A Minireview of Microfluidic Scaffold Materials in Tissue Engineering

Abstract

In 2020, nearly 107,000 people in the U.S needed a lifesaving organ transplant, but due to a limited number of donors, only ∼35% of them have actually received it. Thus, successful bio-manufacturing of artificial tissues and organs is central to satisfying the ever-growing demand for transplants. However, despite decades of tremendous investments in regenerative medicine research and development conventional scaffold technologies have failed to yield viable tissues and organs. Luckily, microfluidic scaffolds hold the promise of overcoming the major challenges associated with generating complex 3D cultures: 1) cell death due to poor metabolite distribution/clearing of waste in thick cultures; 2) sacrificial analysis due to inability to sample the culture non-invasively; 3) product variability due to lack of control over the cell action post-seeding, and 4) adoption barriers associated with having to learn a different culturing protocol for each new product. Namely, their active pore networks provide the ability to perform automated fluid and cell manipulations (e.g., seeding, feeding, probing, clearing waste, delivering drugs, etc.) at targeted locations in-situ. However, challenges remain in developing a biomaterial that would have the appropriate characteristics for such scaffolds. Specifically, it should ideally be: 1) biocompatible—to support cell attachment and growth, 2) biodegradable—to give way to newly formed tissue, 3) flexible—to create microfluidic valves, 4) photo-crosslinkable—to manufacture using light-based 3D printing and 5) transparent—for optical microscopy validation. To that end, this minireview summarizes the latest progress of the biomaterial design, and of the corresponding fabrication method development, for making the microfluidic scaffolds.

Introduction

Recently, a lot of progress has been made in generating microscopic 3D cell culturing environments (e.g., posts, fibers, etc.) in microfluidics devices (Raman et al., 2013; Morrow et al., 2019). Therefore, adopting the microscopic cell manipulation technologies for the creation of macroscopic cultures (aka “microfluidic scaffolds”) can potentially overcome all of the bottlenecks above by providing the following key elements: 1) An Active “Vasculature” for distributing metabolites and clearing waste throughout organ-sized scaffolds; 2) Non-Disruptive Mini-Probing of the cells and of the fluids within them for ex-situ analysis [as was done in Supplementary Figure S1 of (Tong et al., 2020)]; 3) Long-term Live Microscopy validation of the cell/fluid probing/assaying in #2, where possible (e.g., at objective focal length depths within the scaffold, assuming an optically transparent scaffold material); 4) Tissue Modulation via cell and bio-active chemical (e.g., chemo-attractants, growth and differentiation factors, drugs, etc.) delivery, in order to minimize product variability; and 5) Automated Spatiotemporal Control over the tissue development in a closed-loop manner, based on optical and chemical assaying feedback, in order to enable computer-driven culturing. The overall idea is depicted in Figure 1A.

FIGURE 1

Scotti et al., 2010; Farrell et al., 2011).

Hence, given that the foundation for the addressable plumbing and the automation design have been described in our recent publication (Tong et al., 2020), the remainder of this minireview is dedicated to the development of the novel materials for the microfluidic scaffolds. Although many excellent reviews of materials used for microfluidics devices have been published in the past (Huang et al., 2011; Papaefthymiou, 2013; Ren et al., 2013; Hou et al., 2017; Mou and Jiang, 2017; Convery and Gadegaard, 2019; Xie et al., 2020), none of them have focused on the creation of microfluidic scaffolds with an addressable (i.e., with valves) plumbing. Thus, the discussion is focused solely on the materials needed for creating the envisioned device. Furthermore, it does so without assuming a tissue type that the scaffolds would be used to culture. Hence, the reviewed material properties do not account for tissue-specific requirements, such as cell adherence and mechanotransduction. Instead, it is presumed that such customizations would be accomplished by coating the interior pore space of the envisioned device with the tissue-specific proteins via its microfluidic plumbing.

Microfluidic Scaffold—Materials and Manufacturing

According to our literature search, the different types of biomaterials, and corresponding fabrication techniques, developed for the construction of the microfluidic scaffolds are summarized in Table 1.

