Materials strategies and multidisciplinary approaches for low-temperature preservation of 3D cellular systems

BackgroundOvarian tissue cryopreservation and transplantation are novel therapeutic approaches for fertility preservation. However, follicle loss caused by ischemic and hypoxic damage is one of the issues after frozen-thawed ovarian tissue transplantation. Promoting angiogenesis in grafts is the key to restore cryopreserved ovarian function. Mesenchymal stem cells (MSCs) have been reported to facilitate angiogenesis in the cryopreserved ovarian tissue transplantation. However, the risk of embolization, immunogenic effect and tumorigenesis hinders the clinical application of MSCs to human organ transplantation. In this study, we established an in vitro ovarian culture system to restore frozen-thawed ovarian function before transplantation with the application of umbilical cord mesenchymal stem cells (UC-MSCs), and explored the effects of UC-MSCs on frozen-thawed ovaries in vitro ovarian culture system and the mechanisms of UC-MSCs on the angiogenesis of frozen-thawed ovaries.MethodsA simple in vitro three dimensional (3D) ovarian culture system using Matrigel was established to support to an ideal niche, and ovary was alone cultured in the 24-well plate as a control. We also evaluated the effects of UC-MSCs treatment on ovarian function with or without Matrigel support. All thawed ovaries were randomly divided into control group (Matrigel−/UC-MSCs−), Matrigel group (Matrigel+/UC-MSCs−), UC-MSCs group (Matrigel−/UC-MSCs+) and UC-MSCs + Matrigel group (Matrigel+/UC-MSCs+). HE staining was used to detect the histological structure of follicles and TUNEL staining was used to detect cell apoptosis. The number of microvessels was counted to evaluate neovascularization. The mRNA expression of VEGFA, IGF1 and ANGPT2 were detected by RT-PCR. Western blotting was used to measure the expression of GSK-3β, β-catenin and p-β-catenin.ResultsIn the absence of UC-MSCs, 3D culture system supported by Matrigel showed significantly improved follicular development and microvascular number. Additionally, UC-MSCs were also found to effectively improve follicular development and microvascular number regardless of the culture condition used. However, alleviated follicular apoptosis, increased mRNA expression of angiogenesis-related gene and activated Wnt/β-catenin pathway occurred only in the UC-MSCs + Matrigel group. Besides, with the application of IWP-2 in UC-MSCs + Matrigel group, Wnt//β-catenin pathway could be blocked by IWP-2 serving as one of Wnt/β-catenin pathway inhibitors.ConclusionsThis in vitro study showed the beneficial effects of UC-MSCs on thawed ovaries and explored a potential mechanism inducing angiogenesis. In particular, 3D ovarian culture system supported by Matrigel further improved UC-MSCs treatment. The in vitro culture system using Matrigel and UC-MSCs may provide a potential treatment strategy for improving the success rate of thawed ovaries transplantation.

Bio Assembler integrates the state-of-the-art micro robotics and tissue engineering to create 3D tissues in vitro. In Bio Assembler we have different key technologies in different complex structure for creating 3D cellular systems. In this chapter, we have reviewed our recent progress to construct 3D cellular systems. Especially, introduce three key technologies of micro robotics for 3D cellular construction, (1) Cell manipulation by microhand, (2) Construction of 3D lattice by hydrogel fiber and (3) Changeable Cell Culture mold for Advanced Cell Sheet. These proposed new technologies contribute to realize “Bio Assembler”.

In VHF air/ground communication systems, the radio-line-of-sight (RLOS) between a ground station and an aircraft must exist. An aircraft at a certain altitude may be interfered by those signals from an over-horizontal stations using the same frequency band. A simple frequency management method is proposed to solve this problem and to get the efficient frequency-reuse to alleviate the frequency congestion in the air traffic control (ATC), flight services and mobile communication services. The authors apply the concept of the terrestrial mobile cellular system to design the 3D cellular system and find the capacities of systems with different access schemes, such as FDMA (interference free), TDMA, and CDMA. >

Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. The past decade has seen a rapid advancement of the application of 3D cellular systems to cancer research. These 3D tumor models, or tumor organoids, occupy a range of distinct form factors, each with their own strengths and weaknesses, and appropriateness for particular applications. In this chapter we highlight the major categories of tumor organoids and the methods by which they are biofabricated, aiming to provide the reader with an overview of the types of tumor organoids currently employed in cancer research applications.

