Microfluidics‐based 3D cell culture models: Utility in novel drug discovery and delivery research

Abstract The implementation of microfluidic devices within life sciences has furthered the possibilities of both academic and industrial applications such as rapid genome sequencing, predictive drug studies, and single cell manipulation. In contrast to the preferred two‐dimensional cell‐based screening, three‐dimensional (3D) systems have more in vivo relevance as well as ability to perform as a predictive tool for the success or failure of a drug screening campaign. 3D cell culture has shown an adaptive response to the recent advancements in microfluidic technologies which has allowed better control over spheroid sizes and subsequent drug screening studies. In this review, we highlight the most significant developments in the field of microfluidic 3D culture over the past half‐decade with a special focus on their benefits and challenges down the lane. With the newer technologies emerging, implementation of microfluidic 3D culture systems into the drug discovery pipeline is right around the bend.


| I N T R O D U C T I O N
Cell culture, an alternative to organ culture and in vivo animal models, is an integral part of several ongoing studies pertaining to biomedical research including biochemistry, biology, pharmacokinetics, and pharmacodynamic discovery and development of therapeutic drugs, as well as tissue engineering. 1 Cell culture models offer an easily accessible, highly reproducible, and reliable mode of investigation with capability of high throughput screening (HTS). Cell culture studies are essential to make a "go/no-go" decision before proceeding toward further preclinical and human studies. 2 Human body is a very complex system with multitude of cell types interacting with each other for sharing and propagation of crucial information. The physiological cellular network resembles an electronic circuit of a supercomputer which needs integration and coordination of hundreds and thousands of microcomponents (chips) for calculation and analysis of data. Similarly, a cell, basic unit of tissue, works in a coordinated fashion with other cells to carry out its essential functioning.
In diseased conditions, cells start behaving in an abnormal fashion which can be characterized by growth, differentiation, secretion of markers, invasion, migration, or premature death. These abovementioned properties of cells are harnessed to study the effect of drugs, both small and large molecules, and are utilized effectively for discovery and development of new therapeutic molecules. Also, these attributes provide information regarding differences between normal and diseased tissues.
While countless studies delineate the mechanisms of cellular growth and pathogenesis, the actual environment inside the tissues still remains more of an enigma. Broadly speaking, cells grow and arrange themselves in a three-dimensional (3D) format and are elliptical with 100% of their surface area exposed to other cells for vital processes such as cell-to-cell signaling, gene/protein expression, response to external stimuli and growth cycle to name a few. 1 Cell culture, after its discovery in 1907, has observed and underwent many significant changes which has led to a near-perfect modeling of human system. 1 In traditional culture, cells are grown on a flat surface as a monolayer. Culture flasks, wells, and Petri dishes are commonly used to grow them by providing a medium as a source of nutrition at physiological temperature (37 8C). Medium is enriched with serum and glutamine to boost growth and, a cocktail of antibiotics to prevent infections. Depending on the doubling time, cells acquire confluence after a certain period of time and after that they are subcultured to avoid competition among themselves for nutrition. This is done by detaching them from the surface using trypsin and/or ethylenediamine tetraacetic acid (EDTA) and reseeding into new flasks for the further growth of cell line. This protocol is usually termed as "Two-dimensional (2D) Culture." As 2D culture does not mimic the inherent physiological conditions, use of 3D culture systems has come to light to bridge the unfilled gaps. 3 Cells are a product of their 3D complex matrix-based environment which facilitates cellular communications and secretions. In vivo, each cell is 100% exposed to the neighboring cell which is not present in 2D-based culture and hence it limits the predictive accuracy during the experimental and clinical studies. Recently, 3D culture has gained widespread attraction because of its several advantages over 2D culture ( Figure 1).

| A DVA NTAGES OF 3D CELL CULTURE O V E R D C E L L C U L T U R E
The 2D and 3D cell cultures can be compared on the basis of several features which lead to difference in effects including cellular morphology, phenotype, metabolic activity, and cellular functionality.

