microfluidique-organs-sur-puce221

AUTEURS

Écrit par Hans Luboya Kombe avec comme co-auteur Hadrien Vielle sous la supervision de Guilhem Velve Casquillas et Christophe Pannetier.

 

Hans Luboya Kombe

Hans Luboya Kombe étudiant à l’UPEC. Il a effectué des travaux de recherche en culture celulaire 3D et une recherche bibliographique poussée dans le domaine des organes sur puces.

 

Hadrien Vielle

Hadrien Vieille étudiant à l’école polytechnique féminine (EPF). Il a effectué des travaux de recherche en synthèse photochimique et une recherche bibliographique poussée dans le domaine de l’extension de la longévité de la vie.

 

Dr. Guilhem Velve Casquillas

Dr. Guilhem Velve Casquillas est CEO d’Elvesys et Co-fondateur de quatre entreprises innovantes. Il est ancien chercheur en biologie cellulaire (Institut Curie) et en microfluidique (CNRS –ENS).

INTRODUCTION ABOUT 3D CELL CULTURE

3D cell culture methods and applications - a short review-introduction-cells from a 3D cell cultureCell culture consists in growing cells in an artificial environment in order to study their behavior in response to their environment[1]. Different kinds of cell cultures can be found nowadays, and some would be more suited than others depending on its properties and applications.

Amongst them, 3D cell culture has been increasingly used for its new and convenient features compared to other alternative cell culture method. 3D cell culture could be described as the culture of living cells within micro-assembled devices and supports that present a three dimensional structures mimicking tissue and organ specific microarchitecture[2]. In literature, there are some commendable reviews tackling 3D cell culture in-depth such as  John W. Haycock et al. “3D Cell Culture: A Review of Current Approaches and Techniques”, and this review mostly relies on those excellent references.

 

1. 2D VS 3D CELL CULTURE APPROACH

Cell cultures can be realized via 2D or 3D cell cultures techniques, two distinct methods that will briefly be described below.

 

1.1.  A quick look at 2D cell culture


3D cell culture methods and applications - a short review-2D VS 3D CELL CULTURE APPROACH-A quick look at 2D cell culture-2D cell culture image

A SEM 2D cell culture image (from infinitebio.com)

2D cell culture has traditionally been used over the past decades not only to study different cellular types in vitro but also to conduct drug screening and testing. Typically, this monolayer system allows cell growth over a polyester or glass flat surface[3] presenting a medium that feeds the growing cell population. Countless biological breakthroughs occurred thanks to 2D cell culture[4]. However due to its simplicity, this model can’t accurately depict and simulate the rich environment and complex processes observed in vivo such as cell signaling, chemistry or geometry[5]. Consequently, data gathered with 2D cell culture methods could be misleading and non-predictive for in vivo applications[6]. That’s the reason why scientists have recently been working on three-dimensional biomimetic cell cultures, a technique that represents more precisely the actual microenvironment in which cells can thrive in vivo.

 

 

 

1.2.  3D cell culture: comparing 3D cell culture advantages and downsides with 2D cell culture


3D cell culture methods and applications - a short review-2D VS 3D CELL CULTURE APPROACH-Comparing 3D cell culture advantages and downsides with 2D cell culture-retina generated from 3d cell culture

A nascent retina, generated from 3D cell culture (from flickr.com)

As you may already be aware of, there are different type of 3D cell culture, with each kind of them offering different avantages and drawbacks. Unlike 2D cell culture, 3D cell culture facilitate cell differentiation and tissue organization by using micro-assembled structures and a complex environmental parameters[7]. In fact, in a 3D environment, cells tend to be more subjected to morphological and physiological changes contrary to those grown in a 2D environment[8]. This can mostly be explain by the structuring role and the influence of the scaffold that guide the cells behavior. Researchers have found that the geometry and composition of this cellular support can not only influence genes expression[9] but also enhance cell-cell communication[10]. For instance, some genes promoting cell proliferation are repressed in a 3D cell culture, hence avoiding the anarchic proliferation encountered in 2D cell cultures[11].

