**2. Evolution of cells and organ on chip: from 3D culture to organ on chip**

**Figure 6.** (a) Diagram for sample recirculation system on the hybridization chamber and hybridization image of fluo‐ rescence‐labeled target nucleotide [45]. (b) *Left:* Photograph of the microfluidic chip containing shuttle‐flow channels, microvalves and micropumps. The shuttle flow hybridization was realized by controlling the gas ports 1, 2 and 3 auto‐ matically. *Right*: Hybridization specificity assay using four serotypes of Dengue virus under shuttle flow conditions (frequency 2 Hz) in channels. The duration of hybridization process was 90 s and washing time was 30 s [48].

The commercialization of microarray and microfluidic technologies is evolving very fast as demonstrated by the emergence of many start‐up companies due to its state‐of‐art technology. Affymetrix is an example where they generated a new market based on their GeneChip®

Apparently, microfluidics devices have the potential to serve different scientific needs of healthcare and biomedical sectors and as we discussed earlier, their several successful applications have already been reported. The major advantages associated with miniaturized systems are faster/more accurate diagnoses; better epidemiological data for disease modeling; vaccine introduction; and utilization of minimally trained healthcare workers and better use of existing therapeutics but still many hurdles are there in broader applications of microfluidics

However, there is always a silver lining and due to vastly increased interest in global health issues, the current funding climate for the development of diagnostics kits is significantly good.

technology over a 12‐year period.

86 Lab-on-a-Chip Fabrication and Application

systems.

*1.1.2. Challenges for lab‐on‐chip devices*

The process of growing eukaryotic cells *in vitro* was put forth by Harrison in 1907 to investigate the origin of nerve fibers [50] and since then its almost 100 years, these 2D cell cultures have greatly advanced our knowledge of cellular biology. They have been routinely and diligently undertaken in thousands of laboratories worldwide. However, the 2D cell cultures are arguably primitive and do not reflect the anatomy or physiology of a cell or tissue microen‐ vironment in true sense. Two‐dimensional (2D) cell cultures oversimplify the extracellular matrix (ECM) and cell microenvironment and the processes, such as drug delivery, toxicolog‐ ical analysis, gene expression and apoptosis, may not be directly taken up for the *in vivo* experiments from 2D analysis as ECM is completely different in *in vitro* and *in vivo* and cannot be adequately mimicked by 2D cell systems [51, 52]. These limitations of 2D cell culture led to the innovation of 3D cell culture methodologies; the concept that gave birth to the idea of OOC devices. In 3D culture, cells are grown in extracellular matrix, that is, hydrogels, scaffolds or on hanging drops. The cells, growing in third dimension, exhibit enhanced expression of differentiated functions and improved tissue organization but require a multidisciplinary approach and expertise [53, 54].

Generally, spheroids, cell aggregates and cell sheets are the common platforms for 3D culturing [55–60]. Basic objectives for developing 3D cell culture systems vary from engineering tissues for clinical delivery to the development of models for drug screening. It was observed that certain cellular processes of differentiation and morphogenesis for tissue engineering occurred preferentially in 3D instead of 2D.

In one study by Slamon et al., alteration of cellular architecture between 2D and 3D cells was observed in the growth of SKBR‐3 cells that overexpress HER2, an oncogene found to be overexpressed in approximately 25% of breast tumors [61]. Cells grown as 3D spheroids using p‐HEMA‐coated plates had HER2 homodimers form, while in 2D cultures, HER2 formed heterodimers with HER3 [61]. Recently, Choi et al. [62] also reported that human neural stem cells with familial Alzheimer's disease mutations when grown in 3D culture recapitulate both amyloid‐β plaques and neurofibrillary tangles. 3D cell culture more accurately simulates normal cell morphology, proliferation, differentiation and migrations. Similarly, in chemo‐ therapy procedures, a difference in sensitivity to drug exposure was observed in cells grown in 2D or 3D microenvironments [63]. A study by Tung et al. indicated that A431.H9 cells grown in 2D and 3D show differences in viability when treated with the same concentrations of 5‐ fluorouracil (5‐FU) and tirapazamine (TPZ). In the case of 5‐FU, 2D cultures were reduced to approximately 5% viability following a 96‐h treatment (5‐FU; 10 mM), whereas 3D cells treated with the same concentration and duration, showed 75% viability; indicating that these 3D spheroids were more resistant to the antiproliferative effects of 5‐FU [64].