| Morphology
Cells in 2D culture are typically flat with average thickness of 3 mm whereas in 3D culture, cells are ellipsoids having dimensions of 10-30 mm. Cells grown as monolayers do not show altered morphology as observed in diseased conditions such as cancer or inflammation. For example, 3D culture shows clear differences in the morphology, alignment, integrity, and polarization of human bladder carcinoma cells as compared to 2D culture of the same cells. 5 Human retinal cells show more neurite extension in 3D. 6 Vascular smooth muscle cells show more prominent stress fiber formations and focal adhesions in 3D but not in 2D culture. 7 With an added dimension, 3D cell culture offers a more applicable morphological understanding of the cellular environment providing a deeper insight into the cellular responses and the associated changes to their structure.

| Differentiation
Cellular differentiation is well characterized and evidenced in 3D culture. In contrast, 2D culture is not efficient in predicting the differentiation. As shown by Farrell et al., modulation of osteogenesis of adult rat mesenchymal stem cells could be clearly seen in 3D culture, as marked by expression of collagen type I which was not evident when the cell culture was performed in 2D manner. 8 Also, markers indicative of differentiation and other parameters such as duration, phenotypic changes, state of nondifferentiation can be easily visualized under microscope in 3D culture. 9 While 2D methods have been optimized for most conventional studies; this tool falls short when understanding the progression of cellular differentiation. Due to enhanced in vivo relevance that 3D culture offers, this new multifaceted tool allows a more comprehensive study to understand the nascent cellular behavior.

| Viability
Cells in 2D culture are less viable and more susceptible to apoptosis than in 3D culture. Cells behave differently in 3D culture because of more prominent cell-to-cell interactions. 10,11 Smooth muscle cells are more viable in 3D systems, even under suboptimal conditions (depletion of nutrients). 10 Some cartilage cells show differences in growth kinetics when cultured in 3D systems. 12 Also, cancer cells show more differences related to cell death in response to drugs in 2D/3D systems. 13 The 3D culture promotes more interactions among cells allowing them to remain healthier in suboptimal conditions.

| Response to stimuli
As there are several types of stimuli either triggered by adjacent cells or external factors, cells respond to them in different ways when cultured differently. Lin et al. showed that 3D culture showed no effect on human MCF-10A cell morphology and sensitivity after radiation exposure, while they were found to be sensitive in 2D culture. 4 In another study, Merwin et al. outlined that TGF-b did not exert any antiproliferative effects on human endothelial cells in 3D systems. 14 Osteoblasts, when cultured in 2D system, showed less proliferation in response to shear stress as compared to 3D culture. 15 With the addition of 3D cell culture to the life sciences toolbox, it has been easier to differentiate between the normal and stimuli based responses of cells.

| Drug metabolism
Cells metabolize drugs and secrete metabolic products in a far distinctive manner when cultured in 3D systems. H358 cells showed variable cytotoxicity in response to drugs such as paclitaxel, doxorubicin, and vinorelbine in 3D culture as compared to 2D system. 16 Elkayam et al. demonstrated that hepatocytes secrete more urea and albumin and FIGURE 1 Comparison of 2D and 3D cell culture. Cells grown on conventional 2D surfaces adopt a typical flattened morphology covering mainly x-y plane and have a reduced height in the vertical z plane. In comparison, 3D culture allows more cuboidal morphology and 3D structure, particularly in z plane (modified from Ref. 4) show enhanced resistance in response to drugs in 3D culture. Also, they showed increased CYP p-450 activities in response to addition of drugs. 17 Cancer cell lines such as MCF-7, Lovo, and PC-3 showed increased/decreased chemosensitivity in 3D culture utilizing the large and porous biodegradable microparticles as matrix which suggest the significant roles of cellular architecture, variation in phenotypes, and extracellular matrix (ECM) barrier to drug transport phenomena. 18,19 2.6 | Gene expression and protein synthesis Neuroblastoma cells, when grown in 3D culture model, show altered differential expression of about 1,766 genes, including those relevant to cytoskeleton, ECM, and neurite outgrowth, as compared to 2D culture, differences are attributed to influence of culture material on the gene expression, cell spreading, and neurite growth. 5 Vascular smooth muscle cells showed twofold increased expression of 77 genes and reduced expression of 22 genes in 3D systems because of less stress fibers formation and focal adhesions in 3D matrix. 6 Hybridoma cells showed increased production of monoclonal antibodies suggested by reduced apoptosis and resistance to low-serum environments in 3D fibrous matrix. 12 MCF-7 cell line cells showed increased expression of E-cadherin, catenin and p27 and synthesis of collagen owing to different state of cell adhesion and expression of intercellular adhesion molecules in 3D environment. 18

