3d cell culture a review of current approaches and techniques pdf

3d Cell Culture A Review Of Current Approaches And Techniques Pdf

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A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions.

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Three-dimensional 3D culture systems are becoming increasingly popular due to their ability to mimic tissue-like structures more effectively than the monolayer cultures. In cancer and stem cell research, the natural cell characteristics and architectures are closely mimicked by the 3D cell models. Thus, the 3D cell cultures are promising and suitable systems for various proposes, ranging from disease modeling to drug target identification as well as potential therapeutic substances that may transform our lives. This review provides a comprehensive compendium of recent advancements in culturing cells, in particular cancer and stem cells, using 3D culture techniques. The major approaches highlighted here include cell spheroids, hydrogel embedding, bioreactors, scaffolds, and bioprinting.

Three‑dimensional cell culture: A powerful tool in tumor research and drug discovery (Review)

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In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Innovations in biomaterials and stem cell technology have allowed for the emergence of novel three-dimensional 3D tissue-like structures known as organoids and spheroids. As a result, compared to conventional 2D cell culture and animal models, these complex 3D structures have improved the accuracy and facilitated in vitro investigations of human diseases, human development, and personalized medical treatment.

Due to the rapid progress of this field, numerous spheroid and organoid production methodologies have been published. However, many of the current spheroid and organoid production techniques are limited by complexity, throughput, and reproducibility. Microfabricated and microscale platforms e.

Microfabricated and microfluidic devices have been shown to improve nutrient delivery and exchange and have allowed for the arrayed production of size-controlled culture areas that yield more uniform organoids and spheroids for a higher throughput at a lower cost. In this review, we discuss the most recent production methods, challenges currently faced in organoid and spheroid production, and microfabricated and microfluidic applications for improving spheroid and organoid generation.

Specifically, we focus on how microfabrication methods and devices such as lithography, microcontact printing, and microfluidic delivery systems can advance organoid and spheroid applications in medicine. Animal models and conventional two-dimensional 2D cell culture models have long been used to understand human physiology and pathology 1. Though these models have propagated numerous scientific advances, their application in modeling human physiology and pathology is limited.

Animal models are inherently limited in mimicking human-specific biology due to the existing physiological differences between humans and animals. While a monolayer culture of human cells can be a window to human-specific biology, the simplicity of 2D cell culture does not reflect the complexity and cellular diversity of the tissues in vivo.

In addition, our access to adult or human embryonic tissues is minimal due to ethical considerations. These limitations have led to advancements in materials and manufacturing techniques combined with stem cell technology to generate 3D human tissue-like models known as organoids and spheroids 2 , 3. Organoids are three-dimensional cell culture models that self-organize into complex organ-like tissues 4.

Spheroids are 3D culture systems that can be used to model multicellular tumors; more broadly, spheroids can be defined as cell aggregates cultured on nonadherent substrates 5 , 6. Consequently, they have become groundbreaking systems to study human development, disease progression, and treatment, as well as to develop personalized medicine approaches that are not possible with animal models.

Typically, spheroids are formed from cancer cell lines or dissociated cell clusters from tumor tissue in nonadherent substrates Fig. Even though organoid models can be generated from mince tissue containing epithelial cells, a large number of organoid model protocols use stem cells as the cellular source for organoid production Fig.

Stem cells are a particular type of cell and are defined by their ability to self-renew as well as their potential to make more specialized cell types.

Strikingly, stem cells can give rise to differentiated progenies that self-organize into tissues that recapitulate the form and functions of the organ 7. The cells do so by autocrine and paracrine signaling as well as via exposure to a specific extracellular matrix ECM 8. Though several spheroids and organoid production techniques have been introduced recently, there are still some challenges to overcome in their production Table 1.

In particular, the reproducible production of organoids remains challenging, as their production is a complex multistep procedure that depends on multiple variables such as cell type, cellular state, and growth 7.

Spheroid production is hindered by the lack of size uniformity 9. In this review, we will discuss stem cell types, traditional techniques used for the generation of human organoid and spheroid models and their shortcomings.

Our primary focus is on emphasizing state-of-the-art microtechnology-based platforms for the production of organoids and spheroids, their advantages and applications in microfabricated and microfluidic-assisted spheroid and organoid models. Stem cells can be classified into three groups: i embryonic stem cells ESCs , ii induced pluripotent stem cells iPSCs , and iii adult stem cells.