3D cell culture also grants the possibility to grow simultaneously two different cellular populations with co-cultures accurately reproducing cellular functions observed within a tissue unlike co-cultures based on 2D cell culture[11]. Interactions existing between cells of interest and others cell are obviously key element in cell functions. That’s the reason why studies focusing on stromal cell (organ connective cell tissues) that play an important part in cancer have been conducted[11]. Finally, using 3D cell culture make it easier to control and monitor the growing cells micro-environment parameters (temperature, chemical gradients, oxygen rate, pH, etc.) to a certain extent while remaining as close to reality as possible thanks to micro-engineering (microfluidic).

One must bear in mind that 3D cell culture is a relatively new technique that researchers have not yet fully grasped the underlying phenomenon and implications[11]. Unfortunately, this culture method presents some noticeable downsides that would most likely be overcome by technological advances[12]. First, some scaffold matrices incorporate compounds from animal or others unwanted sources (virus, soluble factors) that could interfere with the cell culture[12]. Some other matrices provide good cell adherence, making cell removal all the more difficult. Beside, while 3D cell culture could be a cost saving technique that would skip the animal drug testing step in drugs trials, developing automation and reproducible applications still remains a very costly and meticulous process[13].

Unlike 2D monolayer cell culture, 3D cell culture is a much more satisfactory model mimicking in vivo cells behaviors and organization (morphology and physiology). Assembling multi-layer 3D cells structures can only be made possible by using scaffolds, which are micro-organized cell supports that greatly influence cell differentiation and proliferation. Due to its novelty, this technique is not fully comprehended, hence not so easy to handle. Finally, applications development carried out to improve 3D cell cultures can be costly.

2. SCAFFOLDS TYPES IN 3D CELLS CULTURES

 

Scaffolds are key supporting elements in a 3D cell culture, and depending on the conditions and intended goals, different kinds of scaffolds are currently available.

2.1.  Scaffold-based 3D cell culture technique


3D cell culture methods and applications - a short review-SCAFFOLDS TYPES IN 3D CELLS CULTURES-Scaffold-based 3D cell culture technique-scaffold

Examples of a random organized scaffold structure (from inocure.cz)

As pointed out above, scaffolds can be convenient supports for 3D cell culture. Due to their porosity, scaffolds facilitate oxygen, nutriment and waste transportation. Thus, Cells can proliferate and migrate within the scaffold web to eventually adhere on it[24]. As they keep growing, the maturing cells end up interacting with each other and will eventually turn into structures closed to the tissues they were initially originated from[25]. Most of the time, those aggregates are presented as heterogeneous-sized spheres called spheroids: that’s the cell structure generally employed for drug screening and any other 3D cell culture application[26]. Finally, 3D cell culture that use scaffolds offer bigger surface and are generally larger than those not relying on this support[27].

 

 

 

2.1.1  Scaffolds: categories and general composition


3D cell culture methods and applications - a short review-SCAFFOLDS TYPES IN 3D CELLS CULTURES-Scaffolds categories and general composition-scaffold

Mesoporous silica microparticles scaffold (from flicker)

As mentioned in the previous part, depending on the cell types handled, the adequate scaffolds possessing suitable properties and shapes must be associated with. The scaffold layout should match the tissue of interest, reproducing its structure, scale (macro, micro, Nano) and function. However, the bigger and the more complex a scaffold is, the harder extraction for analysis purpose becomes[14]. Besides, to avoid any hindrances (immunity system, fibrosis, weak growth), no matter the category considered, the scaffold handled must provide cell growth support and present biocompatibility properties[15].

Two different scaffold categories can be found: on the one hand, there are in vitro 3D scaffolds for cell culture and experimental applications (drug and cosmetic testing), on the other hand biomedical engineering scaffolds are selected as support for tissues regeneration applications[16]. In the late category, the scaffold can either be definitively implanted to help tissue reconstruction, either be removed or biodegraded after fulfilling its purpose.

