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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Microtechnology-based methods for organoid models

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells.

We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source.

Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Development of novel biomaterials for the in vitro culture of cells in three-dimensional 3D microenvironments has gained traction in recent years [1] — [6].

The motivation behind this development is to compensate for limitations of current two-dimensional 2D cell culture practices.

In particular, 2D plastic or glass substrates are ubiquitously employed to study many biological processes, despite the obvious structural and mechanical differences with the in vivo microenvironment.

In vivo , cells are found in a complex extracellular matrix ECM whose biochemical and physical properties have a significant impact on numerous critical physiological and pathological processes [7]. Significant morphological and biological differences have already been observed between cells grown on 2D versus 3D microenvironments [8] , [9]. It is has been routinely observed that primary cells isolated from tissues will become progressively flatter when cultured on conventional 2D surfaces [10] , [11].

Conversely, cells cultured on 2D surfaces can regain their 3D morphologies when placed into a 3D culture scaffold [12]. Both synthetic and naturally derived materials are currently employed in 3D culture methods, in order to create tuneable scaffolds engineered with specific biochemical and physical properties. As such, there has a great focus on using cellulose as a candidate biomaterial [12] , [15] — [23]. Cellulose has previously been employed as a permeable dialysis membrane and as diffusion limiting membranes within biosensors [24].

As well, previous studies found that cellulose produced by bacteria could support the proliferation of mammalian cells [20] , [25] , [26]. Synthetically produced cellulose scaffolds have also been employed for 3D mammalian cell culture [2] , [19] , [21] , [23]. Myocytes cultured on these synthetic cellulose scaffolds contained periodic myofibrils, a distinct cytoarchitectural element within mature cardiac myocytes [27].

As well, enhanced connectivity, in the form of increased gap junction density, and electrochemical connectivity, resulted from 3D culture, in comparison to cells grown on glass [27]. These examples suggest that cellulose may be a suitable material to support 3D cell growth. Moreover, cellulose is widely available as it is the most common organic polymer, accounting for 1. Apple hypanthium tissue has an internal structure composed of cell walls that encompass pores and air pockets, facilitating the transport of nutrients and water throughout the fleshy tissue.

These naturally developed characteristics, are important in any scaffold employed for 3D cell culture [29]. In order to act as a 3D scaffold, the apple hypanthium tissue must first be decellularized in order to remove existing nucleic acids, lipids, and proteins, producing a purified cellulose scaffold.

Decellularized tissue can then be repopulated with new cells, in order to produce new functional organs. Hearts, kidneys have been decellularized and reseeded with various cells [33] , [41] — [43]. As well, functional bladders and lungs have be produced and transplanted into animals using this technique [44] , [45]. Importantly, decellularized tissue also maintains a well conserved native ECM architecture and cell-ECM binding domains [41].

In this study, we hypothesized that decellularized apple hypanthium tissue might provide an easily produced scaffold for 3D cell culture. The major aim of this study was to demonstrate that mammalian cells would successfully proliferate within a 3D cellulose scaffold in vitro.

Through modification of an existing decellularization protocol, we generated apple-derived cellulose scaffolds for cell culture. We examined how three mammalian cell types mouse NIH3T3 fibroblasts, mouse C2C12 myoblasts and human HeLa epithelial cells proliferated within these scaffolds, for up to twelve weeks. Phase contrast microscopy, laser scanning confocal microscopy and scanning electron microscopy were used to characterize the structure of the scaffolds, cell growth, cell morphology and the influence of the scaffolds on the actin cytoskeleton.

We also modified the surface biochemistry and mechanical properties of the cellulose scaffolds by collagen functionalization, or chemical cross-linking with glutaraldehyde.

Atomic force microscopy was employed to quantify the effect of these modifications on the mechanical properties of the scaffolds. We demonstrate that the 3 mammalian cell lines used in this study were able to proliferate and remain viable in the 3D cellulose scaffold in vitro, achieving cell densities similar to other synthetic and natural biomaterials.