| Cell functions
Human HepG2 liver cells showed enhanced performance and functional activity in polystyrene scaffold-based 3D culture. 20 In 3D culture of bladder carcinoma cell line, RT112, cells demonstrated well developed cell-cell contacts, a distinct endoplasmic reticulum and marked Golgi apparatus within multicellular spheroid-like structure. 4 Bone marrow stem cells showed enabled calcification and increased alkaline phosphatase activity in 3D network of nanofibers which enabled better attachment, proliferation, and osteogenic differentiation. 21 HER2 over expressing cells were marked with formation of homodimers as opposed to heterodimers in 3D culture which makes them more activated and a switch in signaling pathways close to in vivo systems. 22

| In vivo relevance
Tumors which are characterized with polarized epithelial structures or spheroids with more cell-to-cell contacts are more prominent in 3D culture. 23 In a study by Merwin et al., human endothelial cells demonstrated more tube-like structures mimicking angiogenesis due to enhanced tight junctions and abluminal basal lamina deposition in 3D cultures. 14 In another study, rat hippocampal region was shown to have increased neuron/astrocyte ratio conferred due to stability in 3D-based cultures. 24 Cow articular cartilage cells showed similar in vivo histology in 3D culture. 11 Rat olfactory cells maintained their original spindleshape morphology in 3D collagen scaffolds which provide suitable environment to maintain their morphology and functional phenotypes. 25

| Proliferation
Increased growth rate of mesenchymal stem cells, osteosarcoma cells,

human umbilical vein endothelial cells (HUVEC) and tumor epithelial cells (TEC) cells, and human glioblastoma cells has been reported in 3D
fibrous matrix-based culture models and where they were more protected from shear stress and had lower apoptosis even under nutrient depletion. 9,26,27 However, human neuroblastoma, breast cancer, sheep bone marrow, rat interior tibialis muscle, and airway smooth muscle cells showed decreased proliferation due to differences in morphology, lower contractile protein expression and basal proliferation in 3D cultures. [28][29][30] As discussed above, there has been an argument about the correlation of results obtained from 2D cultures and their relevance to in vivo scenarios, which stems from differential behavior of cells in vitro and in vivo. Naturally in human body, the cells grow in a 3D pattern. In addition to interacting with ECM, the cells interact with other cell types as well, which affects a broad range of cellular functions. 31 Capability to grow cells in a 3D format bridges the gap between in vitro and in vivo conditions, and hence is the most appropriate form of representation of real-life in vivo scenarios. The 3D culture models have become increasingly relevant to the biomedical research, and are continuously being recommended as a "must do" before moving on to the more advanced studies. In the sections below, we will discuss the most common methods used for 3D cell culture, their importance and relevance to biomedical research, and will also try to elaborate on the need for further refinement of the culture models.

