Cell Culture Dishes Research Articles

Integration of bioprinting advances and biomechanical strategies for in vitro lung modelling

Abstract The recent occurrence of the Covid-19 pandemic and frequent wildfires have worsened pulmonary diseases and raised the urgent need for investigating host-pathogen interactions and advancing drug and vaccine therapies. Historically, research and experimental studies have relied on two-dimensional cell culture dishes and/or animal models, which suffer from physiological differences from the human lung. More recently, there has been investigation into the use of lung-on-a-chip models and organoids, while the use of bioprinting technologies has also emerged to fabricate three-dimensional constructs or lung models with enhanced physiological relevance. Concurrently, achievements have also been made to develop biomimetic strategies for simulating the in vivo biomechanical conditions induced by lung breathing, though challenges remain with incorporating these strategies with bioprinted models. Bioprinted models combined with advanced biomimetic strategies would represent a promising approach to advance disease discovery and therapeutic development. As inspired, this article briefly reviews the recent progress of both bioprinted in vitro lung models and biomechanical strategies, with a focus on native lung tissue microstructure and biomechanical properties, bioprinted constructs, and biomimetic strategies to mimic the native environment. This article also urges that the integration of bioprinting advances and biomimetic strategies would be essential to achieve synergistic effects for in vitro lung modelling. Key issues and challenges are also identified and discussed along with recommendations for future research.

Control of myotube orientation using ultrasonication

This study investigated a technique for controlling the orientation of C2C12-derived myotube cells using ultrasonication for future clinical applications of cultured skeletal muscle tissues. An ultrasonicating cell culture dish, comprising a plastic-bottomed culture dish and a circular glass plate (diameter, 35 mm; thickness, 1.1 mm) attached to an annular piezoelectric ultrasonic transducer (inner diameter, 10 mm; outer diameter, 20 mm; thickness, 1 mm), was constructed. A concentric resonant vibrational mode at 89 kHz was generated on the bottom of the dish, and the orientations of myotube cells were quantitatively evaluated using two-dimensional Fourier transform analysis of phase contrast microscopy images captured over a 14 × 10 mm2 area at the center of the dish. Unsonicated myotube cells grew in random directions, but ultrasonication aligned them circumferentially in the culture dish. The timing of treatment was important, with ultrasonication for 48 h before differentiation having a greater impact on myotube orientation than ultrasonication after differentiation. A larger ultrasonic vibration, with an amplitude of over 20 Vpp, resulted in significantly smaller angles of deviation in the circumferential direction than the control. Ultrasonication enhanced the expression of differentiation-related genes and the formation of aligned myotubes, suggesting that it promotes differentiation of C2C12 cells into myotubes.

SCRATCH: A programmable, open-hardware, benchtop robot that automatically scratches cultured tissues to investigate cell migration, healing, and tissue sculpting.

Despite the widespread popularity of the ' scratch assay', where a pipette is dragged through cultured tissue to create an injury gap to study cell migration and healing, the manual nature of the assay carries significant drawbacks. So much of the process depends on individual manual technique, which can complicate quantification, reduce throughput, and limit the versatility and reproducibility of the approach. Here, we present a truly open-source, low-cost, accessible, and robotic scratching platform that addresses all of the core issues. Compatible with nearly all standard cell culture dishes and usable directly in a sterile culture hood, our robot makes highly reproducible scratches in a variety of complex cultured tissues with high throughput. Moreover, we demonstrate how scratching can be programmed to precisely remove areas of tissue to sculpt arbitrary tissue and wound shapes, as well as enable truly complex co-culture experiments. This system significantly improves the usefulness of the conventional scratch assay, and opens up new possibilities in complex tissue engineering and cell biological assays for realistic wound healing and migration research.

Evaluation of conical 9 well dish on bovine oocyte maturation and subsequent embryonic development.