Scaffold developed can be hydrogels, membranes (or tube) and 3D matrices. Materials such as metals, glasses and ceramics can constitute a scaffold though polymers, synthetic or natural derived, are preferably used for an easier control of their chemical and structural surface properties[17].

 

2.1.2.  Hydrogels scaffolds


3D cell culture methods and applications - a short review-SCAFFOLDS TYPES IN 3D CELLS CULTURES-Hydrogels scaffolds-scaffold

Cellulose hydrogel scaffold for 3D cell culture (from
industrynews.zursh.com)

Gels, materials showing good mechanical properties, are one of the most used scaffold since they present a tissue-like stiffness and perfectly mimic the extracellular matrix (ECM) in a certain extent[18]. In fact, like any other scaffold, this porous material acts as a rich extracellular matrix that can store nutriments and soluble factors such as cytokines and growth factors which can navigate through the gel[19]. These soluble factors are secreted by cells that can henceforth communicate otherwise than direct contact.

This substitute to native in vivo ECM indeed contain an import amount of water and natural biomolecules[20] such as alginate, gelatin, hyaluronic acid, agarose, laminin, collagen or fibrin. The gelling mechanism to solidify a gel precursor can sometime be tricky, making the preparation and manipulation of gels a difficult task[21].

As you may know, synthetic and natural biopolymer can also be used as gel for 3D cell culture[22]. Depending on the experimental conditions and the intended goal, different kinds of polymer can be found, ranging from inert to biodegradable (polyester, polyethylene glycol, polyamide, polyglycolic acid, polylactic acid). Polymers are easier to manipulate, offering better and wider possibilities to accurately build a scaffold.

2.1.3.  Other types of scaffold


3D cell culture methods and applications - a short review-SCAFFOLDS TYPES IN 3D CELLS CULTURES-Other types of scaffold-scaffold

Ceramics nanofibers scaffold

As previously mentioned, excluding hydrogels, there are a few other kinds of scaffold that can be found, though the vast majority of them are mostly used as tissue engineering scaffolds. One of the materials, bioglass or bioceramic, is a bioresorbable material that improve the regeneration activity of a nascent tissue[23]. On the other hand, porous metallic scaffolds mostly made of titanium (Ti) and tantalum (Ta), have been designed since metals have high compressive strengths and above all excellent fatigue resistance[23].

Non gel Polymer scaffold commonly used are natural polymers for tissue engineering such as collagen, fibrin, alginate, silk, hyaluronic acid, and chitosan. As for synthetic polymers, there is poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and polycaprolactone (PCL). These polymers are preferentially employed since they produce monomers that are easily removed by the natural physiological pathway when implanted[23]. Lastly, composites are also used to build scaffolds. They are made of two or more distinctly different materials (ceramics combined with polymers for instance) developed to takes advantages of both materials properties to meet mechanical and physiological requirements[23].


3D cell culture methods and applications - a short review-SCAFFOLDS TYPES IN 3D CELLS CULTURES-Other types of scaffold-composite scaffold!

Fabrication process and microscopic view of a composite scaffold (from pubs.rsc.org)

 

2.2.  Scaffold-free 3D cell culture techniques


            To generate spheroids, cells aggregates serving as good physiological models, 3D cultures that don’t rely on solid supports (ECM molecules or biomaterials) can also be made[28]. Spheroids obtained using this technique are most of time smaller and less resistant[28]. The main scaffold-free 3D cells cultures techniques are the forced-floating method, the hanging drop method and the agitation based method.

 

Scaffold free techniques include forced-floating methods that use low adhesion polymer-coated well-plates[29]. Spheroid are generated by filling those well-plates with a cell suspension after centrifugation.