| Hanging-drop method
This is a relatively simple and easy to execute method with a reported reproducibility of 100% for producing one 3D spheroid per drop for several cell lines. 32 As reported by Kelm et al., a small volume (20-50 ml) of single cell suspension at a density of 50-500 cells/well is usually pipetted into wells. 33 After seeding the cells, the plate/tray is inverted which turns the aliquot into a hanging droplet of cells. The cells concentrate at the tip of the drop and remain in place due to surface tension, and the spheroids are tightly packed and overall homogenous in morphology. 34 A disadvantage of this method is that larger volume (>50 ml) cannot be used since the surface tension can no longer hold the droplet. Two newer technologies by 3D Biomatrix and InSphero have tried to answer these issues. The 3D Biomatrix demonstrated a 384-well hanging drop plate which can easily support largescale production of spheroids. 35 InSphero modified the above mentioned plate with "trap" technology which allows easy harvesting of cultured spheroids. 35

| Forced-floating method
Forced-float method is a simple yet reproducible method to produce consistent spheroids. Cells grown by this procedure are prevented from attaching to the surface by several modifications, allowing forcefloating and hence promoting cell-to-cell interactions. 35 Ivascu and Kubbies used this method for the rapid production of cancerous and noncancerous spheroids in different types of well plates. 36 In their method, plates were coated with poly-hydroxyethylmethacrylate (poly-HEMA) which prevented adhesion of cells to the surface. Eight types of breast cancer cells were seeded in which some formed tight spheroids while others produced loose aggregates. This problem was solved by adding reconstituted basement membrane to the suspension of cells and, within 24 hr, compact 3D spheroids were formed with enhanced consistency. A 96-well plate is typically used for this method and sizes of spheroids can be manipulated by simply changing the quantity of seeded cells. Another inexpensive alternative is to use agarose for coating purposes, which also enables long-term culture (>20 days) of spheroids. 37 The main concern is time consumed when coating the plates, as the coating polymer needs to be dissolved and autoclaved prior to use. 38 Few precoated plates such as PrimeSurface, Lipidure, and Sumitomo Bakelite are available in market but it should be noted that these expensive plates increase the overall cost of spheroid production. 39,40

| Matrices and scaffolds
Use of ECM to produce 3D spheroids is a relatively easy method. Sterile ECM is commercially available and can be used to culture the cells; (a) to embed and grow cells within the gel and (b) to grow them on top of the gel. 41 ECM plays an important role in enabling the cells to perform better communication with other cells and cell-ECM interaction is vital for proper cellular functions. 42,43 Various types of ECM are commercially available which helps further in designing the appropriate experiments. BD Biosciences has ECM available as Matrigel which has been extensively used in the production of 3D mammospheres and human hepatocarcinoma cells. 32,44 Matrigel is composed of tumorderived basement membrane proteins such as collagen IV, MMPs, perlecan, entactin, laminin, and growth factors, essential for cell differentiation and propagation of signaling cascade. 43 Breast tissue is a highly branched yet organized complex structure comprising of epithelial cells. 45 Culture of MCF-7 cells in Matrigel showed a stromal structure with better interactions with ECM which helped in enhanced cellular signaling. 32,46 Some disadvantages associated with ECM-assisted culture are nonuniformity of spheroids, expensive for large scale productions and batch-to-batch variability. These problems were rectified using ECM in an array-based system which utilizes soft lithography to produce microstructures which acted as wells in/on which cells can be cultured as spheroids.
For scaffold-based 3D culture, collagen, laminin, alginate, and so forth are used to construct prefabricated scaffolds. These scaffolds consist of a network of fibers through which cells can easily migrate  [31][32][33]47,56) near to other cells and attach. 31 As the cells divide and grow, they fill the interstitial space between the fibers producing a 3D-like morphology. Typically, they are known as hydrogels which offer a porous structure which allows prolonged availability of nutrients, drugs, and oxygen necessary for survival along with removal of waste products. This assembly provides appropriate cell culture conditions for better mobility and organization of cells. 31 Various companies such as GE Healthcare, Solohill, Global Cell Solutions have successfully launched microcarrier beads for 3D culture of cells in bioreactors. 31 The main disadvantage associated with such technique is the special equipment required for this type of culture.

