-throughput anthocyanin production, this study proposes a plant cell factory (PCF) strategy based on suspension cell culture (SCC). In this study, the key gene VdUFGT involved in anthocyanin biosynthesis was cloned from Vitis davidii, and gene overexpression was conducted using Agrobacterium-mediated methods. Notably, the vgt3 transgenic cell line exhibited 4.4-fold increase in anthocyanin content, reaching 286.9 μg/g (FW) in solid medium, along with enhanced levels of flavonoids and proanthocyanidins. Integrative analysis of transcriptomics and metabolomics demonstrated that VdUFGT significantly promoted the expression of genes involved in anthocyanin biosynthesis, leading to the enhanced accumulation of various flavonoids, including anthocyanins. A small-scale plant cell factory was established, achieving an impressive production of 4.11 mg/g (DW) of anthocyanin within just 12 days, which is 8.5 times higher than that of the WT cell line, with the yield of purified freeze-dried anthocyanin powder reaching 3.17%. The plant cell factory, employing genetic engineering and suspension cell culture, offers a safe, ecological, economical, and efficient approach for the high-throughput production of plant natural products such as anthocyanins.

Investigation of miniature goat cheese produced from Cynara scolymus L. pistil cell suspension culture extracts elicited with melatonin and salicylic acid.

The present study aimed to evaluate the potential of enzymes produced by artichoke (Cynara scolymus L.) flower cell suspension cultures elicited by melatonin (5 μm) and salicylic acid (50 μm) on the production and characteristics of miniature goat cheeses. In this study, five types of fresh miniature goat cheese were produced using whole artichoke flower extract, salicylic acid (50 μm) or melatonin (5 μm) treated artichoke suspension cell culture extracts, a culture extract without elicitor treatment (control) and rennin enzyme. The milk clotting activity values of the enzymes were measured in the range 0.52-0.74. The effect of the enzymes on the titratable acidity, water-soluble nitrogen, ripening index, αs-caseins and β-caseins of goat cheese was significant (P < 0.05). The highest level of casein degradation was observed in the cheese produced with the enzyme containing melatonin, followed by the cheese produced with the enzyme containing salicylic acid. For the enzymes produced by the suspension cell culture method, the addition of melatonin and salicylic acid had a slightly positive effect on the proteolytic activity of the extracts. It was also found that the enzymes obtained from artichokes by the suspension cell culture method did not achieve successful cheese production in terms of chemical, textural and biochemical aspects compared to those obtained from animal enzymes. © 2024 Society of Chemical Industry.

The future of single-use plastics in life science: Sterile printing of PLA reduces greenhouse gas emissions by 80% and enables carbon neutrality

The European Union’s Green Deal emphasizes life science and biotechnology as key drivers for achieving a circular economy. However, the prevalent use of non-sustainable single-use plastics derived from petrochemical sources in life science research and development contradicts this ecological goal. This study presents a viable alternative through on-site sterile 3D printing of single-use plastics using poly(lactic acid) (PLA). The sterile printing of PLA demonstrates a substantial reduction in CO2eq emissions per shake flask, decreasing from 260 g CO2eq to 145 g CO2eq. Suitability for cell culture was demonstrated for sterile printed PLA, autoclaved high-temperature PLA and for suspension cell cultures in shake flasks using CHO and insect cells as well as adherent Vero cell cultures in cell culture wells. Further optimization of sustainable shake flasks is achieved by utilizing recycled PLA, resulting in a remarkable reduction to 73 g CO2eq, constituting a 72% decrease in CO2eq emissions compared to conventional single-use plastics. Moreover, by employing geometric optimization to minimize material usage, emissions can be further reduced to 44 g CO2eq, representing an 83% reduction in CO2eq emissions. Anticipated advancements in PLA production suggest future carbon net negative PLA production, potentially achieving zero carbon emissions for single-use plastics using PLA. The exceptionally cost-effective nature of 3D printed single-use plastics, coupled with negligible capital costs for implementation, positions sterile 3D printing as a practical solution aligned with the 2030 Green Deal goals. Moreover, ongoing improvements in bioplastic production underscore the feasibility of meeting the 2050 targets of carbon neutrality for single-use plastics in life science research and development.

