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Multi-culture in microfluidics
Compared to the highly complex microenvironment in vivo, traditional mono-culture of single cell types in vitro only provides limited information about cellular behavior due to the lack of interactions with other cell types. Typically, cell-to-cell signaling can be divided into four categories depending on the distance: endocrine signaling, paracrine signaling, autocrine signaling, and ﻿juxtacrine signaling. For example, in paracrine signaling, growth factors secreted from one cell diffuse over a short distance to the neighboring target cell, whereas in juxtacrine signaling, membrane-bound ligands of one cell directly bind to surface receptors of adjacent cells. There are three conventional approaches to incorporate cell signaling in in vitro cell culture: ﻿conditioned media transfer, mixed (or direct) co-culture, and segregated (or indirect) co-culture. The use of conditioned media, where the cultured medium of one cell type (the effector) is introduced to the culture of another cell type (the responder), is a traditional way to include the effects of soluble factors in cell signaling. However, this method only allows one-way signaling, does not apply to short-lived factors (which often degrade before transfer to the responder cell culture), and does not allow temporal observations of the secreted factors. Recently, co-culture has become the predominant approach to study the effect of cellular communication by culturing two biologically related cell types together. Mixed co-culture is the simplest co-culture method, where two types of cells are in direct contact within a single culture compartment at the desired cell ratio. Cells can communicate by paracrine and juxtacrine signaling, but separated treatments and downstream analysis of a single cell type are not readily feasible due to the completely mixed population of cells. The more common method is segregated co-culture, where the two cell types are physically separated but can communicate in shared media by paracrine signaling. The physical barrier can be a porous membrane, a solid wall, or a hydrogel divider. If the physical barrier is removable (such as in PDMS or hydrogel), the assay can also be used to study cell invasion or cell migration. Co-culture designs can be adapted to tri- or multi-culture, which are often more representative of in vivo conditions relative to co-culture.

Closed channel multi-culture systems
The flexibility of microfluidic devices greatly contributes to the development of multi-culture studies by improved control over spatial patterns. Closed channel systems made by PDMS are most commonly used ﻿because PDMS has traditionally enabled rapid prototyping. For example, mixed co-culture can be achieved in droplet-based microfluidics easily by a ﻿co-encapsulation system to study paracrine and juxtacrine signaling. Two types of cells are co-encapsulated in droplets by combining two streams of cell-laden agarose solutions. After gelation, the agarose microgels will serve as a 3D microenvironment for cell co-culture. Segregated co-culture is also realized in microfluidic channels to study paracrine signaling. Human alveolar epithelial cells and ﻿microvascular endothelial cells can be co-cultured in compartmentalized PDMS channels, separated by a thin, porous, and stretchable PDMS membrane to mimic ﻿alveolar-capillary barrier. Endothelial cells can also be co-cultured with cancer cells in a monolayer while separated by a 3D collagen scaffold to study endothelial cell migration and capillary growth. When embedded in gels, ﻿salivary gland adenoid cystic carcinoma (ACC) cells can be co-cultured with ﻿carcinoma-associated fibroblast (CAF) in a 3D extracellular matrix to study stroma-regulated ﻿cancer invasion in the 3D environment. If juxtacrine signaling is to be investigated solely without paracrine signaling, a single cell coupling co-culture microfluidic array can be designed based on a cellular valving principle.

Open channel multi-culture systems
Although closed channel microfluidics (discussed in the section above) offers high customizability and biological complexity for multi-culture, the operation often requires handling expertise and specialized equipment, such as pumps and valves. In addition, the use of PDMS is known to cause adverse effects to cell culture, including leaching of oligomers or absorption of small molecules, thus often doubted by biologists. Therefore, open microfluidic devices made of polystyrene (PS), a well-established cell culture material, started to emerge. The advantages of open multi-culture designs are direct pipette accessibility and easy fabrication (micro-milling, 3D printing, injection molding, or razor-printing – without the need for a subsequent bonding step or channel clearance techniques). They can also be incorporated into traditional cultureware (well plate or petri dish) while remaining the complexity for multi-culture experiments. For example, the ‘monorail device’ which patterns hydrogel walls along a rail via spontaneous capillary flow can be inserted into commercially available 24-well plates. Flexible patterning geometries are achieved by merely changing 3D printed or milled inserts. The monorail device can also be adapted to study multikingdom soluble factor signaling, which is difficult in traditional shared media co-culture due to the different media and culture requirements for microbial and mammalian cells. Another open multi-culture device fabricated by razor-printing is capable of integrating numerous culture modalities, including 2D, 3D, Transwell, and spheroid culture. It also shows improved diffusion to promote soluble factor paracrine signaling.