3D Cell Culture
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A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

 

 

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Lab-Grown Kidney Buds

Lab-Grown Kidney Buds | 3D Cell Culture | Scoop.it

Mini-kidneys this week (November 17) joined a growing group of functional organ progenitors, or buds, when scientists from the U.S. and Spain described the differentiation of human stem cells into renal progenitor-like cells in Nature Cell Biology. Other groups had previously created liver buds and cerebral organoids using human stem cells.

 

 

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High-throughput drug screening in 3D

Scientists in China have developed a simple microchip that enables quick and inexpensive high-throughput screening of potential drug candidates in 3D cell cultures.
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Three-dimensional insights into dermal tissue as a cue for cellular behavior

Scar formation after injury is a big problem, which influences the skin function and esthetic appearances. Recent researchers have hinted many directions, one of which has shown that scar formation is related to the loss of integrity in dermal tissues. The structure of dermal tissue, which contains mostly collagen, is not only crucial for the mechanical stability of skin, but also acts as a dermal template, providing contact guidance for regulating cell behavior and restoring normal structure and function to skin that has been damaged by injury. These findings suggest a series of questions. How does contact guidance regulate cell behavior? What is the three-dimensional (3D) architecture of the dermal tissue? How does the native 3D architecture influence cell behavior in vivo? In this paper, combing our recent research, we will review the recent advances in this field, that is, the phenomenon of contact guidance and explore the possible mechanism behind it.

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Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells | 3D Cell Culture | Scoop.it

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.

 

 

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Potential of 3D cell-based models for regenerative medicine explored in new webinar

Potential of 3D cell-based models for regenerative medicine explored in new webinar | 3D Cell Culture | Scoop.it
Presented by Dr Elad Katz, a senior scientist at AMSBIO, a new on-demand webinar explores the potential of 3D cell-based models for regenerative medicine and drug discovery.
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Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis(1,2). Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation(3). Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells(4,5). Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis(6,7). Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior(8). However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible(8). For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

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Danish Researchers Create 3D Culture Method And Grow Miniature Pancreas

Danish Researchers Create 3D Culture Method And Grow Miniature Pancreas | 3D Cell Culture | Scoop.it
The new 3D method allows the cell material from mice to grow vividly in picturesque tree-like structures.
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Non-invasive measurement of cell viability in 3-dimensional cell culture construct

Non-invasive measurement of cell viability in 3-dimensional cell culture construct | 3D Cell Culture | Scoop.it

In this work, a non-invasive measurement technique for the quantitative determination of cell viability in a three-dimensional (3D) cell culture construct is proposed. This technique is based on on-site electrical impedance measurement. A microfluidic chip with a 3D culture chamber is fabricated to demonstrate this technique. In vitro 3D cell culture has been interpreted for faithfully representation of the in vivo cellular responses in 3D cell culture construct is normally time-consuming and labor-intensive. In this study, the microfluidic chip consists of a culture chamber, in which a pair of vertical electrodes at its opposite sidewalls was embedded, and a fluidic channel for drug perfusion. Cancer cells encapsulated in agarose gel were loaded into the culture chamber to perform 3D cell culture under the perfusion of culture medium and anti-cancer drug in different concentrations (6, 12, 18, and 24 µg/ml) for 2 days. Since higher drug concentration led to more cell damage or death, the total impedance magnitude of the culture construct was shown to be reasonably proportional to the anti-cancer drug concentration. Moreover, cell proliferation can be also monitored using this technique. The proposed measurement method can determine cell viability without affecting the cellular behaviors during culture. It has a high potential to develop a fast and easy measurement compared with the conventional cellular analysis techniques.

 

 

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In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

Background

Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.

Results

We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.

Conclusions

This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.

 

 

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Three-dimensional neuron–muscle constructs with neuromuscular junctions

Three-dimensional neuron–muscle constructs with neuromuscular junctions | 3D Cell Culture | Scoop.it

This paper describes a fabrication method of muscle tissue constructs driven by neurotransmitters released from activated motor neurons. The constructs consist of three-dimensional (3D) free-standing skeletal muscle fibers co-cultured with motor neurons. We differentiated mouse neural stem cells (mNSCs) cultured on the skeletal muscle fibers into neurons that extend their processes into the muscle fibers. We found that acetylcholine receptors (AChRs) were formed at the connection between the muscle fibers and the neurons. The neuron–muscle constructs consist of highly aligned, long and matured muscle fibers that facilitate wide contractions of muscle fibers in a single direction. The contractions of the neuron–muscle construct were observed after glutamic acid activation of the neurons. The contraction was stopped by treatment with curare, an neuromuscular junction (NMJ) antagonist. These results indicate that our method succeeded in the formation of NMJs in the neuron–muscle constructs. The neuron–muscle construct system can potentially be used in pharmacokinetic assays related to NMJ disease therapies and in soft-robotic actuators.