Elicitation and precursor induced approaches for the enhancement of α−tocopherol production using suspension cultures of Solanum lycopersicum

Elicitation and precursor feeding are the effective strategies for enhancing the synthesis of bioactive compounds in plant cell suspension cultures. The present study aimed to explore an efficient elicitation and precursor feeding protocol and its effect on inducing the accumulation of α-tocopherol in Solanum lycopersicum (tomato) suspension cell culture. The tomato cell suspension cultures were treated with different elicitors (Methyl Jasmonate, Salicylic acid and Yeast extract) and precursors (Homogentisic acid, Tyrosine, Hydroxypyruvic acid and Phytol) and the effect of α-tocopherol production was studied. Significant increase in the α-tocopherol was observed on day 5 upon methyl jasmonate treatment which represented 17.7 fold increase in comparison to the control. The treatment of precursor in combination viz., 150 μM Homogentisic acid + 150 μM Phytol showed the maximum enhancement of α-tocopherol up to 22 fold on day 10 compared to the untreated control. These results suggested that the suspension cultures combining with the optimal precursor feeding and elicitors enhanced the production of α-tocopherol in economically important tomato cell cultures.

Controlling Pericellular Oxygen Tension in Cell Culture Reveals Distinct Breast Cancer Responses to Low Oxygen Tensions.

In oxygen (O2)-controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O2 tension experienced by cells (i.e., pericellular O2). However, it is discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to cellular O2 consumption. A reaction-diffusion model is developed to predict pericellular O2 tension a priori, demonstrating that the effect of cellular O2 consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O2 tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia-inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O2 tension in cell culture incubators is insufficient to regulate O2 in cell culture, thus introducing the concept of pericellular O2-controlled cell culture.

In vitro plant regeneration and bioactive metabolite production of endangered medicinal plant Fritillaria cirrhosa

The bulb of Fritillaria cirrhosa D. Don is widely used for the anti-asthmatic, anti-tussive, and anti-cancer agents, etc., while the yield is limited by an endangered status, a long juvenile phase, and restricted growth habitat. Ancillary approaches to improve the bulb yield by micropropagation and bioactive metabolites production by bioreactor have not been established. Here is reported the plant regeneration, suspension cell culture, and bioactive metabolite production at different treatments. The embryogenic calli were successfully induced via the histomorphological identification. The highest proliferation times (4.11-fold) were observed with a select combination of hormones [NAA (0.2 mg/L) + 6-BA (1.0 mg/L) + GA3 (1.0 mg/L)] and culture conditions (red light and 20 °C), the highest content of imperialine (0.13 mg/g) was observed under blue light, total phenolic (0.52 mg/g) under red light, polysaccharides (36.57 mg/g) and total flavonoids (2.67 mg/g) as well as antioxidant capacity under white light. The plantlets were regenerated within 125 d from the induced embryogenic calli to acclimation and transplantation of seedlings. For the suspension cell culture, a 6.30-, 1.78-, 1.37-, and 1.51-fold increase of proliferation times, imperialine, polysaccharides, and total phenolic contents was observed at 40 d, respectively. Based on the above observations, an effective and complete in vitro approach has been proposed to regenerate plants and produce bioactive metabolites in F. cirrhosa.

Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells

The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted a SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared to the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.

A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane.

The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1705.

Suspension cell cultures of Panax vietnamensis as a biotechnological source of ginsenosides: growth, cytology, and ginsenoside profile assessment.

