Microfluidic On-Chip Biomimicry for 3D Cell Culture: a Fit-For-Purpose Investigation From the End User Standpoint

Figures & data

Figure 1.  Preclinical and clinical models for drug development.

With an increase in system complexity, preclinical models range from 2D petri dish, to 3D in vitro and ex vivo culture systems, to in vivo animal models. Clinical studies are based on human subjects and consist of four testing phases.

CAM: Chicken chorioallantoic membrane assay.

Figure 2.  Key microenvironment homeostasis and spatiotemporal dynamics that are desirable to be modeled in in vitro 3D culture systems.

All microenvironment cues are dynamic, heterotypic and drivers of cell behavior and fate.

ECM: extracellular matrix; GF: growth factor.

Adapted from [82,83].

Figure 3.  Characterization of research area, usage and acceptability of microfluidic culture systems, usage of analytical tools and concerns about new culture technology among biomedical researchers.

(A) The cohort consists of scientists from fundamental, interdisciplinary research and drug discovery industry. (B) Few researchers have previous experience of using complex 3D culture models including microfluidics. (C) Acceptance of 3D culture systems including microfluidics is a predominant response. (D) Popular analytical tools for biomedical scientists include western blotting, PCR, immunofluorescence microscopy, live cell imaging, flow cytometry and ELISA. (E) Potential users are concerned about the reproducibility, standardization, validation, user-friendliness and compatibility of new culture technology.

PCR: Polymerase chain reaction.

Figure 4.  coculture number, extracellular matrix, product type and price expected by researchers.

(A) ACR prefer complex 3D culture systems enabling the coculture of more than four types of cells. ECR require two or three cell types in coculture. (B) ACR prefer 3D scaffold/hydrogel and endogenous ECM. ECR prefer thin coating of ECM molecules and 3D scaffold/hydrogel. (C) ACR prefer more customized culture systems. ECR require standardized products. (D) ACR are willing to pay double or even triple price for 3D culture models. ECR will pay less.

ACR: Advanced-career researcher; ECM: Extracellular matrix; ECR: Early-career researcher.

Table 1. Functions of microenvironment cues and biomimetic examples in various 3D culture systems.

Biomimetic microenvironment cues	Function	3D hydrogel	Polymer scaffold	Microfluidics	Spheroid	3D printing
Spatial organization of cells/ECMs	Reproduce 3D tissue structure	[23,24]	[25,26]	[27,28]	[29]	[30,31]
Chemical gradient	Reproduce gradient of signaling molecules/growth factors	[32,33]	[34]	[35,36]	[37]	–
coculture of heterotypic cells	Enable cell–cell cross-talk	[38,39]	[40,41]	[42–44]	[45,46]	[47,48]
External force stimuli	Enable biomechanical sensing	[49,50]	[51,52]	[53,54]	[55]	[56,57]
ECM	Reproduce structural, mechanical and chemical properties of ECM	[58,59]	[60,61]	[62,63]	[64,65]	[66,67]
Hypoxia	Induce tumor angiogenesis	[68]	[69]	[69–70]	[64]	[71]
Topography	Direct cell motion and confine cell distribution	[72,73]	[60,61]	[74,75]	[76]	[77,78]
Simulated body fluids	Reproduce blood/interstitial flow	–	–	[79–81]	–	–

Typical microenvironment features include spatial organization of cells/ECM, biochemical gradient, co-existence of heterotypic cells, external mechanical stimuli, ECM, hypoxia, topography and body fluids. Individual or a combination of microenvironment cues can be replicated using various 3D culture methods such as hydrogel matrix, fibrous polymer scaffold, microfluidics, multicellular spheroid and 3D printed tissue.

ECM: Extracellular matrix.

Table 2. Characteristics of commercially available 3D culture systems.