Hanging drop methods, a scaffold free techniques adapted by Kelm et al[30], consists in placing a cell suspension aliquot inside a MicroWell MiniTray (Nunc). By inverting the plates (trays), aliquots become droplets presenting cell aggregates on it tips and thus creating compact and homogeneous spheroids.

Last but not least, agitation based approaches using bioreactor can also be a simple alternative method to obtain three-dimensional spheroids. A cell suspension placed into a rotating bioreactor gradually turn isolated cells into aggregates that cannot adhere to the container wall due to the continuous stirring. As a result, a broad range of non-uniform spheroids are eventually generated.

 

Scaffold-free 3D cell culture techniques- from S.Breslin et al.,”Three-dimensional cell culture: the missing link in drug discovery”

 

 

 

Scaffolds are porous support that regulate cell growth and enhance signaling. To achieve this purpose, they facilitate oxygen, nutriments, soluble factors and waste transportation thanks to their singular structures meant to mimic in vivo tissue organization. Scaffold currently made are membranes, matrices and above all hydrogels that present excellent properties. Scaffold used for tissue engineering differ from those found ins 3D cell cultures since presenting distinct features such as biodegradability. Lastly, scaffold-free 3D cells cultures techniques allowing to obtain spheroid are also available: the forced-floating method, the hanging drop method and the agitation based method.

3. CELLS PROPERTIES IN A 3D CELL CULTURE

 

No matter where they originated from, cells grown in a 3D cell culture take on peculiar appearances depending on the tissue they are meant to mimic. They present different properties and interactions discussed below.

 

3.1.  3D cell culture: Cells general appearance


Contrary to 2D cell culture presenting monolayer structures, the three-dimensional method generates multilayer cell aggregates (spheroids) presenting a complex tissue organization similar to what can be observed in vivo[31]. As explained in the previous part, this feat is mainly due to the scaffold structuring role that allows cells to obtain such tissue-like organization. Finally, the general appearance would ultimately depend on the cell types as epithelial tissues grown in 3D culture tend to form polarized sheets[32], just like the skin epidermis.

A 3D Spheroid on a Nanoculture plate (from jsrlifesciences.com)

A 3D Spheroid on a Nanoculture plate (from jsrlifesciences.com)

3D cell culture-CELLS PROPERTIES IN A 3D CELL CULTURE-3D cell culture: Cells general appearance

A SEM Image of a different spheroid (from jsrlifesciences.com)

3.2.  3D cell culture: Cells interactions involved


Cell-to-cell and cell-matrix interactions are both critical parameters to consider in 3D cell cultures. Through direct contact or chemistry, cell can interact with each other and act in synergy to accomplish a defined purpose[33].

3D cell culture methods and applications - a short review-CELLS PROPERTIES IN A 3D CELL CULTURE-3D cell culture Cells interactions involved

cell-cell & cell-matrix interactions (from Tiago G. Fernandes et al.,”High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research”)

Firstly, communication is implemented by cell junctions which are direct intercellular passageway made of protein that form the channel connecting one cell to its neighbors (or one cell to the matrix)[33]. Plus, soluble factors secreted such as cytokines or growth factors are transported to neighboring cells and ECM via direct contact or streamflow, creating a gradient dictated by the molecule lifetime[33]. Those factors would eventually bind a receptor expressed by any other cell (or the same cell for auto regulation), thus triggering a physiological response. Besides, molecules heading to the ECM would be stored by the support and released when needed[33].

Furthermore, cells can additionally thrive thanks to the cell-to-matrix interactions necessary for differentiation and physiological functioning[34] (for a better expression of biomarkers and receptors). This interaction is crucial since some properties gained by the growing cells are only obtainable via the scaffold influence on gene expression and its supporting role on the overall tissue organization[34].

 

3.3  Origin and properties of cells used in 3D cell culture


A broad range of cell types can be sampled to serve as substrate to generate spheroids in a 3D culture. As for tissue engineering, most of the time it requires specific cell types groups such as stem cells, autologous cells, allogenic cells, xenogenic cells, progenitor and multipotent cells. Similarly, 3D culture uses those cells to obtain spheroids. In addition to it, this culture method also covers genetically modified variants of these cells types and also includes cell lines or animal derived-primary cells[35].

