Engineering porous membranes mimicking in vivo basement membrane for organ-on-chips applications

A. Synthetic porous membranes and in vivo basement membranes

The common types of porous membrane used in TBoCs are usually made from synthetic polymers such as polydimethylsiloxane (PDMS), polycarbonate, polyethylene terephthalate, or polytetrafluoroethylene. For their use in TBoCs, these types of synthetic porous membranes have good mechanical characteristics, allowing easy handling, facile integration into a microfluidic chip, and stable cell support. In addition, they are commercially available as they can be mass-produced. However, despite their wide availability and their mechanical strengths in practical applications, their material characteristics and structures are notably different from those of in vivo BMs; this limits the realization of physiological in vivo cellular or organ behaviors.1111. G. Perry, W. J. Xiao, G. I. Welsh, A. W. Perriman, and R. Lennon, Integr. Biol. 10(11), 680–695 (2018). https://doi.org/10.1039/C8IB00138C For example, because the cells cannot degrade or remodel these synthetic porous membranes, their pore sizes fundamentally limit the movement of cells or molecules across them.1010. H. H. Chung, M. Mireles, B. J. Kwarta, and T. R. Gaborski, Lab Chip 18(12), 1671–1689 (2018). https://doi.org/10.1039/C7LC01248A Furthermore, cells can be trapped in the pores of the membrane, leading their phenotype to follow the pore shape, which can be far from the natural and physiological cell phenotype (Fig. 1).1414. W. F. Quiros-Solano, N. Gaio, O. M. J. A. Stassen, Y. B. Arik, C. Silvestri, N. C. A. Van Engeland, A. Van der Meer, R. Passier, C. M. Sahlgren, C. V. C. Bouten, A. van den Berg, R. Dekker, and P. M. Sarro, Sci. Rep. 8, 13524 (2018). https://doi.org/10.1038/s41598-018-31912-6 Importantly, the fact that manufacturing companies sell numerous types of synthetic membrane indicates that no single type of membrane can yet fully replace in vivo BM.figure

In this regard, one of the major engineering issues in the development of porous membranes suitable for physiologically relevant TBoCs is to impose BM-like characteristics on the membrane. Because the cells sense and interact with the underlying porous membrane, its BM-like characteristics can guide and induce them to behave more physiologically. Although the characteristics of in vivo BMs vary according to the types of organs and their physiological conditions, some particular biophysical and biochemical characteristics are commonly observed. These include a collagenous nanofibrillar architecture, a membrane thickness of the order of 10 nm to 1 μm, high permeability of the order of 10−4 to 10−5 cm s−1, stiffness of the order of 100 kPa to 1 MPa, and high enough stretchability to maintain the BM structure under the dynamic motions of organs such as in the peristalsis of intestine tissue.

In terms of biochemical characteristics, in vivo BM is composed of ECM proteins, such as collagens (mainly type IV), laminins, and proteoglycans, with biochemically functional groups.1515. J. Youn, H. Hong, W. Shin, D. Kim, H. J. Kim, and D. S. Kim, Biofabrication 14(2), 025010 (2022). https://doi.org/10.1088/1758-5090/ac4dd7 There have been many efforts to develop porous membranes possessing many aspects of BM-like characteristics based on nano- or micro-fabrication technologies, including soft lithography, spin-coating, silicon etching, femtosecond laser machining, and electrospinning.1111. G. Perry, W. J. Xiao, G. I. Welsh, A. W. Perriman, and R. Lennon, Integr. Biol. 10(11), 680–695 (2018). https://doi.org/10.1039/C8IB00138C These fabrication technologies have been exploited in processing synthetic polymers to create porous membranes having BM-like low thickness, high permeability, or nanofibrous architecture. For example, using a spin-coating process, Rathod et al. developed an advanced PDMS membrane that possesses in vivo BM-like submicrometer thickness and mechanical characteristics.1616. M. L. Rathod, J. Ahn, B. Saha, P. Purwar, Y. Lee, N. L. Jeon, and J. Lee, ACS Appl. Mater. Interfaces 10(47), 40388–40400 (2018). https://doi.org/10.1021/acsami.8b12309 Robert et al. also proposed an ultrathin and transparent silicon membrane made using an etching process.1717. R. N. Carter, S. M. Casillo, A. R. Mazzocchi, J. P. S. DesOrmeaux, J. A. Roussie, and T. R. Gaborski, Biofabrication 9(1), 015019 (2017). https://doi.org/10.1088/1758-5090/aa5ba7 However, although such membranes have some BM-like characteristics, the in vivo BM-like stiffness, ECM protein compositions, and biodegradability are difficult to realize simultaneously due to the innate properties of synthetic polymeric materials (Table I).Table icon

TABLE I. Representative types of artificial membranes and their general properties compared to those of in vivo BM (OO: similar, O: moderate. X: different).