3D culture strategy	Commercialized product	Main feature	Maximum culture time	Chemical gradient	Flow	coculture	ECM	Notes	Ref.
Scaffold-based culture	Biomimesys	Cells on top of hyaluronic acid scaffolds; 96-well format	21 days	No	No	No	3D hyaluronic acid-based hydrogel	Highly porous gel (150–200 μm pores); Non-uniform spheroids (50–250 μm)	[96,97]
	LiverChip	Porous membrane separating two compartments; 12-well format	14 days	No	On-chip micropump-controlled recycling perfusion	Heterotypic cells on opposite sides of a membrane	Polymer membrane mimicking liver sinusoid architecture	Limited to liver organ	[98,99]
Microfluidic chips	OrganoPlates (by MIMETAS)	Microfluidics; 384-well format	15 days	Controllable gradient	Perfusion controlled by Phase guide technology	Heterotypic cells in different regions of microchannels	3D hydrogel insertion	Formation of microvasculature (diameter <100 μm)	[100,101]
	SynVivo	Individually packed microfluidic chips	10 days	Controllable gradient	External pump-controlled perfusion	Heterotypic cells/tissues in different regions of microchannels	2D coating of hydrogel/ECM molecules	Linear/bifurcating channels and tortuous networks (diameter >100 μm)	[102,103]
Scaffold-free cell aggregates	InSphero	Self-assembled multicellular spheroids; 96-well format	Weeks	Gradient as a function of depth within spheroids	No	Heterotypic cells aggregated in spheroids	Endogenous ECM	Tight junction; Controllable spheroid size; Limited by cells’ ability to form spheroids	[104,105]
	ExVive3D (by Organovo)	Layer-by-layer bioprinted tissues; available in multiwell format	28 days	Gradient as a function of depth within micro tissues	No	Controllable spatial distribution of heterotypic cells	Endogenous ECM	Tight junction; Controllable tissue size; Wide choice of cell types	[106,107]

Two categories of 3D culture systems are commercially available. Scaffold-based culture models use 3D hydrogel matrixes or polymeric membranes/scaffolds as miniature cell culture niches. Scaffold-free culture can be realized by microfluidics, self-assembled spheroid or direct-printing technologies. Each 3D culture method is able to reproduce a set of microenvironment features.

ECM: Extracellular matrix.

Table 3. Characteristics of microfluidic vasculature generation technologies.

Microfluidic vasculature generation technology	Cross-section	Geometry	Note	Advantages	Ref.
Photolithographic moulding	Planar	>30 μm	Most developed and standardized technology	Fluid Chemical gradient Coculture	[42]
Injection moulding	Quasi-cylindrical	>20 μm	Template molded in sacrificial material	Fluid Chemical gradient Coculture	[35]
Direct-write assembly	Cylindrical	200–800 μm	Template direct-printed from sacrificial material	Fluid Chemical gradient Coculture	[138]
Viscous finger patterning	Cylindrical	100–700 μm	Less controllable vessel geometry	Fluid Chemical gradient Coculture	[140]
Removable rods	Cylindrical	150 μm	Stiff hydrogel to withstand rod removal	Fluid Chemical gradient Coculture	[141]
Laser micromachining	Cylindrical	8 μm	Precise control over small geometry	Fluid Chemical gradient Coculture	[142]

Microfluidic vasculature mimics the geometry of vascular networks and allows for simulated body fluids, gradient and cell coculture. Various techniques have been reported to pattern microfluidic networks for vasculature generation.

Table 4. Principle, advantages and disadvantages of two vasculature generation strategies.

Vasculature generation strategy	Principle	Advantages	Disadvantages
Prevascularization	Endothelial lining of predefined vasculature	Precise control over vasculature architecture Precise control over flow	Simple geometry Low resolution
Angiogenesis-assisted vascularization	Chemical-induced angiogenesis in endothelial-embedded hydrogel	Complex geometry, dense network Cylindrical lumen Self-organized capillaries	Limited to tumor vasculogenesis Low controllability over flow Slow formation of vasculature

Prevascularization refers to the lining of vascular endothelial cells in the lumen of patterned microvasculature, suitable for mimicking veins and arteries of more than 50 μm in diameter. Angiogenesis-based vascularization refers to spontaneous capillary (diameter <20 μm) formation inside extracellular matrix hydrogels.

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