Lab-on-a-Chip—A Model for Angiogenic Brain Diseases: History Edit

Various factors need to be synergized to model brain angiogenesis in vitro; this involves the coordination of different cell lines (endothelial and the brain cells) [50], the combination of angiogenic factors, and the presence of shear stress and dynamic flow, etc. The conventional monolayer, Transwell, and vessel-like capillary cells cultures grown in a static petri dishes are insufficient for synergizing and completely neglect dynamic flow and shear stress. Hence have proven to be unsuitable for evaluating brain angiogenesis drugs, thus a platform to synergies these factors towards angiogenesis is required. Moreover, the way in which NPs are loaded into well plates was found to alter the cellular adsorption and uptake, respectively [89]. As a result, carefully designed experiments involving NMs are needed. In addition to the above advantages, LOCs also allow the testing of very low drug concentrations in a dose-dependent manner, even in the range of a few nanomoles per liter, which is corresponds to the therapeutic concentration [45]. Further, simultaneous, high throughput screening of different concentration on the same microfluidic platforms is possible [46,49,58]. The possibility of developing precise brain-like vasculature to perform challenging experiments has encouraged a number of researchers to adapt LOCs for their studies, Table 2.

Angiogenic Nanomedicine Screening in LOCs

In order to evaluate the pro/anti-angiogenic effects of NMs, their effect on cell migration, angiogenesis, lumen formation, and their ability to target specific sites needs to be studied. In addition, the cellular uptake, transport via the transcellular/paracellular routes and drug-release kinetics, needs to be evaluated. Various device geometries have been reported for brain vasculature models (Figures 2 and 3); parallel channels (PCs), with interconnecting micro-gaps, [42–45,51,56,90], hollow-microtubes (HTs) [2,47,49,50,52], and others, concentric-ring (CR) channels [54], multi-layered channels [53], hybrid-microchambers [57], and microwells [58]. Each of the above designs has its own advantage in terms of angiogenesis drug screening application.

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Figure 2. The angiogenesis and vasculogenesis microfluidic model, respectively, for potential angiogenic drug screening application. (A) The angiogenesis model [43], i. shows the geometry of the microfluidic device. ii. The device comprises of three parallel microchannel, the endothelial cells are loaded to the top channel (pink), the angiogenic growth factors are added to the bottom channel (blue) and the gradient of the growth factor is generated in the middle channel. 1. Formation of a stable monolayer of the endothelial cells in response to the angiogenic gradient. 2. Cell tip formation followed by 3. The lumen formation. iii. Angiogenic sprouts after four days simulated with different combinations of angiogenic factors, VEGF, phorbol 12-myristate 13-acetate (PMA) and sphingosine-1-phosphate (S1P). iv. a) Angiogenic sprouts after six days of stimulation with VEGF+PMA+S1P, b) VEGF+PMA and b) VEGF+S1P, respectively, and c) close-up of the lumen middle c(i), top c(ii), and cross-section c(iii) and stained against F-actin (red) and nucleus (blue). (B) The vasculogenesis model [42], i. The schematics of the in vitro 3D NVU platform comprising of astrocytes, neurons and the endothelial cells. ii. The perfusable vascular network is formed over a three-day period via vasculogenesis. 1. The vascular network formation in the middle channel, 2. Loading of astrocytes and neurons into the right-side channel. 3. The formation BBB within 5–7 days. BBB: blood–brain barrier.