Given the natural porosity and ease of production of cellulose scaffolds, as well as the ability to modify their mechanical properties, we demonstrate that cellulose scaffolds are a potentially useful biomaterial that can be successfully employed for in vitro 3D cell culture.

Only the outer hypanthium tissue of the apple was used. Slices containing visible ovary-core tissue were not used. The slices were then cut into 2. Apple tissue was then decellularized by using a well-established protocol [41] for removing cellular material and DNA from tissue samples while leaving behind an intact and three-dimensional scaffold.

Individual apple tissue samples were placed in sterilized 2. Samples were shaken for 12 hours at RPM at room temperature Fig. B Uniform 1. Slices containing any of the ovary core of the apple were removed. C The apple slices were cut into uniform 2. E The scaffolds were then coated with Type 1 collagen, chemically cross linked with glutaraldehyde or incubated in PBS.

After 6 hours in the incubator the wells were filled with DMEM and cells cultured for up to 12 weeks. Here, we examined cell proliferation and invasion into native, collagen functionalized, or chemically cross-linked cellulose scaffolds.

The scaffold seeding procedure took place in well tissue culture plates. Each well was individually coated with polydimethylisiloxane PDMS to create a hydrophobic surface in order to prevent the adhesion of cells. A solution of curing agent: elastomer Sylgard , Ellsworth Adhesives was poured into each well. Scaffolds were cut into 0. The samples were placed in the incubator for 6 hours to allow the cells to adhere to the scaffolds. At this point, samples containing mammalian cells were then carefully transferred into new well PDMS-coated tissue culture plates.

For continued cell proliferation, the culture media was exchanged every day and scaffolds were moved into new well plates every 2 weeks. The actin cytoskeleton and nucleus of mammalian cells, cultured on glass or within the scaffolds, were stained according to previous protocols [46] , [47].

Briefly, samples were fixed with 3. Samples were then mounted in Vectashield Vector Labs. In order to simultaneously stain the cellulose scaffold and mammalian cells, we first fixed the samples as described above, and then washed them with PBS 3 times. To label the apple cell walls, we used an established protocol described previously by Trueunit et al. The tissue was rinsed again with water and incubated in Schiff reagent mM sodium metabisulphite and 0.

The samples were then washed with PBS. WGA and Hoechst are live cell dyes that label the mammalian cell membrane and nucleus, respectively. The samples were then transferred onto microscope slides and mounted in a chloral hydrate solution 4 g chloral hydrate, 1 mL glycerol, and 2 mL water. Slides were kept overnight at room temperature in a closed environment to prevent dehydration. The samples were then placed in PBS until ready for imaging. We also labelled samples to test for long-term mammalian cell viability.

Samples were then fixed with 3. Individual Hoechst-positive and PI-positive cells were automatically counted using the particle analyzer function on ImageJ. Confocal imaging was performed on an A1R high speed laser scanning confocal system on a TiE inverted optical microscope platform Nikon, Canada with appropriate laser lines and filter sets. Transmitted light images were acquired on an inverted TiE microscope Nikon, Canada with phase contrast optics.

Brightness and contrast adjustments were the only manipulations performed to images. Scaffolds containing mammalian cells were first fixed with 3. Samples were then gold-coated at a current of 15 mA for 3 minutes with a Hitachi E ion sputter device. SEM imaging was conducted at voltages ranging from 2.

Local mechanical properties were measured by 5—15 force-indentation curves collected at 10—15 randomly chosen locations at a rate of 1 Hz. PUNIAS software was used to fit the first nm of indentation to the Hertz model for a spherical indenter, using a Poisson ratio of 0. As described in the Materials and Methods section, apple hypanthium tissue was cut to uniform size and decellularized following established protocols Fig.

Hypanthium tissue was employed as it is rich in cellulose and contains very few cells [52] , [53] Decellularization protocols were employed to ensure the complete removal of any remaining plant cells, nucleic acids and biomacromolecules. After processing the samples, a highly porous structure is observed with phase contrast microscopy Fig. The apple tissue has evolved as a very porous structure, with cell wall cavities observed throughout the sample, allowing for facilitated nutrient transfer throughout.

