MACS-W:A modified optical clearing agent for imaging 3D cell cultures

Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.

In Vitro Invasive Pattern of Hepatocellular Carcinoma Cell Line HCCLM9 Based on Three-dimensional Cell Culture and Quantum Dots Molecular Imaging

Summary： This study aimed to establish a new in vitro three-dimensional （3D） cell culture and use quantum dots （QDs） molecular imaging to examine the invasive behaviors of hepatocellular carcinoma （HCC） cells. Each well of the 24-well cell culture plate was cover-slipped. Matrigel diluted with se- rum-free DMEM was added and HCCLM9 cells were cultured on the Matrigel. The cell morphological and cell growth characteristics were observed by inverted microscopy and laser confocal microscopy at different culture time. Cell invasive features were monitored by QDs-based real-time molecular imaging techniques. The results showed that on this 3D cell culture platform, HCCLM9 cells exhibited typical multi-step invasive behaviors, including reversion of cell senescence, active focal proliferation and dominant clones invasion. During the process, cells under 3D cell culture showed biological behaviors of spatio-temporal characteristics. Cells first merged on the surface of matrix, then gradually infiltrated and migrated into deep part of matrix, presenting polygonal morphology with stretched protrusions, forming tubular, annular and even network structure, which suggested that HCC cells have the morpho- logical basis for vasculogenic mimicry. In addition, small cell clones with their edges well-circumscribed in early stage, progressed into a large irregular clone with ill-defined edge, while the other cells developed invadopodia. And QDs probing showed MT1-MMP was strongly expressed in the invadopodia. These findings indicate that a novel 3D cell culture platform has been successfully estab- lished, which can mimic the in vivo tumor microenvironment, and when combined with QDs-based mo- lecular imaging, it can help to better investigate the invasive behaviors of HCC cells.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

A Novel <i>in Vitro</i>Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells

In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.

Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Effect of deforolimus and VEGF on angiogenesis in endometrial stromal cells following three-dimensional culture

The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase(ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbedwith a structure resembling the loose connective tissue morphology in a novel 3 D culture system. We confirmed that the 3 D system using CellbedTMaccurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3 D-cultured tongue cancer cell lines than in 2 D cultures. Typically, under conventional 2 D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells.However, in the 3 D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3 D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3 D culture systems using Cellbed? are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Human Endometrial Stem Cells May Differentiate into Schwann Cells in Fibrin Gel as 3D Culture

Damage in central nervous system plays an important role in biological life and causes severe paralysis of limbs and some organs. There are solutions to problems that can be a great revolution in the transplanted spinal cord and nerve injuries. Schwann cells (SCs) have important roles in development, myelination and regeneration in the peripheral nervous system. The applications of SCs in regenerative medicine are limited because of slow growth rate and difficulties in harvesting. Critical to the hypothesis is the experimental fact that human endometrial-derived stem cells (hEnSCs) as multipotent accessible source of cells are known as useful cell candidates in the field of nerve tissue engineering. We decided to use the three-dimensional culture of Schwann cells differentiated from endometrial stem cell in fibrin gel. In this study, we investigate the expression of differentiated Schwann cell markers by exposing of endometrial stem cells with induction media including FGF2/FSK/HRG/RA. Using immunocytochemistry, we show that differentiated cells express S100 and P75 markers. These results show that for the first time, human endometrial stem cells can be differentiated into Schwann cells in 2D and 3D culture. These novel differentiated cells in fibrin gel might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches for nerve repair.

The 3D Cell Culture System in the Study of Tumor-Applications and Prospects

Compared with 2D tumor cell culture, 3D tumor cell culture can better simulate the microenvironment of signal transduction between cells and extracellular matrix. As one of the best cell models in tumor research, it has been widely used in the study of cancer cell morphology, nanotechnology drug delivery system, and anticancer drug screening. The main theme of this paper is to review the previous research of 3D cell culture applying to tumors in vitro and the prospects for the applications of 3D cell culture system.

Investigation of VEGF and PDGF signals in vascular formation by 3D culture models using mouse ES cells

Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.

Application of 3D Scanned Big Data of Large-scale Cultural Heritage Objects Based on Noise-robust Transparent Visualization

Three-dimensional(3D) scanning technology has undergone remarkable developments in recent years.Data acquired by 3D scanning have the form of 3D point clouds.The 3D scanned point clouds have data sizes that can be considered big data.They also contain measurement noise inherent in measurement data.These properties of 3D scanned point clouds make many traditional CG/visualization techniques difficult.This paper reviewed our recent achievements in developing varieties of high-quality visualizations suitable for the visual analysis of 3D scanned point clouds.We demonstrated the effectiveness of the method by applying the visualizations to various cultural heritage objects.The main visualization targets used in this paper are the floats in the Gion Festival in Kyoto(the float parade is on the UNESCO Intangible Cultural Heritage List) and Borobudur Temple in Indonesia(a UNESCO World Heritage Site).