Membrane typeRepresentative materialsECM proteinsNanofibrous architectureStiffnessThicknessEtched membraneSiO2, SiN, polycarbonate (PC), etc.O (additional ECM coating is required)X (flat surface with pores)XOOSpin-coated membranePolydimethylsiloxane (PDMS)O (additional ECM coating is required)X (flat surface with pores)OO (tunable from ∼10 kPa to ∼10 MPa)OElectrospun nanofiber membranePolycaprolactone (PCL), polylactide-co-glycolide (PLGA), etc.O (additional ECM coating is required)OOO (tunable from ∼100 kPa to ∼10 MPa)OOVitrified ECM membraneType-I collagen, Matrigel, etc.OOOOOO

B. Extracellular matrix membranes

Recently, the use of functional or biological materials has emerged as a novel approach to reconstituting BM-like porous membranes. In particular, the natural material properties of animal-derived ECM hydrogels, such as collagen, Matrigel, and Geltrex, have shown great possibilities in the development of BM-like porous membranes. Porous membranes made from ECM hydrogels, referred to as ECM membranes, are usually fabricated based on simple drying methods such as lyophilization or vitrification (Fig. 2).18,1918. M. J. Mondrinos, Y. S. Yi, N. K. Wu, X. Ding, and D. Huh, Lab Chip 17(18), 3146–3158 (2017). https://doi.org/10.1039/C7LC00317J19. C. Y. Wang, N. Tanataweethum, S. Karnik, and A. Bhushan, ACS Biomater. Sci. Eng. 4(4), 1377–1385 (2018). https://doi.org/10.1021/acsbiomaterials.7b00883 ECM membranes inherently possess a natural collagenous nanofibrillar architecture with BM-like biochemical functional groups on their surfaces.figureFurthermore, ECM membranes can be remodeled by cells and have advantages for modeling immune-cell transmigration or cancer metastasis through the barrier tissue. Many researchers have demonstrated the strengths of ECM membranes for developing physiologically relevant OoCs, in vitro disease modeling, and drug testing.2020. H. F. Tsai, A. Trubelja, A. Q. Shen, and G. Bao, J. R. Soc. Interface 14(131), 20170137 (2017). https://doi.org/10.1098/rsif.2017.0137 However, despite the advantages of using ECM hydrogels in membranes, their weak mechanical characteristics undermine their practical applicability. Because dried ECM membranes are usually very brittle, the peeling-off process during their fabrication, handling, and integration requires very high user proficiency. Unlike some types of synthetic porous membranes made from elastomeric polymers (e.g., PDMS), dried ECM membranes are not suitable for realizing the cyclic stretching motions of some barrier tissues due to their tendency to sag, which is followed by deformation. Furthermore, the fabrication processes of ECM membranes are still manual and primitive, and they are usually conducted only at the laboratory scale; this limits their potential for mass production and quality control.

C. Nanofiber-reinforced extracellular matrix membranes

In light of the above-mentioned difficulties, there is a need to develop an advanced porous membrane possessing both the practical applicability of synthetic porous membranes and the BM-like characteristics of ECM membranes; this would lead to an ideal artificial BM for TBoCs. Very recently, a promising approach for developing such an artificial BM has been introduced. Youn et al. developed a novel stretchable ECM membrane reinforced with a very thin and sparse electrospun nanofiber scaffold, named a nanofiber-reinforced ECM (NaRE) membrane (Fig. 3).1515. J. Youn, H. Hong, W. Shin, D. Kim, H. J. Kim, and D. S. Kim, Biofabrication 14(2), 025010 (2022). https://doi.org/10.1088/1758-5090/ac4dd7 The NaRE membrane is composed of collagen nanofibrils entirely covering a silk fibroin (SF)/polycaprolactone (PCL) nanofiber scaffold. Because the major component of the NaRE membrane is type-I collagen, it inherently possesses the characteristics of the ECM. NaRE membranes show not only BM-like biochemical compositions but also the biophysical and structural characteristics of a nanofibrillar architecture; this includes a thickness below 5 μm and permeability of the order of 10−5 cm s−1, values very similar to those of in vivo BM. At the same time, the SF/PCL nanofiber scaffold reinforces the NaRE membrane and gives it superior mechanical robustness compared to pristine ECM membranes. The mechanical robustness of NaRE membranes allowed NaRE membranes to maintain their original structures under microfluidic dynamic environments, even after thousands of cycles of stretching at a strain of 15%.figure

The fabrication process for NaRE membranes is based on the electrospinning of SF/PCL followed by a simple self-assembly process of collagen nanofibrils on the SF/PCL nanofiber scaffold. NaRE membranes can be fabricated in situ, which allows their integration into a microfluidic chip without the requirement for elaborate handling. Furthermore, because the fabrication processes of NaRE membranes can be easily scaled, their mass production appears to be feasible.

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