In recent years, an increasing shift in research focus from 2D cells cultures to 3D cell cultures occurred which in turn translated 2D *in vitro* research to 3D *in vivo* animal models.

#### **2.1. Advantages and limitations of 3D cell culture**


**Figure 7** is schematic of various methods of synthesis of 3D culture, including hanging drop, forced floating method, etc.

It is an evolving field and requires further research for its optimization, and therefore, it is evident that some clarity is needed in selecting the best method for the generation of 3D cells from individual cell lines. Additionally, the best established 3D culture methods currently available produce avascular tumor models that failed to mimic the full architecture of *in vivo* tissues and vascularization aspect of tumor development is left out, which is a huge significant part of true tumorigenesis. These limitations are the prime hurdles in the application of 3D culture as potential drug discovery tools.

overexpressed in approximately 25% of breast tumors [61]. Cells grown as 3D spheroids using p‐HEMA‐coated plates had HER2 homodimers form, while in 2D cultures, HER2 formed heterodimers with HER3 [61]. Recently, Choi et al. [62] also reported that human neural stem cells with familial Alzheimer's disease mutations when grown in 3D culture recapitulate both amyloid‐β plaques and neurofibrillary tangles. 3D cell culture more accurately simulates normal cell morphology, proliferation, differentiation and migrations. Similarly, in chemo‐ therapy procedures, a difference in sensitivity to drug exposure was observed in cells grown in 2D or 3D microenvironments [63]. A study by Tung et al. indicated that A431.H9 cells grown in 2D and 3D show differences in viability when treated with the same concentrations of 5‐ fluorouracil (5‐FU) and tirapazamine (TPZ). In the case of 5‐FU, 2D cultures were reduced to approximately 5% viability following a 96‐h treatment (5‐FU; 10 mM), whereas 3D cells treated with the same concentration and duration, showed 75% viability; indicating that these 3D

In recent years, an increasing shift in research focus from 2D cells cultures to 3D cell cultures

**•** Flexible synthesis approach in 3D cell culture allows facile manipulations for cellular

**•** With 3D cell culture systems, study at different states of disease models can be done in a

**•** 3D culturing is more authentic way of monitoring drug metabolism studies instead of 2D. Due to the presence of layers of cells in 3D culture with tightly bind cells as compare to a monolayer in 2D, drug diffusion to cells by blocking or slowing simulate the real barriers

**•** Scaffolds to support 3D cell with simultaneous growth factor, drug or gene delivery can also

**Figure 7** is schematic of various methods of synthesis of 3D culture, including hanging drop,

It is an evolving field and requires further research for its optimization, and therefore, it is evident that some clarity is needed in selecting the best method for the generation of 3D cells from individual cell lines. Additionally, the best established 3D culture methods currently available produce avascular tumor models that failed to mimic the full architecture of *in vivo* tissues and vascularization aspect of tumor development is left out, which is a huge significant part of true tumorigenesis. These limitations are the prime hurdles in the application of 3D

**•** 3D cell culture has direct applications in tissue engineering and regenerative medicine.

occurred which in turn translated 2D *in vitro* research to 3D *in vivo* animal models.

similar tissue microenvironment that may reduce the need of animal testing.

spheroids were more resistant to the antiproliferative effects of 5‐FU [64].

**2.1. Advantages and limitations of 3D cell culture**

microenvironment modeling.

88 Lab-on-a-Chip Fabrication and Application

for drug action.

be synthesized.

forced floating method, etc.

culture as potential drug discovery tools.