| Agitation-based approaches
The basic principle of this approach is that a cell suspension is placed into a container while keeping the suspension in motion. Gentle stirring of rotation is used to provide motion to cells. Due to this, cells do not adhere to the walls and form cell-to-cell interactions.
1. Spinner flask bioreactors: They consist of a container and a stirring element to hold and continuously stir the cell suspension. 47 Size of the container can be varied and hence spheroids of different sizes can be produced. Medium can be changed periodically to ensure long-term culture of cells. Motion of culture fluids assists in providing nutrients to cells and subsequent removal of waste products. 46 Drawbacks associated with spinner systems are altered physiology of cells due to sheer force of stirring bar, requiring a larger amount of culture medium and inconsistency in the sizes of spheroids formed. 48 These issues can be addressed by first culturing the spheroids in agarose coated wells and transferring to spinner flasks. 49 Some commercially available spinner assemblies are from companies like Wheaton and Corning.
2. Rotating cell culture bioreactors: While the functioning of this system is similar to spinner flasks, the whole container is rotated instead of using a stirrer bar/rod. Initially, when cells are in single cell suspension, the culture chamber is rotated at low speed; however, as the cells begin to form larger aggregates, the speed is increased to maintain the spheroids in suspension. Low sheer force is the main advantage of this system. 50 While this system is simple, allowing easy handling, and large-scale and long-term production of spheroids, there is large variability in the size of the spheroids. 51 Synthecon provides commercial rotary cell culture systems.
3. Other bioreactors: There are few more, although not very popular, bioreactor-based 3D culture systems such as rotary perfusion and compression bioreactors. Rotary perfusion system allows a continuous feeding of the cell chamber from external media bottle; cells are retained in the chamber by molecular weight cutoff membrane. 52 Compression bioreactor provides a controllable mechanical and physiological environment for simulating in vivo conditions in vitro, and is generally used in cartilage engineering. 53

| 3D bioprinting
Also known as additive manufacturing, 3D printing of biocompatible materials, constituent cells and supporting structures into functional living organs is gaining momentum in the field of drug discovery and research.
Although very complex and cumbersome, bioprinted heart, cartilages, bones, skin, and vascular grafts have been employed for transplantation purposes. 54 Further research is going on to make this process high throughput so as to utilize in the mainstream drug discovery cascade.

| SUMMARY OF PITFALLS OF CURRENT 3D CULTURE S YSTE MS
While 3D cell culture systems offer state-of-the-art technology for enabling drug development and several other applications, there are many unmet needs and gaps which need to be filled to get a universal standardized and validated system. 55 Applications of 3D culture differ in academia and industries. Academia focusses on biological relevance whereas industries look for more cost-efficient, automated and easily readout systems.
Below are some drawbacks of the currently available 3D culture systems: 1. Existing systems represent static conditions rather than mimicking the biochemically dynamic characteristics of the tissue.

There is a risk of transmission of infections or diseases from
human-/animal-derived materials used to prepare scaffolds.
3. Reproducibility of scaffold-based culture as there is significant batch-to-batch variation. 4. Protocol optimization to isolate proteins from 3D cultured cells is needed.
5. Synthetic scaffolds, PEG-based, are typically inert in nature and require modifications prior to embedding and cell growth. 6. HTS and processing is difficult as sometimes cells or scaffolds demonstrate autofluorescence.
7. More calibration of scaffold-based 3D culture systems as there is significant interaction of drugs/molecules with the materials used to prepare scaffolds/matrices.