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Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells

Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells | 3D Cell Culture | Scoop.it

Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, PEPCK, and ASGPR-1 was observed. The differentiated phenotype was sustained for more than two weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from embryonic stem cells.

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Gadgets review: Tools for 3D cell culture

Since the biggest shift in cell culture from 2D to 3D, there is a boom of tools providers on the market.
CYTOO 's insight:

Unfortunately, CYTOO is missing... But the company also offers solutions for 3D cell culture!

 

Learn more on www.cytoo.com and in the application notes

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Generation of co-culture spheroids as vascularisation units for bone tissue engineering

Abstract: Cell spheroids represent attractive building units for bone tissue engineering, because they provide a three-dimensional environment with intensive direct cell-cell contacts. Moreover, they allow for co-culture of both osteoblasts and vessel-forming cells, which may markedly increase their survival and vascularisation after transplantation. To test this hypothesis, we generated co-culture spheroids by aggregating different combinations of primary human osteoblasts (HOB), human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) using the liquid overlay technique. Mono-culture spheroids consisting either of HOB or HDMEC served as controls. After in vitro characterisation, the different spheroids were transplanted into dorsal skinfold chambers of CD1 nu/nu mice to study in vivo their viability and vascularisation over a 2-week observation period by means of repetitive intravital fluorescence microscopy and immunohistochemistry. In vitro, co-culture spheroids containing HDMEC rapidly formed dense tubular vessel-like networks within 72 h and exhibited a significantly decreased rate of apoptotic cell death when compared to mono-culture HDMEC spheroids. After transplantation, these networks interconnected to the host microvasculature by external inosculation. Of interest, this process was most pronounced in HOB-HDMEC spheroids and could not further be improved by the addition of NHDF. Accordingly, HOB-HDMEC spheroids were larger when compared to the other spheroid types. These findings indicate that HOB-HDMEC spheroids exhibit excellent properties to preserve viability and to promote proliferation and vascularisation. Therefore, they may be used as functional vascularisation units in bone tissue engineering for the seeding of scaffolds or for the vitalisation of non-healing large bone defects.

 

 

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Three-dimensional Aggregates of Mesenchymal Stem Cells: Cellular Mechanisms, Biological Properties, and Applications

Three-dimensional Aggregates of Mesenchymal Stem Cells: Cellular Mechanisms, Biological Properties, and Applications | 3D Cell Culture | Scoop.it

Mesenchymal stem cells (MSCs) are primary candidates in cell therapy and tissue engineering and are being tested in clinical trials for a wide range of diseases. Originally isolated and expanded as plastic adherent cells, MSCs have intriguing properties of in vitro self-assembly into three-dimensional (3D) aggregates reminiscent of skeletal condensation in vivo. Recent studies have shown that MSC 3D aggregation improved a range of biological properties including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition. Hence, formation of 3D MSC aggregates has been explored as a novel strategy to improve cell delivery, functional activation, and in vivo retention to enhance therapeutic outcomes. This article summarizes recent reports of MSC aggregate self-assembly, characterization of biological properties, and their applications in preclinical models. The cellular and molecular mechanisms underlying MSC aggregate formation and functional activation are discussed, and the areas that warrant further investigation are highlighted. These analyses are combined to provide perspectives for identifying the controlling mechanisms and refining the methods of aggregate fabrication and expansion for clinical applications.

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Three-dimensional perfused cell culture

Three-dimensional perfused cell culture | 3D Cell Culture | Scoop.it

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.

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3D in vitro tissue models and their potential for drug screening

3D in vitro tissue models and their potential for drug screening | 3D Cell Culture | Scoop.it

Introduction: The development of one standard, simplified in vitro three-dimensional tissue model suitable to biological and pathological investigation and drug-discovery may not yet be feasible, but standardized models for individual tissues or organs are a possibility. Tissue bioengineering, while concerned with finding methods of restoring functionality in disease, is developing technology that can be miniaturized for high throughput screening (HTS) of putative drugs. Through collaboration between biologists, physicists and engineers, cell-based assays are expanding into the realm of tissue analysis. Accordingly, three-dimensional (3D) micro-organoid systems will play an increasing role in drug testing and therapeutics over the next decade. Nevertheless, important hurdles remain before these models are fully developed for HTS.

Areas covered: We highlight advances in the field of tissue bioengineering aimed at enhancing the success of drug candidates through pre-clinical optimization. We discuss models that are most amenable to high throughput screening with emphasis on detection platforms and data modeling.

Expert opinion: Modeling 3D tissues to mimic in-vivo architecture remains a major challenge. As technology advances to provide novel methods of HTS analysis, so do potential pitfalls associated with such models and methods. We remain hopeful that integration of biofabrication with HTS will significantly reduce attrition rates in drug development.