3D cell culture methods and applications - a short review-CELLS PROPERTIES IN A 3D CELL CULTURE-Origin and properties of cells used in 3D cell culture-mesenchymal stem cells

Human mesenchymal stem cells attached to fibrin (from flickr.com)

Fearing immune rejection, scientists prefer using autologous or at best allogenic cells though they are not without defects. In fact, while manipulating all the above cell types, one should bear in mind that low availability (a common issue for most cell types in 3D culture), difficulties encountered in their ability to proliferate in vitro or the lack of clinical applications could be major downsides[35]. However, unlike the others cell types, progenitor and multipotent cells showed promising results since not burdened by all the limitations previously mentioned[35]. Indeed, their ability to differentiate into different lineages is a stunning attribute thoroughly studied by many scientists. Researchers are desperately trying to control this process in vitro since there are huge discrepancies between stem cells growin in vivo and in vitro[36]. Stem cells could be induced pluripotent stem cells (IPS) generated in vivo from differentiated cells. They can also be isolated from a vast array of tissues such as the pancreas, cardiovascular system, brain, lung, liver, adipose tissue, and bone marrow[37].

 

 

 

Cells grown in a 3D cell culture can be stem cells, autologous cells, allogenic cells, xenogenic cells, progenitor and multipotent cells. They form multi-layered aggregates called spheroids, showing a tissue-like organization and used in many applications due to their structural proximity to in vivo organ tissue. Lastly, direct and indirect interaction can be observed between the cells or the matrix via cells junctions and soluble factors.

4. 3D CELL CULTURE APPLICATIONS

 

Being amongst other things a tool to study cell behavior in an environment reflecting in vivo conditions, 3D cell culture provide many applications. Here are some of the most well-known and useful one available.

 

4.1.  3D cell culture for tissue engineering


            For general or individual patient use, 3D cell culture has recently been a major breakthrough in the tissue engineering area[37]. In fact, tissue regeneration and reconstruction may have greatly beneficiated from 3D cell culture providing alternative methods to tackle tissue repairing.

3D cell culture methods and applications - a short review-3D CELL CULTURE APPLICATIONS-3D cell culture for tissue engineering

Indeed, instead of using biomaterial, human tissues can be generated working with micro-structured fiber scaffolds in a 3D culture (epithelial-dermal sheets for skin reconstruction for instance)[38]. Unfortunately, tissue engineering can be quite expensive, and regulation around this application are not well defined in some countries[39].

Despites all that, 3D culture remains a trustworthy approach to conduct researches on stem cells and cell differentiation[40]. Understanding convoluted mechanisms such as how osteoblasts turn into osteocytes is now possible and reproducible. In this case, osteogenesis can be triggered by stem cells expressing collagen I markers amongst others[40] (CBFA-1, alkaline phosphatase, osteonectin, osteopontin and JNK2). Thus, cells produced that have gained the desired properties could be injected inside a bone lesion in order rebuilt a damaged tissue[41].

 

 

4.2.  microfluidic 3D cell culture : Organs-on-chips 3D cell culture methods and applications - a short review-3D CELL CULTURE APPLICATIONS-microfluidic 3D cell culture  Organs-on-chips


With the development of microfluidic technologies that allow accurate micro-environmental parameters control (microfluidic), long term and controlled 3D cell culture models were created by using biocompatible microfluidic chips that facilitate tissue manipulation and study. Those organs-on-chips are biomimetic systems that replicate key functions of living organs[42] by  mimicking the microstructures, the dynamic mechanical properties and the biochemical functionalities of organs. Organs-on-chips have changed the way 3D cell culture are done by improving existing methods and by bringing new possibilities:

 

  • 3D cell culture methods and applications - a short review-3D CELL CULTURE APPLICATIONS-microfluidic 3D cell culture  Organs-on-chips-lung-on-chip

    A breathing Lung-on-chip

    To better replicate a living tissue organization and functioning, microstructures made of collagen or polymer-based membraneare built within the chips micro-channels. Unlike traditional 3D cell culture, those structures can actually recreate a function observed in vivo. For instance, a human breathing lung-on-a-chip is a model of the alveolar–capillary. It integrates a flexible polymer membrane allowing movements just like inside a living human lung[48].