The Conical 9 well dish (C9 well dish) is characterized by a decreasing cross-sectional area towards the base. This design was hypothesized to enhance embryonic development by emulating the in vivo physical environment through density modulation. Comparative analyses revealed no significant difference in nuclear maturation rates between the C9 well dish and the 5-well dish. Reactive oxygen species (ROS) generation was lower in the C9 well dish compared to the 5-well dish; however, this difference was not statistically significant. On the second day of in vitro culture, the cleavage rate in the C9 well dish was 4.66% higher, although not statistically significant, and the rates of blastocyst development were similar across both dishes. No significant differences were observed in the intracellular levels of glutathione (GSH) and ROS, as well as in the total cell number within the blastocysts between the dish types. The expression of mitogen-related factors, TGFα and IGF-1, in the blastocysts was consistent between the dishes. However, PDGFβ expression was significantly lower in the C9 well dish compared to the 35 mm petri dish. Similarly, the expression of the apoptosis factor Bax/Bcl2l2 showed no significant differences between the two dishes. Despite the marked difference in PDGFβ expression, its impact on blastocyst formation appeared negligible. The study also confirmed the feasibility of culturing a small number of oocytes per donor, collected via Ovum Pick-Up (OPU), with reduced volumes of culture medium and mineral oil, thus offering economic advantages. In conclusion, the present study indicates that the C9 well dish is effective for in vitro development of a small quantity of oocytes and embryos, presenting it as a viable alternative to traditional cell culture dishes.

High-Quality Three-Dimensionally Cultured Cells Using Interfaces of Diblock Copolymers Containing Different Ratios of Zwitterionic N-Oxides.

To control three-dimensional (3D) cell spheroid formation, it is well-known the surface physicochemical and mechanical properties of cell culture materials are important; however, the formation and function of 3D cells are still unclear. This study demonstrated the precise control of the formation of 3D cells and 3D cell functions using diblock copolymers containing different ratios of a zwitterionic trimethylamine N-oxide group. The diblock copolymers were composed of poly(n-butyl methacrylate) (PBMA) as the hydrophobic unit for surface coating on a cell culture dish and stabilization in water, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) as the precursor of N-oxide. The zwitterionic N-oxide converted from 0 to 100% using PDMAEMA. The wettability and surface zeta potential varied with different ratios of N-oxide diblock copolymer-coated surfaces, and the amount of protein adsorbed in the cell culture medium decreased monotonically with increasing N-oxide ratio. 3D cell spheroid formations were observed by seeding human umbilical cord mesenchymal stem cells (hUC-MSCs) in diblock copolymer-coated flat-bottom well plates, and the N-oxide ratio was over 40%. The cells proliferated in two-dimensions (2D) and did not form spheroids when the N-oxide ratio was less than 20%. Interestingly, the expression of undifferentiated markers of hUC-MSCs was higher on surfaces that adsorbed proteins to some extent and formed 50-150 μm spheroids in the range of 40-70% of N-oxide ratio. We revealed that a moderately protein-adsorbed surface allows precise control of spheroid formation and undifferentiated 3D cells and has potential applications for high-quality spheroids in regenerative medicine and drug screening.

How to Keep Myofibroblasts under Control: Culture of Mouse Skin Fibroblasts on Soft Substrates

During the physiological healing of skin wounds, fibroblasts recruited from the uninjured adjacent dermis and deeper subcutaneous fascia layers are transiently activated into myofibroblasts to first secrete and then contract collagen-rich extracellular matrix into a mechanically resistant scar. Scar tissue restores skin integrity after damage but comes at the expense of poor esthetics and loss of tissue function. Stiff scar matrix also mechanically activates various precursor cells into myofibroblasts in a positive feedback loop. Persistent myofibroblast activation results in pathologic accumulation of fibrous collagen and hypertrophic scarring, called fibrosis. Consequently, the mechanisms of fibroblast-to-myofibroblast activation and persistence are studied to develop antifibrotic and prohealing treatments. Mechanistic understanding often starts in a plastic cell culture dish. This can be problematic because contact of fibroblasts with tissue culture plastic or glass surfaces invariably generates myofibroblast phenotypes in standard culture. We describe a straight-forward method to produce soft cell culture surfaces for fibroblast isolation and continued culture and highlight key advantages and limitations of the approach. Adding a layer of elastic silicone polymer tunable to the softness of normal skin and the stiffness of pathologic scars allows to control mechanical fibroblast activation while preserving the simplicity of conventional 2-dimensional cell culture.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Bidirectional phase retrieval: Protecting the imaging of cells and tissues from interference of noise on the carrier

Quantitative phase imaging plays a crucial role in biological imaging, with many phase retrieval technologies being particularly important. However, cells and tissues must be placed on certain carriers during imaging, such as glass slides, cell culture dishes, and cell culture boxes. Therefore, current unidirectional phase retrieval technologies are not suitable for biplane samples, causing the phase of the object to always be coupled with the phase of noise. As a solution, this study proposes bidirectional phase retrieval to independently analyze the phase of both planes in the sample. Considering that most biological samples can be treated as two planes: one representing the object and the other serving as noise. By decoupling the phase of the two planes, this approach protects biplane biological samples from interference caused by the noise plane, leading to more accurate phase retrieval. This innovation holds significant value for the advancement of biological imaging.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes

BackgroundOsteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes.ResultsHuman monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates.ConclusionOur study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Thermoresponsive block-copolymer brush-modified interfaces for effective fabrication of hepatocyte sheets

Hepatic tissue engineering has been investigated for the treatment of liver disease. One promising tissue is the hepatocyte sheet, cultured in temperature-responsive cell culture dishes. However, the preparation of hepatocyte sheets requires precoating of the dish with fetal bovine serum (FBS), which is not desirable for clinical applications. To overcome this issue, we developed a thermoresponsive polymer-modified glass with an affinity for hepatocytes. Poly(N-p-vinylbenzyl-O-β-d-galactopyranosyl-(1→4)-d-guliconamide)-b-poly(N-isopropylacrylamide) (PVLA-b-PNIPAAm) was prepared through two steps of atom transfer radical polymerization (ATRP). The prepared copolymer brushes were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, gel permeation chromatography, contact angle measurements, and fluorescence labeled-fibronectin and -lectin adsorption assays. These characterizations showed that the smooth block copolymer layer was successfully modified by ATRP. The feasibility of using the prepared polymer brush-grafted glass as a hepatocyte sheet fabrication dish was investigated using primary rat hepatocytes. The PVLA-b-PNIPAAm brushes exhibited hepatocyte adhesion and proliferation without FBS precoating. By reducing the temperature from 37 °C to 20 °C, the hepatocyte sheet was recovered from the copolymer brush. These results indicated that the prepared PVLA-b-PNIPAAm brushes can be utilized for effective hepatocyte sheet fabrication without the need for FBS precoating.

Heart rhythm in vitro: measuring stem cell-derived pacemaker cells on microelectrode arrays.

Cardiac arrhythmias have markedly increased in recent decades, highlighting the urgent need for appropriate test systems to evaluate the efficacy and safety of new pharmaceuticals and the potential side effects of established drugs. The Microelectrode Array (MEA) system may be a suitable option, as it provides both real-time and non-invasive monitoring of cellular networks of spontaneously active cells. However, there is currently no commercially available cell source to apply this technology in the context of the cardiac conduction system (CCS). In response to this problem, our group has previously developed a protocol for the generation of pure functional cardiac pacemaker cells from mouse embryonic stem cells (ESCs). In addition, we compared the hanging drop method, which was previously utilized, with spherical plate-derived embryoid bodies (EBs) and the pacemaker cells that are differentiated from these. We described the application of these pacemaker cells on the MEA platform, which required a number of crucial optimization steps in terms of coating, dissociation, and cell density. As a result, we were able to generate a monolayer of pure pacemaker cells on an MEA surface that is viable and electromechanically active for weeks. Furthermore, we introduced spherical plates as a convenient and scalable method to be applied for the production of induced sinoatrial bodies. We provide a tool to transfer modeling and analysis of cardiac rhythm diseases to the cell culture dish. Our system allows answering CCS-related queries within a cellular network, both under baseline conditions and post-drug exposure in a reliable and affordable manner. Ultimately, our approach may provide valuable guidance not only for cardiac pacemaker cells but also for the generation of an MEA test platform using other sensitive non-proliferating cell types.

Development of Organoid-Based Cytotherapeutics for Taste Loss Induced By Anti-Cancer Chemotherapy and Radiotherapy