**Figure 7.** Schematic of 3D culture synthesis methods. These methods include forced‐floating of cells; hanging drop methods; agitation‐based approaches; the use of matrices or scaffolds; and microfluidic systems [53].

#### **2.2. From 3D culture toorgans on chips: a giant leap toward biomedicine revolution**

In previous section, we discussed the role of 3D cell culture and its significant impact on different fields. The next important step of 3D microfabrication is evolution of integrated OOC microsystems with the ability to mimic key structural, functional, biochemical and mechanical features as well as interactional effect of microenvironment on cell and tissues *in vivo* of living organs in a single device [65]. By definition, OOC devices are microfluidic devices for culturing living cells in continuously perfused, micrometersized chambers in order to model physio‐ logical functions of tissues and organs [66].

Cellular behavior and its interaction with *in vivo* microenvironment is still an unsolved mystery. Advancements in the field of 3D OOC opened entirely new possibilities to create *in vitro* models that reconstitute more complex, 3D, organ‐level structures, with integrated chemical signals and important dynamic mechanical cues. OOC devices not only mimic the cells biomechanical and biochemical behavior in *in vivo* tissue but also predict the interactional effects of microenvironment on cells and tissue functions [58]. This unique ability of OOC devices makes them a potential candidate for drug discovery programs and a boon for healthcare segment. Though this state‐of‐art innovation is in its nascent state, preliminary data obtained had shown promising future of OOC devices with wide applications in biomedical sciences. As a proof of concept, researchers have fabricated two stacked PDMS cell culture chambers separated by permeable synthetic membrane to study polarized functions of various epithelial cells of intestine [67, 68], lung [69], kidney [70], heart [71], etc.

#### **2.3. Basic microfabrication techniques and material for OOC devices**

To mimic *in vivo* organ‐specific microenvironment, OOC devices required high precision and accuracy. Microfabrication techniques are the preferred methodologies to fabricate OOC devices due to feasibility of constructing tissue‐specific environment at microscale. Typical techniques include replica modeling, soft lithography and microcontact printing [52, 66, 72]. **Figure 8** is a schematic representation of these techniques.

**Figure 8.** Schematic of microfabrication techniques. (a) Replica modeling. (b) PDMS stamp for formation of microchan‐ nels [158]. (c) Microcontact printed protein for cell pattering [159].

Replica molding techniques have been used to replicate complex surface relief patterns to produce biomimetic structures that mimic organ‐specific microarchitecture. Lee et al. designed the replica modeling techniques to recreate the artificial liver sinusoid and natural endothelial barrier layer in liver. [73] This was an important breakthrough that successfully reconstituted a tissue‐tissue interface that was a critical element of whole liver organ structure, and was not possible in conventional 3D ECM gel cultures. In other report by Esch et al [74], photolithog‐ raphy was explored to recreate the key aspects of villi structure on microfluidic chambers covered by 3D shaped, porous membranes for models of the gastrointestinal tract epithelium by two‐exposure step fabrication process. As shown in **Figure 9**, complete crosslinking was used to fabricate the chamber and partial with SU‐8 to form the porous membrane. This microdevice could create better *in vitro* models of human barrier tissues, such as the gastroin‐ testinal tract epithelium, the lung epithelium or other barrier tissues with multiorgan "body‐ on‐a‐chip" devices for drug‐screening application.

An array of PDMS microchambers interconnected by 1 μm wide channels was similarly used to enable growth and *in vivo*‐like reorganization of osteocytes in a 3D environment that replicated the lacuna‐canalicular network of bone [76]. In a similar approach, Sudo et al. came up with the idea of a microdevice incorporating ECM gels microinjected between two parallel microchannels to investigate vascularization of liver tissues in 3D culture microenvironments [76], while a compartmentalized microfluidic system for coculturing of neurons and oligo‐ dendrocytes to study neuron‐glia communication during development of the central nervous system was developed by Park et al. [77]

chambers separated by permeable synthetic membrane to study polarized functions of various

To mimic *in vivo* organ‐specific microenvironment, OOC devices required high precision and accuracy. Microfabrication techniques are the preferred methodologies to fabricate OOC devices due to feasibility of constructing tissue‐specific environment at microscale. Typical techniques include replica modeling, soft lithography and microcontact printing [52, 66, 72].