  • Manipulating small amounts of fluids in micro-channels thanks to microfluidic enabled organs-on-chips to have of a precise flow control on different scales to “irrigate” the cell culture. Thus, it is possible to create a spatio temporal gradient by bringing nutrients and other elements necessary to the cells[48].
  • Organs-on-chips can also facilitate the creation of  a compartmentalized microfluidic system that enables controlled co-culture and to reconstitute a tissue–tissue interface[48]. Thus, many disease models can develop : For instance, communication between different tissue types in case of malignant breast and brain tumors, or behavior of breast cancer cells when becoming invasive carcinom[48].

This new technology suit perfectly the picky and complex 3D cell culture requirements. Indeed, it helps emulating tissue interfaces to mimic organ functioning while thoroughly monitoring and regulating the microenvironment (chemical signals, fluids flowing, mechanical phenomena)[43]. Hosting a three-dimensional cell culture, the micro-channels are linked to holes in which fluids running through are mixed[44]. Those pathways are accurately controlled by microfluidic output devices (flow measurement and control systems) that regulate the flow in this micro-environment.

By using a microfluidic chip, the controlled cell growth inside micro-channels is guided by a suitable support presenting adequate mechanical, chemical and surface properties[45]. At the end of the day, the result obtained with organs-on-chips are well-organized tissue that are ever more representative of an in vivo organ structure and its processes. Thanks to this time saving biomimetic model, drug development researches are easily conducted in order to study human physiological response on an organ scale or on a systemic scale (multiple organs-on-chips).

 

4.3. Use of 3D cell culture for drug testing


            Drug discovery studies have generally been carried out using animal models for more than 30 years[46]. At first, this practice was a manageable routine task in the pharmaceutical industry. However, as time went by, the more drug molecules were available the more expensive high-throughput drug screening became and the longer the time required to conduct these test was. Along with this phenomenon, arose ethical controversies concerning animal drug testing that didn’t even translate well into human applications. Since then, 3D culture has solved those issues in a certain extent by providing drug responses quite similar to what happens in vivo unlike 2D or animal cell culture[47]. In fact, some studies carried out showed that cells grown in 3D culture can be more resistant to drugs treatments while showing promising results using other culture methods[47].

3D cell culture methods and applications - a short review-3D CELL CULTURE APPLICATIONS-Use of 3D cell culture for drug testingConsequently, 3D cell culture can also be described as a cost-effective/time saving culture technique for drug screening since it considerately reduces drug trials periods while making them more precise or and targeted[47]. For instance, using micro-engineering applications (organs-on-chips), cancer therapeutic are getting better, improving the benefit-risk balance by targeting more precisely a particular cell type, a defined bio-mechanism, a precise receptor, etc. Unfortunately, there are still too many drug tests that keep failing due to their inability to provide progression-free survival[47]. Indeed, as closer to in vivo as they may be, there is still a gap between 3D cell culture generated tissues and actual natural ones.

 

 

 

3D cell culture present many interesting applications. Amongst them, tissue engineering specializes in repairing damaged tissues by injecting new one generated by 3D cell culture. This culture methods tries to reduce the gap between in vitro and in vivo drug testing models as much as possible. As a result, there are more and more targeted cancer treatment available. Micro-engineering applications such as organs-on-chips and microfluidic have greatly contributed to the improvement of drug testing process. They provide an accurate control over 3D cell culture microenvironment and allow to study organs physiology more precisely and easily than before.