Panax vietnamensis is a valuable medicinal plant and a source of a broad spectrum of biologically active ginsenosides of different structural groups. Overexploitation and low adaptability to planation cultivation have made this species vulnerable to human pressure and prompted the development of cell cultivation in vitro as a sustainable alternative to harvesting wild plants for their bioactive components. Despite high interest in biotechnological production, little is known about the main factors affecting cell growth and ginsenoside biosynthesis of this species under in vitro conditions. In this study, the potential of cell cultures of P. vietnamensis as a biotechnological source of ginsenosides was was assessed. Six suspension cell lines that were developed from different sections of a single rhizome through a multi-step culture optimization process and maintained for over 3 years on media with different mineral salt base and varying contents of auxins and cytokinins. These cell lines were evaluated for productivity parameters and cytological characteristics. Ginsenoside profiles were assessed using a combination of the reversed-phase ultra-high-performance liquid chromatography-Orbitrap-tandem mass spectrometry (UHPLC-Orbitrap-MS/MS) and ultra-performance liquid chromatography-time of flight-mass spectrometry (UPLC-TOF-MS). All lines demonstrated good growth with a specific growth rate of 0.1-0.2 day-1, economic coefficient of 0.31-0.70, productivity on dry weight (DW) of 0.30-0.83 gDW (L·day)-1, and maximum biomass accumulation varying from 10 to 22 gDW L-1. Ginsenosides of the protopanaxadiol (Rb1, Rb2/Rb3, malonyl-Rb1, and malonyl-Rb2/Rb3), oleanolic acid (R0 and chikusetsusaponin IV), and ocotillol (vinaginsenoside R1) groups and their isomers were identified in cell biomass extracts. Chikusetsusaponin IV was identified in P. vietnamensis cell culture for the first time. These results suggest that suspension cell cultures of Vietnamese ginseng have a high potential for the biotechnological production of biomass containing ginsenosides, particularly of the oleanolic acid and ocotillol groups.

Transcriptional Changes in Damask Rose Suspension Cell Culture Revealed by RNA Sequencing.

Damask roses (Rosa x damascena) are widely used in cosmetics and pharmaceutics. Here, we established an in vitro suspension cell culture for calli derived from damask rose petals. We analyzed rose suspension cell transcriptomes obtained at two different time points by RNA sequencing to reveal transcriptional changes during rose suspension cell culture. Of the 580 coding RNAs (1.3%) highly expressed in the suspension rose cells, 68 encoded cell wall-associated proteins. However, most RNAs encoded by the chloroplasts and mitochondria are not expressed. Many highly expressed coding RNAs are involved in translation, catalyzing peptide synthesis in ribosomes. Moreover, the amide metabolic process producing naturally occurring alkaloids was the most abundant metabolic process during the propagation of rose suspension cells. During rose cell propagation, coding RNAs involved in the stress response were upregulated at an early stage, while coding RNAs associated with detoxification and transmembrane transport were upregulated at the late stage. We used transcriptome analyses to reveal important biological processes and molecular mechanisms during rose suspension cell culture. Most non-coding (nc) RNAs were not expressed in the rose suspension cells, but a few ncRNAs with unknown functions were highly expressed. The expression of ncRNAs and their target coding RNAs was highly correlated. Taken together, we revealed significant biological processes and molecular mechanisms occurring during rose suspension cell culture using transcriptome analyses.

Review on the Impact of Impeller‐induced Hydrodynamics on Suspension Cell Culture for Production of Biopharmaceuticals

AbstractHydrodynamic effects continue to play a crucial role in the design and operation of bioreactors for cultivating mammalian cells. Despite intensive studies in the past the link between hydrodynamics and biological process behaviour is still not fully understood. Common methods to describe dependencies, which relay mostly on averaged hydrodynamic parameters, does not seem to be suitable, as they do not incorporate the hydrodynamic heterogeneity. Within this review first a brief introduction to hydrodynamic effects is given, followed by an evaluation of common process design approaches with respect to hydrodynamic effects and recommendations for process development.

Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

In vitro encapsulation and expansion of T and CAR-T cells using 3D synthetic thermo-responsive matrices.

Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.

Recombinant Protein Production in Suspension Mammalian Cells Using the BacMam Baculovirus Expression System.

Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of "insect" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.