Read More: http://informahealthcare.com/doi/abs/10.1517/17460441.2013.852181
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Stem Cell Differentiation and Lumen Formation in Colorectal Cancer Cell Lines and Primary Tumors

Single cancer stem–like cells (CSC) from colorectal cancers can be functionally identified by their ability to form large lumen-containing colonies in three-dimensional Matrigel cultures. These colonies contain the three types of differentiated colorectal epithelial cells, and single cells obtained from them can reproduce themselves and form tumors efficiently in immunodeficient mice. In this study, we show how hypoxia affects these CSC-derived lumens to control differentiation of stem-like cells and enterocytes via the homeobox gene CDX1. Lumens were identified by F-actin staining and they expressed many characteristics associated with normal differentiated intestinal epithelium, including brush border enzymes, polarization, and tight junctions. RNA interference–mediated silencing of CDX1 reduced lumen formation. Inhibitory effects of hypoxia on lumen formation and stem cell differentiation, including suppression of CDX1 expression, could be mimicked by inhibiting prolyl-hydroxylases that activate HIF1, suggesting that HIF1 is a critical mediator of the effects of hypoxia in this setting. Cell line–derived lumens were phenotypically indistinguishable from colorectal tumor glandular structures used by pathologists to grade tumor differentiation. Parallel results to those obtained with established cell lines were seen with primary cultures from fresh tumors. This in vitro approach to functional characterization of CSCs and their differentiation offers a valid model to study colorectal tumor differentiation and differentiation of colorectal CSCs, with additional uses to enable high-throughput screening for novel anticancer compounds. Cancer Res; 73(18); 5798–809. ©2013 AACR.

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A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis | 3D Cell Culture | Scoop.it

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

 

 

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A novel method for 3D culture of central nervous system neurons

A novel method for 3D culture of central nervous system neurons | 3D Cell Culture | Scoop.it

Neuronal signal transduction and communication in vivo is based on highly complex and dynamic networks among neurons expanding in a 3 dimensional (3D) fashion. Studies of cell-cell communication, synaptogenesis and neural network plasticity constitute major research areas for understanding the involvement of neurons in neurodegenerative diseases such as Huntington’s, Alzheimer’s and Parkinson’s disease and in regenerative neural plasticity responses in situations such as neurotrauma or stroke. Various cell culture systems constitute important experimental platforms to study neuronal functions in health and disease. A major downside of the existing cell culture systems is that the alienating planar cell environment leads to aberrant cell-cell contacts and network formation and increased reactivity of cell culture contaminating glial cells. To mimic a suitable 3D environment for the growth and investigation of neuronal networks in vitro has posed an insurmountable challenge. Here we report the development of a novel electrospun, polyurethane nanofiber based 3D cell culture system for the in vitro support of neuronal networks, in which neurons can grow freely in all directions and form network structures more complex than any culture system has so far been able to support. In this 3D system, neurons extend processes from their cell bodies as a function of the nanofiber diameter. The nanofiber scaffold also minimizes the reactive state of contaminating glial cells.

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3D-Culture System for Heart Regeneration and Cardiac Medicine

3D cultures have gained attention in the field of regenerative medicine for their usefulness as in vitro model of solid tissues. Bottom-up technology to generate artificial tissues or organs is prospective and an attractive approach that will expand as the field of regenerative medicine becomes more translational. We have characterized c-kit positive cardiac stem cells after long-term cultures and established a 3D-nanoculture system using collagen scaffolds. By combining informatics-based studies, including proteomic analyses and microarrays, we sought to generate methods that modeled cardiac regeneration which can ultimately be used to build artificial hearts. Here, we describe the use of biodegradable beads or 3D cultures to study cardiac regeneration. We summarize recent work that demonstrates that, by using a combination of molecular analyses with 3D cultures, it is possible to evaluate concise mechanisms of solid tissue stem cell biology.

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High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA

High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA | 3D Cell Culture | Scoop.it

In the past, creating three-dimensional (3D) tumor spheroids that were suitable for high-throughput screening (HTS) was a difficult and often expensive process. We describe how to couple easy, controllable 3D spheroid formation in Perfecta3D® Hanging Drop Plates with the acumen® eX3 high-content imager for rapid, multicolor, whole-well quantification. This process provides researchers with a highly efficient method to achieve physiologically relevant tumor models in an HTS format.

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Introducing 4D tissue engineering

We’ve heard about 3D bioprinting, 3D cell culture and 3D tissue engineered constructs. Creation of three-dimensional tissue constructs in the last decade allowed to advance regenerative medicine tremendously! The most sophisticated 3D stem cell culture could be turned into organ-like structures. Well, we recently ditch 2D, but in the last few years we also realize that 3D is still not a limit. What if we add one more dimension, which allows cells in suspension to self-organize autonomously? Self-assembly is a fourth dimension of tissue engineering.

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Novel 3D Cell Culture Systems

Novel 3D Cell Culture Systems | 3D Cell Culture | Scoop.it

To predict drug response or toxicity, the pharmaceutical industry is increasingly performing smaller-scale validation studies of experimental drug compounds in novel 3D cell culture models intended to mimic more closely the structure, activity, and extracellular environment of tissues in vivo.

 

 

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