Hepatospheres:Three dimensional cell cultures resemble physiological conditions of the liver

Studying physiological and pathophysiological mechanisms in the liver on a molecular basis is a challenging task.During two dimensional(2D) culture conditions hepatocytes dedifferentiate rapidly by losing metabolic functions and structural integrity.Hence,inappropriate 2D hepatocellular models hamper studies on the xenobiotic metabolism of the liver which strongly influences drug potency.Also,the lack of effective therapies against hepatocellular carcinoma shows the urgent need for robust models to investigate liver functions in a defined hepatic microenvironment.Here,we summarize and discuss three-dimensional cultures of hepatocytes,herein referred to as hepatospheres,which provide versatile tools to investigate hepatic metabolism,stemness and cancer development.

Recent advances in chemically defined and tunable hydrogel platforms for organoid culture

Recent developments in organoid culture technologies have made it possible to closely recapitulate intrinsic characteristics of different tissues under in vitro conditions.These organoids act as a translational bridge between the traditional 2D/3D cultures and the in vivo models for studying the tissue development processes,disease modeling,and drug screening.Matrigel and tissue-specific extracellular matrix have been shown to support organoid development,efficiently;however,their chemically undefined nature,non-tunable properties,and associated batch-to-batch variations often limit reproducibility of the assembly process.In this regard,chemically defined platforms offer wider opportunities to optimize and recreate tissue-specific microenvironment.The present review delineates the current research trends in this sphere,focusing on material perspective and the target tissues(e.g.,neural,liver,pancreatic,renal,and intestinal).The review winds up with a discussion on the current limitations and future perspective to provide a basis for future research.

Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish

In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Nevertheless,functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish.In this study,we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers(Bostrychus sinensis),which are prone to ovotesticular or sterile testicular development,and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional(3D)culture system.Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae.Furthermore,melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway,and thus increased the efficiency in functional sperm production.The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

3D printed lactic acid bacteria hydrogel:cell release kinetics and stability

In this study,a new type of 3 D printed living biological hydrogel was developed by integrating lactic acid bacteria(LAB)into biocompatible and non-toxic polymer materials.Interestingly,the living materials loaded with LAB can be freeze-dried and reused for more than 100 times.The bio-hydrogel can be used to co-culture different LAB and keep its fermentation performance stable in long-term use.The release kinetics model and response surface method were used to simulate and optimize the bacteria release mode in the bio-hydrogel.The results show that the release of bacteria from hydrogel is regulated by the coupling of Fickian diffusion and polymer swelling.The stability of LAB hydrogel was evaluated by reuse experiments.The images of confocal microscopy and scanning electron microscope showed that the bacteria with high cell viability were distributed in the hydrogel and intact structure of the living hydrogel was maintained after 100 times of reuse as yoghurt starter.In conclusion,the 3 D printed LAB bio-hydrogel developed in this study has the advantage of reuse and sustainability,which is expected to open up a new way for the preparation of food culture starter.

Emerging roles of 3D-culture systems in tackling tumor drug resistance

Drug resistance that affects patients universally is a major challenge in cancer therapy.The development of drug resistance in cancer cells is a multifactor event,and its process involves numerous mechanisms that allow these cells to evade the effect of treatments.As a result,the need to understand the molecular mechanisms underlying cancer drug sensitivity is imperative.Traditional 2D cell culture systems have been utilized to study drug resistance,but they often fail to mimic the 3D milieu and the architecture of real tissues and cell-cell interactions.As a result of this,3D cell culture systems are now considered a comprehensive model to study drug resistance in vitro.Cancer cells exhibit an in vivo behavior when grown in a three-dimensional environment and react to therapy more physiologically.In this review,we discuss the relevance of main 3D culture systems in the study of potential approaches to overcome drug resistance and in the identification of personalized drug targets with the aim of developing patient-specific treatment strategies that can be put in place when resistance emerges.

Fabrication of three-dimensional islet models by geometry-controlled hanging drop method

Hanging-drop method has been widely used to fabricate three-dimensional (3D) in vitro tissue models due to its advantages such as being easy to perform, inexpensive, and permitting precise control of cell spheroid formation. The geometry of hanging drop may play a critical role on the formation of cell spheroids, which, however, has not been explored. In this study, we developed a modified hanging-drop platform that enables the production of cell spheroids in a high-throughput manner by controlling hanging drop geometry with defined spreading ring. The surface tension force is proportional to the spreading ring and gravitational force is determined by the droplet volume, and the geometry can be determined by the balance between surface tension and gravity.β-TC-6 cell spheroids with optimized diameters were fabricated as 3D in vitro islet models. The models show morphology similar to primary islets and have functionality that more closely resembles primary islets than two-dimensional cell culture. The developed platform holds great potential for engineering well-controlled in vitro tissue models for various applications such as physiological and pathological studies, drug screening, as well as transplantation for treatment purpose.

Digital Preservation:A New Approach to Protecting Historical and Cultural Towns and Villages

Digital technology has penetrated all aspects of modern life and is pushing society forward by profoundly changing the way people live and think.It can not only eternalize cultural heritage,but also allow relevant knowledge to be spread more quickly and effectively.China is home to a diverse array of historical and cultural towns and villages,which are an important cultural heritage.Digital preservation means leveraging technologies such as mapping,remote sensing,computers,3 D imaging,virtual reality,plus physical and chemical techniques to collect data to monitor,record,restore,and rebuild with the goal of preserving both their physical shapes and cultural legacies.