Human ESCs can be derived from spare embryos that are not used for fertility treatments. After isolation, human ESCs can be propagated virtually to unlimited numbers while maintaining the potential to generate any differentiated cell type in the adult body, a remarkable property known as pluripotency. Similar to ESCs, iPSCs are pluripotent cells that are generated by reverting differentiated somatic cells to embryonic pluripotency through cellular reprogramming.

When given the right signaling cues, both ESCs and iPSCs can be instructed to form 3D organoids from a variety of tissues such as the optic cup, liver, and brain 17 , 18 , In addition to their unrestricted developmental potential, iPSCs allow for cellular reprogramming of somatic cells from specific individuals to generate their genetically matched personalized organoid models. This approach holds great promise for precision medicine.

Unlike ESCs and iPSCs, adult stem cells are multipotent cells that can generate a few specialized cell types in the body. Tissue-specific adult stem cells are essential for maintaining homeostasis of the adult tissues by generating specialized cell types of that tissue.

This property can be used to coax adult stem cells into forming 3D organoid models that closely resemble their tissue of origin. A notable example is single intestinal stem cells that can generate organoid models with a structure strikingly similar to that of intestinal epithelium and can be expanded in vitro indefinitely Similar adult stem cell-derived organoid models have been generated from other tissues such as mammary glands, lung, and prostate The choice of stem cells for organoid production, to a large extent, depends on the downstream applications, tissue accessibility, and expertise of the researchers.

In the following sections, we will focus on current approaches for organoid production, their shortcomings and the application of microtechnology-based methods to improve organoid production Table 1. This method has been utilized to generate mouse and human prostate organoids, human ovarian tissues, and human and mouse hepatocyte organoids 22 , 23 , In this method, extracted adult stem cells are plated on Matrigel, a commonly used ECM protein mix, and maintained under culture conditions.

When a specific cell type basal or luminal is desired, cells are stained with antibody, sorted with a fluorescence-activated cell sorting FACS system, and subsequently plated on separate Matrigel dishes. This method generates genetically and phenotypically similar organoids In these models, the fact that cell—ECM interactions drive cell organization is exploited.

The ECM is usually replicated with different natural or artificial hydrogels, which include Matrigel, alginate, collagen, laminin, fibrin, and polyethylene glycol PEG 2 , In this technique, ECM agents can be plated, crosslinked, or mixed with the cell suspension. This method provides the ability to monitor cell biological processes such as cell adhesion, migration, and chemotaxis in a tissue-like setting A drawback of this method is the reproducible generation of a scaffold that represents the composition of the ECM that is naturally present in the tissue The composition of the natural hydrogel is closer to that of the in vivo ECM; however, the production of natural hydrogels is not highly reproducible.

As a result, each batch of the hydrogel can have different mechanical properties, which in turn can affect and alter formation of the organoids 2. The production of purified ECM components such as collagen and laminin is reasonably reproducible, but they do not represent the complexity of the ECM in the tissue. Synthetic hydrogels allow for more defined mechanical and biochemical properties. However, they require the addition of agents that upregulate cellular processes such as adhesion and growth Figure 2a shows a schematic of the steps involved in this ECM scaffold method.

As an example Fig. In addition, organoid LEMgel promotes the expression of mature hepatocyte markers to a level closer to that of human liver when measured by quantitative RT-PCR.

In another example, primary lung cancer cells were grown on agarose via the liquid overlay method In this study, cells were embedded in collagen before the formation of a 3D model of lung cancer.

These models can be potentially useful in testing the efficacy of anticancer drugs. A comprehensive schematic on the methods used for the generation of organoids or spheroids including a extracellular matrix scaffold, b spinning bioreactor, c hanging drop, d low-adherent cell culture plates, and e magnetic levitation method.

Altered and reproduced with permission of MDPI These liver organoids were achieved by seeding hepatocarcinoma, human mesenchymal, and endothelial cells in a liver-derived 3D ECM hydrogel termed LEMgel.

Reproduced with the permission of Wiley This particular example showed how embryoid bodies were formed from pluripotent stem cells and placed into the spinning flask to produce the kidney organoids. Reproduced with the permission of MDPI Suspension cultures are an alternative three-dimensional 3D construct method. Suspension cultures make use of incorporating agents that increase the suspension viscosity or use agitation systems. For example, the addition of carboxymethyl cellulose increases the viscosity of suspension cultures 30 , Spinner flasks or bioreactors are used for suspension cultures that make use of agitation to avoid cell attachment to petri dish surfaces For spinner flasks, cells are placed in a container that is constantly stirred usually by a stirring bar.

Bioreactors consist of rotating cell culture containers instead of stirring bars The shear force that cells experience in the bioreactor method can potentially affect cellular physiology Because bioreactors come in different sizes, spheroids of different sizes are possible.