Naturally Derived Materials

The first line of materials reviewed here belongs to those derived from natural sources, such as: Alginate from seaweed, Gelatin from animal skin, Silk from worm cocoons, Matrigel from extracellular matrix and Fibrin from blood. These are attractive for scaffold manufacturing due to their biocompatibility with cell and tissue growth.

Extracellular Matrix (ECM) and Cell Wall Materials (e.g., Fibrin, Matrigel, and Alginate) (Miller et al., 2012): 3D perfusable vascular networks were created by casting of Fibrin, Alginate, or Matrigel hydrogel solutions onto a dissolvable carbohydrate-glass lattice via a 3D printed sacrificial molding technique. All three gels were polymerized either chemically or thermally (as opposed to via photo-crosslinking). Once a hydrogel was solidified, the sacrificial lattice was dissolved out, generating a complex 3D vascular network without the need for any manual alignment or bonding. The optical properties of Alginate and Matrigel, with an RI ∼1.335–1.34.(Choi et al., 2015; Funane et al., 2018), are close to that of water; while the Fibrin’s RI of 1.53–1.62 (Kirichenko et al., 2021) is slightly higher than that of glass. Despite these attractive properties, however, these hydrogels are too soft for microfluidic valve fabrication: the elastic modulus of Alginate is ∼0.1–10 KPa (Candiello et al., 2013), of Matrigel ∼0.14–1.3 KPa (Kiss et al., 2011) and of Fibrin ∼9.29–30 kPa (Murphy and Leach, 2012; Bachmann et al., 2020). Thus, despite having an excellent elongation at break (e.g., ∼400% for Alginate and 52% for Fibrin (Underwood et al., 2001)), the mechanical properties of these hydrogels are significantly weaker than that of the PDMS (increasing the likelihood of device collapse and deformation). Furthermore, they have shown to degrade too rapidly in vivo: ∼1 week (Benavides et al., 2015; Shkand et al., 2016; Trujillo et al., 2019).

Silk Fibroin (Zhao et al., 2016): A microfluidic Silk Fibroin hydrogel scaffold has been created via soft lithography using PDMS stamps and sacrificial molding. To the best of our knowledge, this is the only transparent (RI = 1.34 (Shan et al., 2018)) biodegradable material in Table 1 that has actually been used to create functional microfluidic valves. The Silk Fibroin was crosslinked via enzymatic reactions. Next, new layers were manually cast onto a semi-crosslinked ones to achieve good bonding. It has an excellent tunable degradation half-life from hours to years. However, it has not been shown that the material can be used to generate valve sizes for single cell manipulation—the construct’s feature sizes in (Zhao et al., 2016) were quite large: 800–1,200 μm, with a layer thickness of ∼2,500 µm. Therefore, the fabrication method needs to be optimized to achieve a finer feature resolution of ∼100 µm for precise cell and fluid manipulations within the microfluidic scaffolds. Another drawback of Silk Fibroin is that its elastic modulus of 1 kPa - 1 MPa (Zhao et al., 2016) and elongation at break of 15–35% (Gu et al., 2020) are lower than that of the PDMS. In fact, a PDMS support was used with the device in (Zhao et al., 2016).

Gelatin (Pimentel et al., 2018)/Gelatin Methacrylate (GelMA) (Zhang et al., 2016): 3D perfusable vascular networks were fabricated from Gelatin/GelMA by casting an aqueous hydrogel solution on a 3D printed sacrificial molding. The mold was then dissolved within the solidified hydrogel to generate complex 3D vascular networks without any manual alignment or bonding. Both of the materials are transparent, with optical properties close to that of water (RI = 1.35–1.39 (Fu et al., 2017; Rinawati et al., 2018)). After being crosslinked chemically, Gelatin had an elastic modulus of ∼0.3–20 kPa (Zhao et al., 2016) and an elongation at break of ∼70% (Lee et al., 2019). GelMA, on the other hand, is a UV crosslinkable material. Its elastic modulus is around 3.08–184.52 KPa (Wu et al., 2019), and its elongation at break can be as large as 80% (Shin et al., 2012). Unfortunately, both Gelatin and GelMA have a really short degradation half-life: ∼25 days for gelatin (Saito and Tabata, 2012) and ∼3 h for GelMA (Zhu et al., 2019). Finally, like the other naturally derived hydrogels, Gelatin/GelMA are generally so soft that they require other materials to serve as mechanical support(Huang et al., 2011).