Introduction Ulcerative mucositis and taste loss are common side effects induced by cancer therapy, including chemotherapy and radiation therapy. These conditions cause significant oral pain and loss of taste, impacting food intake and physical health abnormalities such as malnutrition, as well as depression and mental health deterioration, which can affect prognosis. To address these challenges, an appropriate treatment for oral mucositis is required. While various types of oral mucositis treatments have been developed (e.g., patch, spray, ointment), most show indirect healing effects such as pain control and inflammation relief. Thus, a direct method for treating oral lesions should be developed. This research was supported by the Korean Fund for Regenerative Medi- cine (KFRM) grant funded by the Korea Government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (21C0712L1-11). Methods To compensate for the difficulty in supplying human tissue, we conducted preliminary experiments using mice to establish protocols for manufacturing human oral tissue-derived cell therapeutics. we devised an organoid-derived cell sheet transplantation. First, we generated organoids derived from oral mucosa and prepared a cell sheet using a temperature-responsive cell culture dish. The harvested cell sheet was transplanted to the lesion site to enable structural and functional recovery of the oral mucosa and gustatory organs. Results We have developed a cell-therapeutic platform based on regenerative medicine technology to recover oral lesions in cancer chemotherapy and radiotherapy patients. Specifically, we developed a method for producing autologous cell therapeutics. The oral mucosa tissue of a patient scheduled for anticancer treatment was collected, and epithelial cells were obtained from the tissue. Using these cells, we generated organoids with differentiated taste bud cells, and isolated cells from the organoids were seeded onto temperature-responsive cell culture dishes and cultured beyond confluence for the formation of a sheet. We performed immunostaining to detect various types of taste cells in the cell sheet. We also confirmed the success of cell sheet transplantation and the subsequent healing effect. Conclusion Improving the prognosis and acquiring the will to recover of cancer chemotherapy and radiotherapy patients through repairing taste function and rapid healing of lesions are crucial. We have developed a cell therapy technology that enables the structural and functional recovery of the oral mucosa and gustatory organs, providing a promising solution to treat the side effects of anti-cancer treatment. Our research provides an R&D initiative for the treatment and prevention of intractable diseases by culturing, manufacturing, and transplanting living autologous cells.

A assembled plate column bioreactor for adherent cell culture

The ideal bioreactor should be highly efficient, cost-effective and able to promote cells adhesion growth. In this study, we constructed a plate-tower bioreactor with S-loop in each layer based on multilayer plate bioreactor and plate column theory. Human hepatocellular carcinomas(HepG2) and human normal liver cell L02 (L02) cells were employed evaluation of bioreactor performance. The results show that cells were able to adhere to the plates and the density reached to 1～1.2 × 105/cm2. When circulating flow rate of the reactor is 8 ml/min and the oxygen supply concentration is 40 %, the sufficient material and gas exchange in bioreactor, the amount of albumin, urea and cytochrome P450(CYP450) content in the hyperoxia group were 3.31, 2.58 and 2.6 times higher than that of the control group, there was no obvious cell damage. This condition up the regulation of genes in more metabolic pathways and improved the metabolic capacity of hepatocytes. The single-layer area and the number of bioreactors can be flexibly adjusted, and the manufacturing cost of single-layer is close to that of ordinary plastic cell culture dishes. Either batch or continuous mode can be used in the bioreactor. In view of the above characteristics, the bioreactor is more conducive to practical application.

Fabrication of a Magnetically Driven Cell-Stretching Device for Predefined Cell Alignment in Vitro

Various devices have been developed that use stretching silicone sheets to evaluate cellular mechanotransduction. However, few studies have explored predefined cell alignments using mechanical stimuli for engineering applications, including cell sheets and drug screenings. Therefore, we proposed a magnetically driven cell-stretching device for predefined cell alignment in vitro, which consisted mainly of a circular silicone membrane with a neodymium magnet and standard cell culture dish. As the proposed device was incorporated into a cell culture dish, there may be a small risk of contamination in long-term incubation experiments. The device was fabricated by assembling a polydimethylsiloxane membrane and silicone ring. The fabricated device showed that the membrane strain increased with increasing voltage application to the electromagnet, and indicated that cell alignment occurs when strain exceeds 0.8%. Following cyclic stimulation of cells adhered to a membrane for 4 h in a CO2 incubator with 1.05% strain at 0.1 Hz, cell alignment with the predefined direction increased by 20.4% compared to that before stimulation. The findings imply that the proposed device may be utilized for predefined cell alignment.

Diya – A universal light illumination platform for multiwell plate cultures

Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for invivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform- Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast, and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for invivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Biocompatible coatings based on photo-crosslinkable cellulose derivatives

Materials derived from renewable resources have great potential to replace fossil-based plastics in biomedical applications. In this study, the synthesis of cellulose-based photoresists by esterification with methacrylic acid anhydride and sorbic acid was investigated. These resists polymerize under UV irradiation in the range of λ = 254 nm to 365 nm, with or, in the case of the sorbic acid derivative, without using an additional photoinitiator. Usability for biomedical applications was demonstrated by investigating the adhesion and viability of a fibrosarcoma cell line (HT-1080). Compared to polystyrene, the material widely used for cell culture dishes, cell adhesion to the biomaterials tested was even stronger, as assessed by a centrifugation assay. Remarkably, chemical surface modifications of cellulose acetate with methacrylate and sorbic acid allow direct attachment of HT-1080 cells without adding protein modifiers or ligands. Furthermore, cells on both biomaterials show similar cell viability, not significantly different from polystyrene, indicating no significant impairment or enhancement, allowing the use of these cellulose derivatives as support structures for scaffolds or as a self-supporting coating for cell culture solely based on renewable resources.