Automated Microfluidic System for Suspension Cell Culture Using Lab-on-Chip Devices

Automated Microfluidic System for Suspension Cell Culture Using Lab-on-Chip Devices

Obtaining a primary suspension cell culture of Dracocephalum palmatum Stephan ex Willd

Dracocephalum palmatum Steph. grows on the southern slopes of the Oymyakon plateau in Yakutia (Northeast of Russian Federation) in conditions of harsh continental climate with continuous permafrost. The aboveground phytomass of the plant contains various complexes of secondary metabolites including polyphenolic compounds. It is a potential source of secondary metabolites needed for practical use in the pharmaceutical industry. The aim of the study is to obtain a primary suspension cell culture of Dracocephalum palmatum, growing in the conditions of the Cold Pole — Oymyakon. The work includes optimization of the nutrient medium for introducing calluses into a suspension culture, analysis of the dynamics of biomass growth of the obtained suspension culture, and morphological characteristics of the cells of the suspension culture. The callus cell cultures of Dracocephalum palmatum, cultivated on Murashige-Skoog (MS) medium with the addition of 0.5 mg/L α- naphthylacetic acid (NAA) and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), were most successfully transitioned into suspension culture. The maximum biomass growth of cell suspension culture was observed when cultivated in liquid MS medium with the addition of 2,4-D (0.5 mg/L), 6- benzylaminopurine (0.5 mg/L), and NAA (0.5 mg/L). The primary cell suspension culture of Dracocephalum palmatum, cultivated for 22 days, had an increase in wet weight of 9,2084 g, dry weight — 0,34135 g, and contained dedifferentiated aggregates of parenchyma-like cells and single round-shaped cells. Samples of the obtained cell suspension culture of Dracocephalum palmatum will be used for the analysis of secondary metabolites and for the development of optimal cultivation conditions in a bioreactor.

Triple click chemistry for crosslinking, stiffening, and annealing of gelatin-based microgels.

Microgels are spherical hydrogels with physicochemical properties ideal for many biomedical applications. For example, microgels can be used as individual carriers for suspension cell culture or jammed/annealed into granular hydrogels with micron-scale pores highly permissive to molecular transport and cell proliferation/migration. Conventionally, laborious optimization processes are often needed to create microgels with different moduli, sizes, and compositions. This work presents a new microgel and granular hydrogel preparation workflow using gelatin-norbornene-carbohydrazide (GelNB-CH). As a gelatin-derived macromer, GelNB-CH presents cell adhesive and degradable motifs while being amenable to three orthogonal click chemistries, namely the thiol-norbornene photo-click reaction, hydrazone bonding, and the inverse electron demand Diels-Alder (iEDDA) click reaction. The thiol-norbornene photo-click reaction (with thiol-bearing crosslinkers) and hydrazone bonding (with aldehyde-bearing crosslinkers) were used to crosslink the microgels and to realize on-demand microgel stiffening, respectively. The tetrazine-norbornene iEDDA click reaction (with tetrazine-bearing crosslinkers) was used to anneal microgels into granular hydrogels. In addition to materials development, we demonstrated the value of the triple-click chemistry granular hydrogels via culturing human mesenchymal stem cells and pancreatic cancer cells.

Phenotypic Characterization of 2D and 3D Prostate Cancer Cell Systems Using Electrical Impedance Spectroscopy.

Prostate cancer is the second leading cause of death in men. A challenge in treating prostate cancer is overcoming cell plasticity, which links cell phenotype changes and chemoresistance. In this work, a microfluidic device coupled with electrical impedance spectroscopy (EIS), an electrode-based cell characterization technique, was used to study the electrical characteristics of phenotype changes for (1) prostate cancer cell lines (PC3, DU145, and LNCaP cells), (2) cells grown in 2D monolayer and 3D suspension cell culture conditions, and (3) cells in the presence (or absence) of the anti-cancer drug nigericin. To validate observations of phenotypic change, we measured the gene expression of two epithelial markers, E-cadherin (CDH1) and Tight Junction Protein 1 (ZO-1). Our results showed that PC3, DU145, and LNCaP cells were discernible with EIS. Secondly, moderate phenotype changes based on differences in cell culture conditions were detected with EIS and supported by the gene expression of CDH1. Lastly, we showed that EIS can detect chemoresistant-related cell phenotypes with nigericin drug treatment. EIS is a promising label-free tool for detecting cell phenotype changes associated with chemoresistance. Further development will enable the detection and characterization of many other types of cancer cells.