Synthetic Materials

The second group of materials reviewed here are artificially designed to have better mechanical properties than their naturally derived counterparts. Specifically, most of the latter materials are too soft to make microfluidic valves. Hence the synthetic materials have been made to improve stiffness and stretchability, and are therefore, generally better candidates for the microfluidic scaffolds.

Poly-lactic-co-glycolic acid (PLGA) (King et al., 2004): Patterned 2D PLGA layers were with complex branching pore networks has been created via soft lithography. Once microstructured the films were fabricated, manual layer-by-layer alignment and thermal bonding were used to stack them into a 3D scaffold. Thus, given the use of manual fabrication, we are not aware of this material being compatible (i.e., photo-crosslinkable) with SLA 3D printing. Besides that, PLGA has an elastic modulus of 1.4–2.8 MPa (Barrett and Yousaf, 2009), which makes sufficiently rigid (like PDMS). Furthermore, its RI of 1.47 (Shan et al., 2018) is close to the range of water and glass (1.33–1.52), making it suitable to be used with optical microscopy. Moreover, PLGA has an attractive degradation half-life of 5–6 weeks (Gentile et al., 2014). However, its maximum elongation at break is just 10%, making PLGA making it likely that the microfluidic valves made from this material could tear during use.

Poly(glycerol sebacate) (PGS) (Bettinger et al., 2006): Microfluidic scaffolds made out of up to five layers of PGS have been created using soft lithography with manual alignment and thermal bonding. The material is reported to be transparent, though we were unable to find out its exact RI. It also has a good elastic modulus of 0.77–1.9 MPa (Vogt et al., 2021) and a high elongation at break 267% (Barrett and Yousaf, 2009). However, it is known to degrade too fast (a half-life of ∼21 days (Pomerantseva et al., 2009)) for long-term tissue regeneration. Therefore, more work needs to be done to slow down its degradation time and to make it crosslinkable for light-based fabrication.

Poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate) (APS) (Wang et al., 2010): Single layer APS vascular networks were created via soft lithography in conjunction with thermal curing and bonding. The material is transparent with an RI = 1.5 (Thomas et al., 2012) and has a degradation half-life that is tunable between 6 and 100 weeks (Bettinger et al., 2009). Additionally, with an elastic modulus of 0.56–4.34 MPa (Bettinger et al., 2009; Wang et al., 2010) and an elongation at break of 21–151% (Bettinger et al., 2009), APS’ mechanical properties can be made sufficiently close to that of the PDMS. However, given that it has not been made photo-crosslinkable and has only been used with 2D manufacturing methods, better approaches need to be developed for fabricating 3D microfluidic scaffolds with valves out of the APS.

Poly(octamethylene maleate (anhydride) citrate) (POMaC) (Zhang et al., 2018): A multilayer POMaC scaffold with a branching microchannel network was created via 2D soft lithography, in conjunction with ultraviolet light (UV) photo-crosslinking, manual alignment and inter-layer bonding using UV irradiation. Like PGS, the POMaC scaffold was shown to be transparent, though its RI has not been measured. Additionally, the material has excellent mechanical properties: an elastic modulus of 0.03–1.54 MPa and an elongation at break of 48–534% (Tran et al., 2010). Consequently, 25–50 µm thin vascular walls of the POMaC scaffold proved to be elastic enough to support pulsating blood flow in an artificial cardiac tissue implanted into a rat. Furthermore, in a different application, Digital Micromirror Device Projection 3D printing was used to photo-crosslink the material(Cha et al., 2014). Hence, POMaC is one of the best candidate materials that has all (less the missing RI) of the desired properties in Table 1.