| M I C R O F A BR I C A TE D ME T H O D S F O R 3D C U L T U R E
Microfluidic technology came into existence in the 1990s and offered a great and more versatile platform for biological applications. 56 The 3D cell culture techniques, in particular, have been revolutionized by the integration of microfluidic technology. It is also known as Lab-on-a-chip or micro total analysis system and has been used for countless biomedical applications such as in drug discovery and development, toxicity, cell culture, genetic assays, protein studies, intracellular signaling, stem cells, tissue engineering, pathogen detection, to name a few. 57

| Glass-/silicon-based systems
Glass-based systems offer enhanced optical properties which are advantageous in high resolution microscopy. These systems can be used multiple times and in long-term studies due to capability of glass to provide a well-defined stable surface with reproducible and reliable electroosmotic flow. 61 Jang et al. used Tempax glass to prepare a 3D continuous-perfusion microchip system for culturing osteoblasts. Photolithographic etching method was used to design and synthesize the device, which was further tested for drug screening applications for more than 10 days. 62 In another study, Lin et al. Silicon-based systems, on the contrary, are not in wide-spread use because of their high cost and complicated fabrication procedures. Ling et al. used an SU-8 mold mask on a silicon wafer for preparing an agarose based system which was used to deliver essential nutrients and oxygen to cells in hydrogels. 65

| Polymer-based systems
Various polymers such as PDMS, polycarbonate, polystyrene, polymethyl methacrylate (PMMA) have been used as biocompatible substrates for microdevices. Polymer-based platforms are dominated by PDMS because of its permeability to oxygen and cost effectiveness. 66,67 Over the past few decades, microfluidic 3D cell culture has adopted several names, depending on the structural differences, such as microwells, microchannels, micropillars, and cell retention chambers. 68 PDMS has been extensively used to design these devices to facilitate dynamically perfused 3D culture. All of these devices were optimized for the flow of medium and perfusion of oxygen throughout the culture regions which reflected more in vivo like conditions. Typically, there are two ports in the device; (a) one inlet port through which medium is injected to provide the essential growth factors and oxygen; and (b) one outlet port which is used to eject the remaining medium along with metabolic degradation by-products. 69 In some advanced devices, medium-infused channels have been integrated with microwells which enables spatial and temporal investigation of several factors regulating cell differentiation. 70 Some naturally originated polymers such as agarose, fibrin, and collagens have also been used to create microfabricated cell-laden devices for 3D culture. Ling et al. cultured multiple cell types including hepatocytes, and AML 12 in an agarose-based device created by utilizing soft lithography technique. 65 In this regard, they fabricated 1 cm thick cellladen agarose replica molds by cooling a hot solution of agarose to 70 8C. Cell suspension was then mixed and agarose-cell mixture was poured onto a silicone master for gelation.

| Paper-based 3D culture platforms
Whitesides group, after observing requirements of specialized engineering approaches and instrumentation in silica/glass and polymer-based substrates, came up with a relatively simple and cost effective approach, that is, paper-based microfluidic systems. 71 In this technique, chromatographic papers are used to pattern hydrophobic barriers by wax printing.
Then, suspensions of cells are impregnated on the papers. Multiple papers are stacked over each other to mimic the 3D architecture. These papers can later be destacked for layer-by-layer molecular analysis. 72 Several reports suggest culture of cancerous and endothelial cells by this fashion to validate their proliferation profiles. 73 Recently, paper-based devices have been compared with 3D spheroid culture of MDA-MB 231 cells. Whatman filter papers were used to pattern 96 multilayer array consisting cells and were further tested against different cell based assays to provide information regarding their migration ( Figure 5). 71 Spheroids and stacked paper-based 3D culture of cells provided a comparative evaluation of cell density, complex gradients, and proliferation.

| M A T R I CE S F O R MI CR O F L U I D I C 3D C U L T U R E SY S T E M S
Matrix utilized to support 3D culture, also known as Scaffold, plays a pivotal role in assuring better and reproducible growth, differentiation and cell-to-cell signaling. Microfluidic technology has utilized gel-based and gel-free matrices for various applications in the field of biology.