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Figure 3. (A) Hollow microtube model [2], i. photograph of the PDMS microfluidic device. ii. schematic illustration (center), and immunofluorescence micrographs (right) of the two-channel microfluidic Organ Chip with endothelial cells cultured on all surfaces of the basal vascular channel, and astrocytes and pericytes on the upper surface of the central horizontal membrane in the apical parenchymal channel. iii. At the top pannel, z-stack images of the pericytes (yellow, F-actin staining) and astrocytes (white, glial fibrillary acidic protein (GFAP) staining) in the top channel of the BBB Chip are reconstituted and shown from above; at the bottom panel, a side view of similar stacked images for the lower vascular channel containing endothelial cells (blue, ZO-1 staining). (B) (Reprinted with permission from the publisher) i. The hybrid brain-BBB device, displaying the metabolic flux across the BBB and the brain cells, [57], brain endothelial cells (magenta) are cultured on all four walls of the lower vascular compartment and a mixture of brain astrocytes (blue) and pericytes (yellow) in the top compartment of both BBB chips; neuronal cells (green) and astrocytes (blue) are cultured in the lower compartment of the brain chip. Cell culture medium is flowed into the upper perivascular compartment of BBB chip as an artificial cerebrospinal fluid (CSF) (blue), and cell culture medium mimicking blood is flowed separately through the lower vascular compartment. ii. and iii. The reconstruction of the human BBB chip from confocal fluorescence microscopic images. ii. The endothelial cell monolayer stained for VE-Cadherin (purple), and a mixture of pericytes (F-actin, yellow) and astrocytes (GFAP, blue), (scale bar, 75 μm). iii culture of neurons (β-III-tubulin, green) and astrocytes (GFAP, blue) (scale bar, 100 μm). Figure 3B is reproduced with permission from Maoz, B.M.; Herland, A.; Fitzgerald, E.A.; Grevesse, T.; Vidoudez, C.; Pacheco, A.R.; Sheehy, S.P.; Park, T.E.; Dauth, S.; Mannix, R.; et al. A linked organ-on-chip model of the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells. Nat. Biotechnol. 2018, 36, 865–874. [57].

The PC type LOC (Figure 2), modeling 2D monolayer vascular network, is the most commonly used to model angiogenesis [43,45] (Figure 2A) and vasculogenesis [42,44] (Figure 2B). Usually, five or three microchannels are fabricated (Width, W, 500–1000 um), in the case of vasculogenesis model. The middle channel is loaded with the endothelial cells, and supplied with culture medium through the adjacent channels and the brain cells or fibroblast cells and their respective culture mediums are supplied separately (Figure 2B). In the case of angiogenesis model, the middle channel is supplied with culture medium and the adjacent channels are loaded with endothelial cells (Figure 2A).

The PC type devices enable the study of both the inner and outer surface of the vessels and are best suited for observing angiogenesis. The real-time drug effect on the cell morphology [44,45], cell migration [43–45], lumen growth [45], angiogenic sprouting [43,45], vessel anastomosis [43,44], can be observed and quantified in this model. The lumen perfusability is easily achieved in the 2D monolayer and allows for the injection of drugs into the vasculature through the inlet ports. The formation or inhibition of tumor-associated blood vessel, in response to anti-angiogenic drugs can be precisely quantified [45]. In an in vitro brain spheroid, it is not possible to access the lumens to perform perfusability assays. In such cases, the brain spheroids can be introduced form outside into the vascularize device and anastomosed with the preexisting vasculature, thereby enabling lumen access [44,46]. One of the main drawbacks is that it not possible to install electrodes into the PC devices for TEER measurements.

The HT models (Figure 3A) were used to create 3D BBB, with easy-to-access lumens and are useful for studying the apical side of the BBB. The endothelial cells are loaded into the hollow tube, the cells adhere to the tube walls and form a lumen-like structure. In some cases, the brain cells are grown on the outer surface of the tube and were coupled with the endothelial cells via microchannels [47,52], micropores [2] or extracellular matrix [49,50]. The real-time uptake of NMs and their effect on the TJs [47] and transmigration of the neutrophils [47], antibodies [49], and Q-dots functionalized with Angiopep-2 [2], were easily quantified in this model. The BBB permeability during pathological conditions involving neuroinflammation [47,50] and ischemic obstruction were observed in the HT model [47]. A microrod model, i.e., an inside-out geometry of the HT model, demonstrated that BMEC resists elongation in response to both curvature and shear stress [91]. The cell curvature is directly proportionate to the number of adjacent cells around the perimeter and eventually increase the TJs [91], mimicking more closely the in vivo condition.

Apart from the above mentioned advantages, HT models eliminate the artificial paracellular leaks that occurs in Transwells [52], allowing precise measurement of the NMs perfusability [2]. Further, the HT models not only capture the shear stress and the vessel curvature, it is the most convenient model to integrate electrodes for a real-time measurement of TEER without destroying the cells [2,53].