Pluronic F127 di-acrylate (F127-DA) (Shen et al., 2019): A single layer perfusable microchannel network was created from the F127-DA hydrogel via a serial soft lithography using 3D printed molds and PDMS stamps. The material was photo-crosslinked under UV irradiation. It is transparent and is non-swelling at 37°C, which helps the microfluidic construct to maintain its desired channel morphology. Furthermore, it can be stretched to an incredible 11x (or 1,100%) of its original length before breaking. Additionally, the F127 hydrogel is transparent with an RI lower than 1.4 (Galy et al., 2020). However, its elastic modulus of just ∼75 kPa, making failure more likely due to the mechanical properties of this material significantly weaker than that of the PDMS. For example, the microfluidic construct made from this material has been shown to have a burst limit at ∼600 mmHg (11 psi) due to leakage from its tubing connections. Hence, the F127-DA is potentially unsuitable for microfluidic valve fabrication, since a bursting limit pressure of ∼2x (i.e., 20–25 psi) is needed to maintain microfluidic valve integrity during operation(Kellogg et al., 2014; Tong et al., 2020). Furthermore, F127-DA has a relatively short degradation half-life of ∼21 days. Therefore, more work would need to be done on improving its mechanical and degradation properties, before the material can be used for 3D microfluidic scaffolds with valves.

Photocrosslinkable poly(ethylene glycol) diacrylate (PEGDA) (Grigoryan et al., 2019): Multiple 3D perfusable multi-vasculature networks were created out of PEGDA using rapid prototyping SLA 3D printing. The construct was crosslinked automatically via UV exposure, and no manual bonding was needed. Similar to POMaC, PEGDA is a transparent material with an RI = 1.35 (Choi et al., 2013) which is close to that of water. Furthermore, it can be synthesized to exhibit a wide range of mechanical flexibility values varying from relatively soft ∼0.1 MPa to very stiff ∼18 MPa (Skornia et al., 2007; Chen et al., 2018). And PEGDA has an elongation at break ranging from ∼21 to above 65% (Chen et al., 2018; Feng et al., 2020). It’s one downside, however, is a short degradation half-life of just ∼25 days. Nonetheless, with future work, it can likely get tuned to the appropriate levels. Therefore, PEGDA is also considered to be a potential candidate for the microfluidic scaffolds, which meets most of the criteria outlined in Table 1.

Conclusion

In conclusion, microfluidic scaffolds hold the potential to revolutionize the biomanufacturing of artificial tissues and organs. However, an easily manufacturable biomaterial capable of supporting cell growth for extended periods of time, while maintaining the flexible microfluidic valves and optically transparent properties, needs to be found to make this happen. We have reviewed the potential candidate materials available in the literature and have determined that most naturally derived hydrogels (e.g., Gelatin/GelMA, and ECM/Cell Wall hydrogels) generally lack the mechanical properties for generating sturdy microfluidic valves. For example, Silk fibroin had to be used in conjunction with supports made from other (stronger) materials, like PDMS and Acrylic. They also tend to be leaky, which makes the hydrogels unlikely candidates for fabrication of the 3D microfluidic scaffolds with non-rudimentary plumbing. Consequently, synthetic materials (e.g., PLGA, PGS, APS, POMaC, F127-DA, and PEGDA) have been specifically designed to have the mechanical properties that are more tailored towards the microfluidic valve fabrication, while still retaining the biocompatibility and degradation profiles of an implantable material. Among these, the best overall candidate materials for making the microfluidic plumbing with valves appear to be POMaC and PEGDA, because they fit most of the criteria in Table 1. While the former appears to meet all the requirements, we were not able to find data on its transparency. Meanwhile, the latter has the drawback of having a relatively short degradation half-life of ∼25 days. However, even with this duration, the material can still be used for tissues that do not require long-term mechanical support. Ideally, though, we anticipate that a more stable version will soon be designed to accommodate all tissue types. Lastly, despite some of the materials being photocrosslinkable, all the microfluidic scaffolds reviewed in this manuscript were made either via solvent casting or manual stacking/bonding of 2D layers fabricated via soft lithography. Thus, the manufacturing approaches need to be improved also, ideally to use automated high-resolution approaches, such as stereolithography 3D printing. Once such a material is designed, the advanced microfluidic scaffolds will, without a doubt, begin to play a more dominant role in tissue engineering and biomanufacturing.

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