Cellulose scaffolds were then fixed and dehydrated for SEM imaging. Samples were cut horizontally down the mid-section revealing the interior surface. A highly porous and relatively robust scaffold is clearly observed Fig. In all cases, the cellulose scaffold was the only apparent feature observed in all images, as no other identifiable structures were witnessed i.

A Phase contrast image of cellulose cell wall structure in a decellularized apple tissue sample. The dark lines correspond to distinct cellulose structures which form a three dimensional matrix.

B SEM image of a similar cellulose scaffold revealing its three dimensional nature and large cavities. We employed two post-decellularization functionalization protocols in order to examine the ease of modification of the mechanical properties of these cellulose scaffolds.

The two modifications included functionalization of the scaffold with type I collagen, or chemically cross-linking of the scaffold with glutaraldehyde. These modifications allowed us to control the biochemical environment of the scaffolds, and alter their mechanical properties. AFM was used to quantify the local elasticity of the scaffolds in response to each treatment.

Phase contrast microscopy revealed the presence of mammalian cells in each of the scaffolds compared to the cellulose scaffold presented in Fig. The native tissue and unmodified scaffolds do not display any significant difference in mechanical properties. Phase contrast images of C2C12 cells after two weeks of growth reveal the presence of cell colonies.

3D cell culture

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. As 3D culture systems become more mature and relevant to human and animal physiology, the ability to design and develop co-cultures becomes possible as does the ability to integrate stem cells.


The intention of this review is to provide a general overview of the common approaches and techniques for designing 3D culture models.


3D Cell Culture

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 ….

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source.

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.

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.

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

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. In addition, the progress of employing 3D cell culture systems as a platform for cancer and stem cell research was addressed, and the prominent studies of 3D cell culture systems were discussed.

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. As the intersection between tumor cell biology and tissue engineering, 3D in vitro tumor models simulate the in vivo physiological microenvironment, and may be useful at the pre-clinical development stage to identify potentially successful prototypes and eliminate failures at an early stage. This means that it has potential to bridge the gap between traditional monolayer cell culture and tumor cytology experiments in vivo.

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

Duplicate citations

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.

Рассказ канадца показался ему полным абсурдом, и он подумал, что старик еще не отошел от шока или страдает слабоумием. Тогда он посадил его на заднее сиденье своего мотоцикла, чтобы отвезти в гостиницу, где тот остановился. Но этот канадец не знал, что ему надо держаться изо всех сил, поэтому они и трех метров не проехали, как он грохнулся об асфальт, разбил себе голову и сломал запястье. - Что? - Сьюзан не верила своим ушам. - Офицер хотел доставить его в госпиталь, но канадец был вне себя от ярости, сказав, что скорее пойдет в Канаду пешком, чем еще раз сядет на мотоцикл. Все, что полицейский мог сделать, - это проводить его до маленькой муниципальной клиники неподалеку от парка.

Проехав еще полмили, Сьюзан подверглась той же процедуре перед столь же внушительной оградой, по которой был пропущен электрический ток.

 - Немедленно. Фонтейн поднял голову и произнес с ледяным спокойствием: - Вот мое решение. Мы не отключаемся. Мы будем ждать. Джабба открыл рот.

Хейл сердито посмотрел на обезумевшего сотрудника лаборатории систем безопасности и обратился к Сьюзан: - Я сейчас вернусь. Выпей воды. Ты очень бледна.  - Затем повернулся и вышел из комнаты.

За небольшую плату они обеспечивают анонимность электронной почты, выступая в роли посредников. Это все равно что номерной почтовый ящик: пользователь получает и отправляет почту, не раскрывая ни своего имени, ни адреса. Компания получает электронные сообщения, адресованные на подставное имя, и пересылает их на настоящий адрес клиента. Компания связана обязательством ни при каких условиях не раскрывать подлинное имя или адрес пользователя.

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