**Figure 8.** Schematic of microfabrication techniques. (a) Replica modeling. (b) PDMS stamp for formation of microchan‐

Replica molding techniques have been used to replicate complex surface relief patterns to produce biomimetic structures that mimic organ‐specific microarchitecture. Lee et al. designed the replica modeling techniques to recreate the artificial liver sinusoid and natural endothelial barrier layer in liver. [73] This was an important breakthrough that successfully reconstituted a tissue‐tissue interface that was a critical element of whole liver organ structure, and was not possible in conventional 3D ECM gel cultures. In other report by Esch et al [74], photolithog‐ raphy was explored to recreate the key aspects of villi structure on microfluidic chambers covered by 3D shaped, porous membranes for models of the gastrointestinal tract epithelium by two‐exposure step fabrication process. As shown in **Figure 9**, complete crosslinking was used to fabricate the chamber and partial with SU‐8 to form the porous membrane. This microdevice could create better *in vitro* models of human barrier tissues, such as the gastroin‐ testinal tract epithelium, the lung epithelium or other barrier tissues with multiorgan "body‐

epithelial cells of intestine [67, 68], lung [69], kidney [70], heart [71], etc.

**2.3. Basic microfabrication techniques and material for OOC devices**

**Figure 8** is a schematic representation of these techniques.

90 Lab-on-a-Chip Fabrication and Application

nels [158]. (c) Microcontact printed protein for cell pattering [159].

on‐a‐chip" devices for drug‐screening application.

**Figure 9.** Porous SU‐8 membranes that are anchored to and span across microfluidic chambers. The membranes are either flat (a and b), or they were dried over sacrificial silicon pillars and take on the shape of the pillars (c and d). (b) A higher magnification scanning electron microscopy image of a flat membrane with 3 μm pores. (d) Close‐up of the 3D‐ shaped membrane imaged in (c). The image reveals the membrane's porous character. The sacrificial silicon pillars can be removed via xenon difluoride etching 3D cell culture of gastrointestinal epithelial cells (Caco‐2) that were grown for 8 days (a, b, c) and 21 days (d, e, f) on porous SU‐8 membranes that were dried on silicon pillars (50 μm wide and 200 μm high) [74].

From their inception, production of these microdevices relied on silicon microfabrication and micromachining techniques. Although widely explored and applied, silicon micromachining is rather complex, costly with limited accessibility to specialized engineers. To overcome these practical hurdles, researchers developed microfluidic systems made of the silicone rubber, poly(dimethylsiloxane) (PDMS), that are less expensive and easier to fabricate, which opened entirely new avenues of exploration in cell biology. [6]

PDMS has several unique properties that make it a perfect choice for the fabrication of microdevices for the culture of cells and tissues. First, PDMS possesses superior gas permeability and flexibility for adequate oxygen supply to cells in microchannels, which eliminates the need for separate oxygenators, commonly required in silicon, glass and plastic device and is particularly important to maintain differentiated function of primary cells of high metabolic demand [54, 78]. PDMS microfluidic systems enabled the formation of viable and functional human tissues.

Excellent optical transparency is prime advantage of PDMS that enabled real‐time monitoring of nitric oxide production and variation in pulmonary vascular resistance in a microfluidic model and cell morphology, tissue repair and reorganization. [79–81]

Moreover, control of cellular parameters is another important phenomenon in designing OOC devices and recent advances in microfabrication techniques have significant contribution toward efficient monitoring and control of cellular responses and study of broad array of physiological factors that wasn't possible with 3D static cultures. Electrical, chemical, me‐ chanical and optical probes for direct visualization and quantitative analysis of cellular biochemistry, gene expression, structure and mechanical responses also can be integrated into virtually any microfabricated cell culture devices and more relevant data can be obtained with these advanced OOC devices. [54, 66]