However, there is a drawback that they are heterogeneous in shape. While both spinner flasks and bioreactors induce shear forces on cells, the shear force is not as significant as in the spinner flasks Figure 2b depicts the procedure for developing spheroids using the spinning bioreactor method.

As an example, Fig. These kidney organoids were derived from IPSCs grown in low-attachment plates to form embryonic bodies The simplicity of this method allows for scalable production of kidney organoids that mimic gene expression and cell biological features of the kidney in vivo.

Brain tissues, referred to as cerebral organoids, were also formed using a spinning bioreactor and embedded neuroectodermal tissues in Matrigel droplets. In this method, embryonic bodies were formed from human pluripotent stem cells grown in low FGF signaling media. Embryonic bodies were induced to form 3D neuroepithelial tissues with striking similarity to the in vivo cortex, expressing markers of different cortical layers with the same spatial pattern of brain development.

Neurons in the cerebral organoids showed neuronal activity, as measured by calcium imaging. This approach allowed microcephaly to be modeled by preparing cerebral organoids from iPSCs of a patient with microcephaly caused by a genetic mutation Additionally, using a rotating wall vessel, spheroids of transformed lung cells bZR-T33 were formed over a period of several weeks, exhibiting immunostaining profiles that are similar to those of human lung tissues Spinning bioreactors allow for batch production of spheroids with a large size range.

The hanging drop method is an air—liquid interface technique Fig. These cells are initially suspended in a droplet of medium and placed on the back surface of a petri dish lid. Droplets are held there as a result of surface tension forces and gravity 36 , The suspended cells and lids are then placed on the petri dish, which contains phosphate buffered saline PBS to avoid the evaporation of droplets The emergence of hanging drop plates HDPs , which create an array of spheroids in a dish, has streamlined the production of spheroids with this method 38 , This platform has also been combined with liquid-handling robotics, enabling the simultaneous manufacturing of a large number of 3D constructs For this method, there are several advantages including simplicity, consistency, lack of requirement of matrices, ability to upscale for high-throughput production, and ability to produce spheroids from a small population of cells.

Microtechnology-based methods for organoid models

As one of the basic techniques utilized to study tumor cell biology, the continual development of tumor cell culture techniques is vital. Traditional cell culture methods use a two-dimensional 2D monolayer. With continuous improvements being made, this method has become a standard technology in life sciences at present. However, due to the inherent flaws of traditional 2D culture, it fails to correctly imitate the architecture and microenvironments of in vivo , which makes 2D-cultured cells different from cells growing in vivo in terms of morphology, proliferation, cell-cell and cell-matrix inter-connections, signal transduction, differentiation and other aspects 1 , 2. In order to improve these simulations of cell microenvironments in vivo , 3D culture has become the next frontier of cell biology research.

Sign in Sign up. Figure 2. Schematic representation of the 3-D cell culture through magnetic levitation. From [ 1 ]. Cells Material and method Results Reference Mouse testicular sperm cells Soft Agar matrix and perfusion bioreactor Complete spermatogenesis [ ] Primary human hepatocytes Multi-compartment capillary membrane-based bioreactor Perfusion culture Maintained stable cell function for 10 days. Applicable system for pharmacological studies based on hepatic drug metabolism.

Overview DOI: Cell culture in two dimensions has been routinely and diligently undertaken in thousands of laboratories worldwide for the past four decades. However, the culture of cells in two dimensions is arguably primitive and does not reproduce the anatomy or. However, the culture of cells in two dimensions is arguably primitive and does not reproduce the anatomy or physiology of a tissue for informative or useful study. Creating a third dimension for cell culture is clearly more relevant, but requires a multidisciplinary approach and multidisciplinary expertise. When entering the third dimension, investigators need to consider the design of scaffolds for supporting the organisation of cells or the use of bioreactors for controlling nutrient and waste product exchange.

3D Cell Culture: A Review of Current Approaches and Techniques

Data correspond to usage on the plateform after The current usage metrics is available hours after online publication and is updated daily on week days. Open Access. Issue OCL. Stem Cells Int.

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In conclusion, I would recommend this book as being very useful to a wide audience …. Chaudhuri, ChemBioChem, Vol. No other words can better tell the reader about this compendium of the most innovative culture techniques we hold to try to reach the ultimate goal of cell culture …. For those who wish to enter the field, some introductory reviews explain the present-day challenges in 3D culture while more detailed protocols talk about bioreactors and agarose constructs.


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