| Gel-supported culture
Mass transport is the concerning factor in the development of effective 3D culture, which is addressed by perfused microfluidic-engineered scaffolds. 74 Of particular interest, hydrogels hold great potential in the development of complex and clinically relevant 3D cellular architecture. 74 The scaffold must promote healthy development of cells, through the transport of respiratory gases, essential nutrients exchange, as well as be amenable to changes in shear-stress when being optimized for structural features. Extracellular proteins such as collagen, fibrin, hyaluronic acid, Matrigel, fibronectin, agarose, poly (ethylene  75 Such factors, hyaluronic acid and collagen, have been used to promote the growth of endothelial cells to study the influence of VEGF on their proliferation and migration. 75 The concentration, perfusion, and diffusion rates were comfortably monitored via microfluidic channels, allowing for easy access to manipulate and study the cells.
Hydrogels provide a number of optimization parameters such as pore size, fiber thickness, gradients, and cell seeding which can be manipulated to develop a robust 3D culture system. 76 80 The neural layers thus generated were more realistic and close to their native counterparts.

| Gel-free systems
Hydrogels often vary in their composition and properties which limit the transport of nutrients and oxygen through thick and dense hydrogels thus leading to possibilities of reduced viability of cells within 3D culture systems. 11 Efforts have been made to get rid of gel-based culture systems to tackle these issues ( Figure 6). In one such attempt, polyethyleneimine-hydrazide, an intercellular polymeric linker, has been used to culture human cancerous cells. 81 Cells were modified for better interaction with hydrazides which led to aggregation of cells without the use of hydrogel matrices.
Microwells for the 3D culture of cells have been employed to decrease the dependency on gel based matrices. 70 In microwells, cells are perfused from bottom of a polycarbonate-based well and medium is supplied upward through the culture wells.
Spheroids can also be cultured in a 3D format using gel-free systems by employing microfluidic approach. 82

| Tissue engineering: organ-on-a-chip technology
Tissue engineering focusses on development of tissues/organ substitutes that maintains/restores/improves the functioning of a tissue or whole organ (Figure 7). Microfluidics technology has emerged as a robust platform for tissue engineering. 86   3. Liver-on-a-chip: Most of the drugs are withdrawn from research pipeline because of severe dose-related toxicity, especially liver toxicity, that is, hepatotoxicity. While in vitro models exist for identifying drug-induced liver toxicity, their utility is drastically limited. Therefore, it is the need of hour to develop an efficient, reliable, accurate, and inexpensive tool for testing liver toxicities. 93 Microfluidics has shown potential to solve the problem by offering on-chip liver tissue models which can maintain metabolic activity and phenotype of the poorly viable hepatocytes. Khetani and Bhatia developed a multiwall micropatterned coculture system comprising of hepatocytes along with endothelial cells, stellar cells, Kupffer cells, and fibroblasts. 94 This chip was able to maintain phenotypic functions for several weeks. It also simulates the morphology of lobules to provide hepatocyte functionality. They   Vunjak-Novakovic and coworkers utilized 3D culture system to study the cell-cell interactions human mesenchymal stem cells and HUVEC. 111 They developed this coculture in a spatially controlled 3D fibrin hydrogel system. They found that stem cells show strong distance dependent migration toward endothelial cells and formed a stable vascular network eventually.

Neural cells:
Neurons play an important role in the signal transduction throughout the brain system and this property has been harnessed to study various neurological disorders such as Alzheimer and Parkinson disease. Neural cells can act as drug testing TABLE 1 Summary of applications of 3D cell culture with reference to organ-on-a-chip technology, 3D cellular aggregates and tissue models for the development and characterization of nanoparticles Culture of brain slices is plagued with necrotic problems. Potter and coworkers developed an interstitial microfluidic perfusion system for supplying oxygenated nutrient medium to brain slices and found that they were viable and functional even after 5 days in vitro while maintaining the in vivo architecture. 115 5.