The CR model is advantageous in mimicking tumor angiogenesis conditions, such as radial oxygen gradient on-chips [54], and are suitable for analyzing drugs targeting tumor mediated-angiogenesis. Another recently reported hybrid BBB-brain on-chip device recapitulated brain-blood/CSF flow and the structural hierarchy of the brain tissues (Figure 3B) [57]. The device screened the effect of Methamphetamine, (anti-angiogenic and psychostimulant drug) on the reversible disruption of BBB. The drug uptake, influx and efflux of the drug across the BBB were demonstrated for their potential to screening NMs.

Design and Fabrication of Brain-Angiogenesis LOCs

LOCs are made of optically transparent polymers, namely, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), SU-8, etc., and fabricated by soft lithography from SU-8 patterned molds. The media reservoir, inlets and outlets can be created with a biopsy punch and other fabrication methods. The polymer chip can be bonded to glass coverslips and petri plates using the plasma bond technique and can be sterilized by autoclave and/or UV. Through simple fabrication techniques, electronics can be integrated into the LOC for various applications. For example, electrodes were fabricated to generate an electric field for evaluating barrier functions, such as permeability [51], and TEER [2,53] respectively. Further, the QDs were used to measure brain cell temperature [92]. In addition, stable drug concentration profiles can be easily generated in a microfluidic platform by controlling the volumetric flow rates across multiple-channels [45,58]. Because of these features, non-destructible, real-time, and simultaneous measurement of various factors, such as BBB permeability, TEER, etc., is possible.

Establishing Brain Vasculature on Chips

In order to vascularize the device, the endothelial cells (see Table 2) are co-cultured with one or more cells constituting the NVU. Although, a mono-culture of endothelial cells alone can produce vasculature [42,44,46,50], a more realistic model requires the co-culturing of astrocytes [43,45,47,51] and pericytes [50], or both [2,49,57]. In recent models, neurons [42,57] and tumor cells [46,54] were also included. Direct contact with the above non-endothelial cells, in particular astrocytes and pericytes were found to produce stable BBB, with high TEER and in vivo like permeability, for a longer time period.

Until recently, primary and immortalized cells from both animals and humans, respectively, were used to develop brain angiogenesis models. The recent development of the gene editing technology, such as CRISPR/Cas9, led to the use of Human induced pluripotent stem cells (HiPSC) derived from both healthy and diseased humans [93]. Although it is very challenging to maintain and standardize HiPSC protocols, microfluidics platforms once again have aided in sustaining more complex, dynamic and controlled environment for using HiPSC [2]. The HiPSCs demonstrated superior barrier function and low inflammatory response when compared with the primary cells [2].

In general, it takes around 3–4 days to vascularize a microfluidic device, the endothelial cells are loaded along with the extracellular matrix, and the cells differentiates and polarizes in response to the growth factors (e.g., VEGF) that is supplied by the culture media or released from the co-cultured cell lines. A monolayer or 3D vascular network is formed depending on the device geometry. Following this, vessel-like structure with a lumen is formed and the perfusability is achieved in a few days and the functional BBB develops within the next 5–7 days [42]. The vasculature inside the LOC can be maintained for many weeks and the BBB integrity for up to a full week [2]. The choice of cell lines, composition of the co-cultures, cell loading sequence, culture media flow rate, and mechanical property of the cell matrix, can be easily optimized based on the final applications. Apart from developing the 2D vascular network, 3D brain micro-spheroids (diameter of 100–600 um) were also used to model tumor angiogenesis [44,46,54,55]. The spheroids were grown through one of these techniques, namely, 3D bio-printing [54,55] or the hanging droplet technique [94]. They can also be grown using the micro-wells [58] or 96-well plate and later transferred into the LOC [44]. Recently, a microfluidic platform that can be integrated with a 96-well plate to develop tumor spheroid angiogenesis model was reported [46].