Cell Mechanical Characterization Based on On-chip Robotics.- Dimensionless Evaluation of Cell Deformability with High Resolution Positioning in a Microchannel.- Real-time Capillary-level Microchannel Flow Analysis Using a Full-pixel Frame-straddling Micro-PIV System.- High-throughput Measurements of Single Cell Rheology by Atomic Force Microscopy.- Discrimination of Cells with Specific Antigens Expressed on Membrane Based on the Dielectrophoresis.- Analysis of Physical Characteristic of Hematopoietic Cells.- Cell Manipulation and Cellular Parts Assembly for Constructing 3D Cellular Systems.- High-throughput Cell Assembly Featuring Heterogeneous Hydrogels Produced by Using Microfluidic Devices.- On-Chip Fabrication, Manipulation and Self-Assembly for Three-Dimensional Cell Structures.- Fabrication of 3D Cellular Tissue utilizing MEMS Technologies.- Photofabrication Techniques for 3D Tissue Construct.- Cell Detachment for Engineering Three-Dimensional Tissues.- Quantitative Evaluation of Cell-Hydrogel Adhesion by Advanced Optical Techniques.- Cell Scooper: A Device for the Rapid Transfer of Living Cell Sheet.- Hydrogel-based Microenvironment for Modulating Gland Tissue Morphogenesis.- Bone Related Cell-stimulating Scaffolds Materials and a 3D Cellular Construct for Hard Tissue Regeneration.- The Visualization of Human Organogenesis from Stem Cells by Recapitulating Multicellular Interactions.- Bionic Simulator Based on Organ-Explant-Chip.- Tempo-spatial Dynamics of Cellular Mechanics.- Four-dimensional Analysis for a Tumor Invasion.- Three-dimensional Mineralized Tissue Formation of Cultured Bone Marrow Stromal Cells.- Cell Sociology Illuminated by Reconstructing Functional Tissue with Cell Sheet Based Technology.

Spheroids are 3D cellular systems largely adopted as model for high‐throughput screening of molecules and diagnostics tools. Furthermore, those cellular platforms also represent a model for testing new delivery carries for selective targeting. The coupling between the 3D cell environment and the nanovectors can be explored at the macroscale by optical microscopy. However, the nanomaterial‐cell interplay finds major action at the single cell and extracellular matrix level with nanoscale interactions. Electron microscopy offers the resolution to investigate those interactions; however, the specimen preparation finds major drawbacks in its operation time and preciseness. In this context, focused ion beam and scanning electron microscopy (FIB–SEM) allows for fast processing and high resolution of the cell‐nanomaterial interface. Here, in fact, a novel approach is shown to prepare large‐area 3D spheroid cell culture specimens for FIB–SEM. Sectioning procedures are explored to preserve the peculiar structure of spheroids and their interaction with magnetic nanovectors. The results pave the way for advanced investigations of 3D cellular systems with nano and micromaterials relevant to tissue engineering, bioelectronics, and diagnostics.

In vitro models fall short of replicating the complex in vivo processes including cell growth and differentiation. For many years, molecular biology research and drug development have relied on the use of cells grown within tissue culture dishes. These traditional in vitro two-dimensional (2D) cultures fail to recapitulate the 3D microenvironment of in vivo tissues. Due to inadequate surface topography, surface stiffness, cell-to-cell, and cell-to-ECM matrices, 2D cell culture systems are incapable of mimicking cell physiology seen in living healthy tissues. These factors can also place selective pressure on cells that substantially alter their molecular and phenotypic properties. With these disadvantages in mind, new and adaptive cell culture systems are necessary to recapitulate the cellular microenvironment in a more accurate manner for drug development, toxicity studies, drug delivery, and much more. Newly developed biofabrication technologies capable of creating 3D tissue constructs can open new opportunities for cell growth and developmental modeling. These constructs show great promise in representing an environment that allows cells to interact with other cells and their microenvironment in a much more physiologically accurate manner. When transitioning from 2D to 3D systems, there is the need to translate common cell viability analysis techniques from that of 2D cell culture to these 3D tissue constructs. Cell viability assays are critical in evaluating the health of cells in response to drug treatment or other stimuli to better understand how these factors effect the tissue constructs. As 3D cellular systems become the new standard in biomedical engineering, this chapter provides different assays used to assess cell viability qualitatively and quantitatively in 3D environments.

Random packing of digitized particles

Owing to complex microenvironmental conditions, it is challenging to reflect the actual biological responses of tissues or the body in a two-dimensional (2D) cellular system. In the present study, a low-attachment-cultivation technique was employed to establish a highly sensitive 3D human-hamster hybrid (AL) model to study the mutagenic effects of environmental pollutants. The results showed that the established 3D system has apparent organizational characteristics. The average diameter and average cell number of the 3D cells were approximately 240 μm and 1500, respectively. The expression of stemness and cell-junction genes (biomarkers for 3D cells) was higher than that in 2D cells. The present study analyzed the mutagenic effects of the environmental carcinogens arsenite and silver nanoparticles using the established 3D system to demonstrate its efficiency in mutagenic assessment. The results showed that the mutagenic effects of arsenite (10 μM) and silver nanoparticles (10 μg/mL) were 70 ± 3 and 99 ± 7 per 105 survivors, respectively. These values were much lower than those from 2D AL cells and comparable to those from the in vivo system. These results suggest that the developed 3D-cell-culture model based on the 2D AL cellular system more effectively reflects the actual gene-mutation frequency of mutagens in vivo.