Singlet oxygen-generating cell-adhesive glass surfaces for the fundamental investigation of plasma membrane-targeted photodynamic therapy

Recently, plasma membrane-targeted photodynamic therapy has attracted attention as an effective cancer immunotherapeutic strategy. However, the released photosensitizers do not only adhere to the plasma membrane but may also be internalized in the cytosol, in endosomes/lysosomes, hindering investigations of the effects of photosensitizers attached to the plasma membrane. In this study, we developed a cell culture dish with singlet oxygen-generating cell-adhesive glass surfaces that allows investigation of the effects of photosensitizers attached to the plasma membrane. For cell adhesion, poly[N-(3-aminopropyl)methacrylamide] conjugated with hematoporphyrin PA–HpD was immobilized on the glass surfaces. Singlet oxygen was produced from the PA–HpD-immobilized glass surface upon laser irradiation at 635 nm. When murine colon adenocarcinoma 26 (Colon-26) cells were cultured on the PA–HpD-immobilized surface, the cells were swollen and ruptured, leading to effective apoptotic cell death using laser irradiation at 635 nm. In addition, microvesicles of approximately 10 μm in diameter were released from the plasma membrane into the culture medium. These phenomena were due to the oxidation of lipids in the cellular membrane, caused by the plasma membrane-targeted photodynamic therapy. In contrast, these phenomena were not observed on poly[N-(3-aminopropyl)methacrylamide]-immobilized glass surfaces. These results indicate that cell culture dishes with singlet oxygen-generating cell-adhesive glass surfaces can be used to investigate fundamental mechanisms in plasma membrane-targeted photodynamic therapy.

Beating heart cells: Using cultured cardiomyocytes to study cellular structure and contractility in laboratory exercises.

Heart muscle cells, or cardiomyocytes, exhibit intrinsic contractility in vitro. We found that commercially-available mammalian cardiomyocytes serve as an excellent model system for studying the cytoskeleton and cellular contractility, fundamental topics in undergraduate cell and molecular biology courses. Embryonic rat cardiomyocytes were plated on cell culture dishes or glass coverslips and visualized using an inverted phase-contrast microscope. The cardiomyocytes began contracting within 1-2 days after plating and continued to contract for many weeks, allowing their use in multiple laboratory sessions. Following background reading and instruction, students fixed and triple-stained the cardiomyocytes to examine the relative distributions of actin filaments and microtubules and the position of nuclei. Analysis and image capture with fluorescence microscopy provided striking examples of highly organized cytoskeletal elements. Students then designed experiments in which cardiomyocyte intrinsic contractility was explored. Changes in contraction rates were examined after treatment with signaling molecules, such as epinephrine. The addition of epinephrine to the culture medium, within a usable concentration window, increased the rate of contraction. These adaptable exercises provide undergraduate cell and molecular biology students with the exciting opportunity to study cardiomyocytes using standard cell culture and microscopy techniques.

Design of thermo-responsive cell culture dishes using poly(N-isopropylacrylamide)-block-polystyrene copolymers for cell sheet technology

The main objective of this work was to assess the cell sheet fabrication of a newly designed thermo-responsive polymer. Thermo-responsive diblock copolymers of poly(N-isopropylacrylamide)-block-polystyrene (PNIPAM-b-PS) with different block ratios were synthesized via sequential reversible addition-fragmentation chain transfer (RAFT) polymerization. The prepared copolymers were characterized by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance and gel permeation chromatography techniques in terms of molecular weight and composition. Physical adsorption was employed to prepare thermo-responsive films. Employing atomic force microscopy (AFM) and water contact angle (WCA) measurements, we found that the PNIPAM-b-PS diblock copolymer coatings showed a temperature-triggered phase transition in water through the reversible hydration of PNIPAM segments. Thermo-responsive PNIPAM-b-PS coatings were utilized as functional substrates for human dermal fibroblasts (HDFs) adhesion and detachment. By adjusting PNIPAM to PS segments ratio, HDFs successfully detached from the PNIPAM-b-PS spin-coated tissue culture polystyrene (TCPS) substrates in a temperature-induced manner.