Important Features of Vascularized LOCs Vs. Petri Dish Models

Dynamic Flow and Shear Stress in Brain-Angiogenesis LOCs

Dynamic flow and shear stress play an important role in the cell differentiation, the expression of the membrane proteins, signal pathways and other barrier properties such as TEER. These factors determine the extent to which the brain-like conditions are mimicked on-chip. Physiologically or pathologically relevant expression of protein/peptide receptors involved in the transcellular and paracellular pathways is very important for the clinical analysis of the NMs. The stress promotes glycalyx formation in the endothelial cells, eventually affecting the charge on the membrane, further the non-uniform shear stress affects the endothelial permeability [95]. Therefore, shear stress is an important factor while studying the cellular uptake of NMs. Unlike in the static petri dish culture, dynamic flow and shear stress can be generated on microfluidic platforms. LOC provides precise control over the flow rate and shear stress when integrated with a syringe pump [90]. In an LOC, it is possible to dynamically flow culture media and other fluids at a physiological flow rate and create shear stress on the BMVECs (6 dyne cm−2 at 100 μL h−1). It is also feasible to recreate blood-like viscosity (3–4 cP) by adding microbeads.

Recently, various studies have reported the significance of these mechanistic factors in mimicking the characteristic features of brain vasculature in LOCs. It was demonstrated that, unlike other endothelial cells, BMEC resists elongation, in response to both curvature and shear stress [91]. Likewise, the TJ proteins were upregulated in a dynamic flow culture, when compared to a static culture [52]. Currently, iPS-BMVECs are used in brain studies as these cells exhibit high levels of TEER (~3000–5000 Ω·cm2) within 24–48 h of culture [2]. However, in a static culture, the TEER levels can be maintained only for ~2 days and the junction protein expressions are not high enough, limiting their use in drug screening applications. A transendothelial impedance as high as ~ 25,000 Ohm, was achieved in a dynamic flow LOC and were maintained for a period of one week [2]. In the same study, the expression of P-gp (permeability glycoprotein, efflux transporter) under dynamic flow (100 μL h−1), enabled the observation of a 2.7 fold increase in DOX flux [2].

Lumen Perfusability in Brain-Angiogenesis LOCs

It is important that the vasculature is perfusable, in order to mimic the circulation of nutrients and metabolites in the brain, which in turn helps the lumen formation and maintenance [45]. The lumen perfusability was found to increase the neural activity, that was measured by the Ca2+ oscillation [44]. While lumen formation can be achieved in petri plate, only LOCs allow direct accesses to them via the chip’s access ports. This enables studying perfusability [96], intra lumen flow [44], drug kinetics, drug translocation across the BBB and their effect on angiogenesis [45,46], in a clinically relevant manner.

The lumen structures with diameters in the range of 1–50 mm can be modeled in an LOC [96], and the intra lumen flow can be generated using a hydrostatic pressure head, simply by connecting the inlet with a larger media reserve [44] and to syringe pumps [50]. The lumens are modeled either by forming a vascular network with innate lumens [42–44,46,49] or by recreating the lumen structure, devoid of any vascular network, as described in the HT models [2,47–50,52]. The perfusability and the integrity of the vasculature were evaluated using microbeads and the dextran, 20 kDa and 70 kDa FITC with Stokes’ radii of 23 and 60 Å, respectively [42–44,46,49].

Compartmentalization in Brain-Angiogenesis LOCs

In a conventional petri dish, it is difficult to maintain both endothelial and brain cells simultaneously that require different growth conditions. LOCs enable (simplify) complex system level experiments of multi-culture cell system by providing confined culturing compartments for different cell types, along with their optimal culture media, and tunable brain-like interfaces between them to regulate their interaction. These tunable brain-like interfaces are realized by discontinuous micropillars [42–45,56], micropores [2,52,53], or extracellular matrix [49] and can be coupled or uncoupled as required to control the migration of molecules and cells between different channels. Direct [42,44,50] or indirect [45,46,52,53] contact between the endothelial cells and the brain cells is achieved through the microstructures.

Better control over the experiments by channelizing and enhancing cell signals, provide a clear understanding about the distinct contribution of various cellular factors. The cell migration [45,46,56], angiogenic sprouting, in response to chemical clues/gradients [42–44,46], respectively, and other complex brain cell interactions were modeled in LOCs. For example, the astrocyte migration towards the vascular network, resulting in the enhancement of BBB [42], similarly migration of the endothelial cells in response to growth factor gradient, were observed [43,45,46]. Recently, a hybrid device, displaying the metabolic flux across the BBB and the brain cells were reported [57]. Two BBB chips were connected on either side of a brain chip and for the first time and a BBB influx/efflux through the artificial CSF was demonstrated (Figure 3B). This device replicated the in vivo CSF and blood, flow on-chip. In the brain chip part of the device, by restricting the active flow only to the upper compartment, the flow velocity on the neurons in the lower compartment was zero, similar to the in vivo condition.