Neural cells reside in a three-dimensional (3D) microenvironment where the structure of the nerve circuitry is shaped by the morphology and stiffness of the surrounding extracellular matrix. In this environment, regular exposure to varying mechanical forces triggers biochemical signals during cell mechanotransduction, which plays a crucial role in physiological processes and disease progression. This highlights the need for a flexible 3D platform that integrates cell culture with real-time observation of mechanotransduction. Herein, a 3D flexible and stretchable gold electrode was developed by using a 3D polydopamine-coated polydimethylsiloxane (PDMS)/Ni foam (PDA/PDMS/Ni foam) scaffold as the template. The 3D porous network, combined with the biocompatibility and electrochemical properties of gold nanostructures, enables the electrode to serve as a scaffold for culturing PC12 cells, promoting dopaminergic neural networks with high cell viability. Moreover, the flexible and stretchable 3D Au electrode functioned as a highly sensitive electrochemical sensor for monitoring dopamine (DA) released from the PC12 cell neural networks under mechanical stimuli, which represents the first exploration of continuous DA stimulation at the cellular level in Parkinson's disease. This study presents a promising approach for designing multifunctional 3D stretchable electrochemical sensing platforms, offering insights into mechanotransduction-related neuronal signaling and advancing our understanding of neural function within 3D cellular systems.

Universal, statistically scale-invariant regime in 3D cellular systems

BackgroundA major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.Methodology and Principal FindingsWe hypothesize that delivering progenitor cells in an aggregate form would serve to mimic the endogenous state with proper cell-cell contact, and may aid the survival of implanted cells. Microwell methodologies allow for the culture of homogenous 3D cell aggregates, thereby allowing cell-cell contact. In this study, we find that the culture of cardiac progenitor cells in a 3D cell aggregate augments cell survival and protects against cellular toxins and stressors, including hydrogen peroxide and anoxia/reoxygenation induced cell death. Moreover, using a murine model of cardiac ischemia-reperfusion injury, we find that delivery of cardiac progenitor cells in the form of 3D aggregates improved in vivo survival of implanted cells.ConclusionCollectively, our data support the notion that growth in 3D cellular systems and maintenance of cell-cell contact improves exogenous cell survival following delivery into myocardium. These approaches may serve as a strategy to improve cardiovascular cell-based therapies.

In the field of three‐dimensional (3D) cell culture and tissue engineering, great advance focusing on functionalized materials and desirable culture systems has been made to mimic the natural environment of cells in vivo. Mechanical loading is one of the critical factors that affect cell/tissue behaviors and metabolic activities, but the reported models or detection methods offer little direct and real‐time information about mechanically induced cell responses. Herein, for the first time, a stretchable and multifunctional platform integrating 3D cell culture, mechanical loading, and electrochemical sensing is developed by immobilization of biomimetic peptide linked gold nanotubes on porous and elastic polydimethylsiloxane. The 3D scaffold demonstrates very good compatibility, excellent stretchability, and stable electrochemical sensing performance. This allows mimicking the articular cartilage and investigating its mechanotransduction by 3D culture, mechanical stretching of chondrocytes, and synchronously real‐time monitoring of stretch‐induced signaling molecules. The results disclose a previously unclear mechanotransduction pathway in chondrocytes that mechanical loading can rapidly activate nitric oxide signaling within seconds. This indicates the promising potential of the stretchable 3D sensing in exploring the mechanotransduction in 3D cellular systems and engineered tissues.

Brain organoid technology has transformed both basic and applied biomedical research and paved the way for novel insights into developmental processes and disease states of the human brain. While the use of brain organoids has been rapidly growing in the past decade, the accompanying bioengineering and biofabrication solutions have remained scarce. As a result, most brain organoid protocols still rely on commercially available tools and culturing platforms that had previously been established for different purposes, thus entailing suboptimal culturing conditions and excessive use of plasticware. To address these issues, we developed a 3D printing pipeline for the fabrication of tailor-made culturing platforms for fluidically connected but spatially separated brain organoid array culture. This all-in-one platform allows all culturing steps—from cellular aggregation, spheroid growth, hydrogel embedding, and organoid maturation—to be performed in a single well plate without the need for organoid manipulation or transfer. Importantly, the approach relies on accessible materials and widely available 3D printing equipment. Furthermore, the developed design principles are modular and highly customizable. As such, we believe that the presented technology can be easily adapted by other research groups and fuel further development of culturing tools and platforms for brain organoids and other 3D cellular systems.

Quantification of collagen contraction in three-dimensional cell culture.