| 3D tissue models for nanoparticles' development and characterization
Field of nanomedicine or nanoparticles based therapeutics has seen significant spur of advancements mainly focused on development and characterization of customized carrier systems specifically designed to deliver payload of active molecules and diagnostic agents for sustained, pre-programmed and/or targeted applications. [145][146][147] An ideally agents with diverse physicochemical properties. 148,149 Despite such substantial and alluring advantages that nanoparticulate carrier systems have to offer, the "bench-to-bedside" transition of these nanotechnology based formulations has remained very limited so far. 150 This less than impressive commercial success for such advanced nanocarriers could potentially be attributed to the challenges faced during their characterization and subsequently in their bulk scale consistent production. Characterizing the performance of nanoparticles at initial stages of development involves use of conventional in vitro models, mainly 2D cell culture models. Attempts of transition from such over-simplified models which generally provide over-promising outcomes face significant difficulty in verifying results of efficiency and performance of nanoparticles in more complicated in vivo settings.
Information regarding cellular interaction of nanoparticles can be collected using in vitro cell culture models, whereas data related to efficacy and toxicity of nanoparticles can be obtained using animal models. Overall, the use of 3D tissue engineered microfluidic platforms represent an innovative step forward to make high-throughput drug screening and characterization of nanocarriers both faster and inexpensive, while generating information that better relates to human physiology in comparison to conventional in vitro or preclinical animal models.
Nevertheless, several key challenges still need to be overcome to fully incorporate different biological, structural and mechanical features and complexities of an organ in such models. Collective efforts are required to focus on developing a whole body-on-a-chip model capable of reproducing both normal and pathophysiological variations. Although some progress has been made in this direction so far, a lot of work is still needed to fully explore and realize scope and applicability of such technology in studying drugs and nanoparticle for better clinical translation.

| FU TUR E PE RSPECTIVES
Combination of microfluidics and 3D cell culture has great potential to provide efficient methods for biomedical applications, tissue engineering, and drug screening in physiologically relevant micromilieu. However, there are many challenges which need immediate attention. For instance, there is limited access to the cultured cells in microsystems which further becomes tough and complicated while sampling. This requires development of dedicated methods and devices for functional studies and screening. Even after this, commercialization of mature and ready-to-use devices, and making them available to scientists is challenging because of the technical hurdles. In this review, we have described different approaches and techniques for microfluidic 3D cell culture, each of them has its own strengths and weaknesses with respect to mimicking the various aspects of 3D culture. For example, there are a number of methods for 3D spheroid generation but discrepancies occur as different cell lines behave differently when cultured using the same method. Transition from 2D to 3D not only adds one more dimension in terms of shape and structure but also in terms of acquired data, that is, high content imaging of a 3D model acquire stacks of images at high resolution at higher speeds, and hence increasing by a thousand to a hundred thousand the data acquired during one single experiment. So, new ideas and methods must be considered to improve and build on the current drug development process, and achieve success. In the near future, we expect new research in microfluidic 3D cell culture to extend in two directions mainly to improve its robustness and parallelism and to facilitate readout. The first one should be the development of automated, high throughput, reproducible, reliable, cost-effective, and easy-to-use microfluidic 3D cell culture systems. The second direction should be the smooth and hassle-free integration of complicated microfluidic systems holding great in vivo relevance. Furthermore, it is highly expected coming era will see new developments and discoveries in the field of tissue engineering with the advances in microfluidics cell culturing techniques.

| C O N C L U S I O N S
Undoubtedly, 3D culture is a blessing for scientists but there are few issues that if addressed, could change the whole perspective of scientific community. Complexity associated with access to cultivated cells and further sampling for assays is a big problem with microfluidic based 3D culture systems. The current systems lack control of dynamics and spatial presentation of various signals, which requires meticulous attention. There is also a strong need of cost effective and easy-to-use systems as technical issues cast a dark shadow over this novel and fruitful technology. Although, organ-on-a-chip and human-on-a-chip have drawn attention, integration of complicated microsystems that can closely mimic the in vivo environments still need further optimization.
A perfect combination of bioinformatics, systems biology, and engineering may help in overcoming these challenges.