Table 2. List of labs-on-a-chip (LOCs) modeling brain vasculature.

S.No.

Model

Drug Screening

Dynamic Flow

Lumen perfusability

Vessel Dia.

Endothelial Cells

Brain Cells

Other Cells

TEER

Ref.

1

GBM (spheroid)-angiogenesis (PC)

BVZ, Sunitinib,Cetuximab

Y

Y

-

HUVEC

U87MG

hLF

NA

2019, [46]

2

BBB (HT)

-

Y

Y

W = 200 μm, H = 100 μm

hCMEC/D3

hA

-

NA

2019, [52]

3

GBM-angiogenesis (CR)

TMZ

Static

N

-

HUVEC

U87MG

-

NA

2019, [54]

4

BBB (HT)

Dox, Cetuximab, Q-dot-Angiopep-2

Y

Y

W = 1000 μm, H = 200 μm

iPS-BMVEC

hP, hA

-

Impedance, ~25,000 Ω

2018, [2]

5

Angiogenesis 3D (PC)

-

Y

Y

D = 25 μm

HUVEC

 

-

-

NA

2018, [43]

6

BBB (HT)

Antibody MEM-189

Y

Y

NA

TY10

hBPCT*, hA

-

NA

2018, [49]

7

GBM spheroid (Microwell)

TMZ, BEV

Static

N

NA

-

GBM cell*

-

NA

2018, [58]

8

Vasculogenesis (PC)

-

Static

Y

-

HUVEC

E17-brain cells

hLF

NA

2017, [42]

9

Vasculogenesis (spheroid) (PC)

-

Y

Y

D = 60 μm

HUVEC, iPS-EC

hNSC

-

NA

2017, [44]

10

Hybrid-Brain

(others)

Methamphetamine

Y

N

NA

hBMVEC

hP, HIP-009, hA

-

NA

2017. [57]

11

BBB (HT)

-

Y

Y

D = 600-800 μm

hBMVEC

hP, hA

-

NA

2016, [50]

12

Angiogenesis (PC)

Bortezomib

Y

Y

-

HUVEC

 

-

NA

2015, [45]

13

BBB (HT)

-

Y

Y

H = 50 μm

RBE4

 

-

NA

2015, [47]

14

BBB

(others)

Mannitol

Y

N

NA

b.End3

 

C8D1A

-

Resistance, 250 Ω cm2

2012, [53]

Yes-Y, Parallel channel-PC, Hollow microtube channel-HT, Co-centric rings-CR, not applicable-NA, Cell line description: HUVEC-Human Umbilical Vein Endothelial Cell (primary), hCMEC-Human Cerebral Microvascular Endothelial Cell (immortal), iPS-BMVEC-induced pluripotent stem cell-derived human brain microvascular endothelial cell, TY10-human spinal cord microvascular endothelial cell (immortal), iPS-EC- induced pluripotent stem cell-derived human endothelial cell, hBMVEC- Human brain microvascular endothelial cells (primary), RBE4-rat brain endothelial cell (immortal), b.End3-mice brain endothelial cell (immortal), U87MG-human glioblastoma cell (immortal), hA- Human Astrocyte (primary), hP-Human brain Pericyte (primary), hBPCT-human brain pericytes (immortal), hNSC- human neuronal stem cell, HIP-009-Human Hippocampal Neural Stem, C8D1A-mice astrocyte cell (immortal), and hLF-human Lung fibroblast (primary). * Patient derived cells.

LOCs for the Synthesis of NMs

Another major challenge for NMs is the difficulty in reproducing particle synthesis, especially in the case of complex NPs [97]. Microfluidic platforms allow the control of various critical synthesis parameters (temperature, flow rate, reaction rate, etc.) and are used to rapidly synthesize therapeutic NPs in the desired size range, shape and composition [64,98]. Recently, LOCs for producing monodispersed liposomes [99], chitosan NPs [100], protein NPs [101], lipid NPs [102] and other organic NPs [103], some of them with higher EE, were reported [99,102,103].

 

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