CONCLUSION ABOUT 3D CELL CULTURE

Cells naturally grow, mature and differentiate in a three dimensional environment. Using 3D culture is an accurate way to reproduce this process in vitro. That’s the reason why scientists have been studying a wide range of cell types including stem cells, autologous cells, allogenic cells, xenogenic cells, progenitor and multipotent cells. Unlike 2D monolayer cell culture, 3D cell culture model can almost perfectly mimic in vivo cells behaviors and organization (morphology and physiology). Assembling multi-layer 3D cells structures can be made possible by using scaffolds or scaffold-free methods. Whether or not scaffold are used, the resulting cells composing spheroids can interact between themselves and the matrix/support via direct and indirect interaction thanks to cells junctions and soluble factors. Nowadays, many applications such as tissue engineering stem from 3D cell culture that also contributes to the improvement of drug testing thanks to microfluidic organ-on-chips. Nevertheless, as 3D cell culture scientists are still trying to get the hang of.

Acknowledgement : This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 690876

REFERENCES

[1] «Three-Dimensional Cell Culture     Rasheena Edmondson et al. ».

[2] Huh, Hamilton, e Ingber, «From 3D Cell Culture to Organs-on-Chips».

[3] «3D Cell Culture: A Review of Current Approaches and Techniques      John W. Haycock».

[4] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[5] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.»  Maddaly Ravi et al

[6] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[7] Huh, Hamilton, e Ingber, «From 3D Cell Culture to Organs-on-Chips».

[8] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[9] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.»

[10] «Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses Daniel J.»  Daniel J. Maltman and Stefan A. Przyborski

[11] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[12] «What are the differences, advantages/disadvantages to 3-D matrices?      cell culture dish»

[13] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[14] «3D Biomatrix White Paper      3D Cell Culture 101».

[15] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[16] «3D Biomatrix White Paper      3D Cell Culture 101».

[17] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[18] «3D Biomatrix White Paper      3D Cell Culture 101».

[19] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[20] Huh, Hamilton, e Ingber, «From 3D Cell Culture to Organs-on-Chips».

[21] «3D Biomatrix White Paper      3D Cell Culture 101».

[22] «Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses Daniel J.»

[23] «Recent advances in bone tissue engineering scaffolds      Susmita Bose et al. ».

[24] «Three-dimensional cell culture: the missing link in drug discovery Susan Breslin Q1 and Lorraine O’Driscoll».

[25] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[26] «Three-Dimensional Cell Culture».

[27] «Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses Daniel J.»

[28] «3D Biomatrix White Paper      3D Cell Culture 101».

[29] «Three-dimensional cell culture: the missing link in drug discovery Susan Breslin Q1 and Lorraine O’Driscoll».

[30] «Kelm, J.M. et al. (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety».

[31] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.» Maddaly Ravi et al.

[32] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[33] «Cell Culture Models in Microfluidic Systems     Ivar Meyvantsson and David J. Beebe».

[34] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.»  Maddaly Ravi et al.

[35] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[36] «Developments in three-dimensional cell culture technology aimed at improving the accuracy of in vitro analyses Daniel J.»

[37] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[38] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[39] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[40] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.»

[41] «3D Cell Culture: A Review of Current Approaches and Techniques       John W. Haycock».

[42] «Three-Dimensional Cell Culture».  Susan Breslinand Lorraine O’Driscoll

[43] Huh, Hamilton, e Ingber, «From 3D Cell Culture to Organs-on-Chips».

[44] «Cell Culture Models in Microfluidic Systems     Ivar Meyvantsson and David J. Beebe».

[45] Huh, Hamilton, e Ingber, «From 3D Cell Culture to Organs-on-Chips».

[46] «3D Cell Culture Systems – Advantages and Applications   Maddaly Ravi et al.»

[47] Edmondson et al., «Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors».

[48] «From 3D cell culture to organs-on-chips       Dongeun Huh et al. ».