Co-fabrication of chitosan and epoxy photoresist to form microwell arrays with permeable hydrogel bottoms
Introduction
Microfabricated platforms allow the study of biological systems with unparalleled control of the physical and chemical microenvironment to which a cell or group of cells is exposed. Such technology permits precise customization of the shape, size, and surface chemistry of the area used for cell growth [1], [2], [3], [4]. By achieving such control, researchers gain the ability to interrogate the mechanisms and regulation of fundamental cellular processes and to create novel bio-inspired platforms [5], [6], [7], [8], [9]. To date, numerous types of microscale platforms have been developed for the high-throughput analysis of cells, but several limitations to these technologies exist. Droplet microfluidics can rapidly assay large numbers of isolated cells, but long-term culture of cells in droplets is difficult due to reduced delivery of oxygen and nutrients [10], [11]. In addition, analytical reagents must be pre-loaded into the droplet with the cells and analyses subsequent to the initial screening are technically difficult to set up [12]. Micropallet or microraft arrays, consisting of a large number of individual pedestal elements, can be used as substrates on which cells can be grown. However, biochemical crosstalk (in the form of secreted paracrine signaling molecules distributed by the culture medium) and cell migration from one pedestal to another can lead to confounded data and mixing of clonal cell populations [11], [13]. Additionally, such platforms can be problematic for nonadherent cells [11], [14], [15]. Microwell arrays can be used to array cells at low density into physically separate wells. However, confounding biochemical crosstalk and intermixing of clonal cell populations can nonetheless pose a problem for these platforms as well, since maintenance of cells beyond several hours necessitates that cells be exposed to a relatively large common media reservoir. While certain techniques, such as overlaying a physical barrier onto microwells seeded with cells, can reduce biochemical crosstalk and cell migration, cell survival and viability can be compromised over the long term as cells quickly consume the small quantities of culture media with which they are seeded, a consequence of the solid, impermeable substrates, such as PDMS or glass, upon which microwell arrays are generally fabricated [16].
Therefore, a platform that allows for user-customized interrogation of discrete numbers of cells, isolated so as to ensure the independence of measured responses, and yet still arrayed in a fashion that permits high-throughput study, would be a useful tool for cellular analyses. Our lab recently developed and characterized freestanding photoresist films [17]. Given the ease with which such films could be micropatterned, we sought to combine this approach with a hydrogel-based support that would yield arrays of microscale wells, each with a permeable bottom.
Chitosan is a cationic polysaccharide derived from chitin that has begun to see increased use in a number of biologically-relevant applications [18], [19], [20], [21]. Many of the chemical and physical properties of chitosan have been well characterized [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. Chitosan has been shown to be biocompatible to a number of cell types, including endothelial, hepatic, bone, and peripheral nerve cells [33], [34], [35], [36], [37]. Chitosan can be manipulated to form hydrogels, porous scaffolds, and dry films [21], [30], [38]. Studies have shown that capsules and films prepared from chitosan are permeable at the microscale and may have a potential role in drug and gene delivery systems [39], [40], [41], [42], [43]. A key feature of chitosan is the free amine group in its molecular structure, which protonates in a pH-dependent manner and induces swelling of the chitosan matrix and an increase in its network pore size. In addition to being responsible for chitosan's pH-dependent solubility, the amine group also allows the conjugation of exogenous molecules to the polysaccharide chain [20], [44], [45]. Recently several research groups have devised strategies to pattern chitosan at the microscale, including on surfaces for bioreactor applications and as a semi-permeable microscaffold in a microfluidic device for the study of bacterial biofilms [20], [44], [46], [47]. Due to its biocompatibility, pH-dependent solubility, and demonstrated use in certain microscale platforms, chitosan presented potential.
This report describes the co-fabrication of the biocompatible materials chitosan and 1002F epoxy photoresist to create a freestanding, two-layer hybrid film of permeable, hydrogel-bottomed microwell arrays. Several material properties of these films, including optical characteristics, surface chemistry, and permeability are analyzed. Finally, the application of these hybrid films to studies of cellular heterogeneity is described. Platforms co-fabricated from chitosan and epoxy photoresist can be used to reduce both high-molecular weight biochemical crosstalk between cells and mixing of different clonal populations, and will enable high-throughput cellular studies with increased ability to customize dynamic interrogations when compared to the materials in currently available technologies.
Section snippets
Materials
Chitosan (medium molecular weight), γ-Butyrolactone (GBL), photoresist developer (propylene glycol methyl ether acetate, PGMEA), toluidine blue, FITC-dextrans (average molecular weights 4, 10, 20, 40, and 70 kDa), rhodamine B-dextran (70 kDa), glutaraldehyde (70% v/v), and mineral oil were purchased from Sigma–Aldrich (St. Louis, MO). EPON resin 1002F photoresist was obtained from Miller–Stephenson (Sylmar, CA). UVI-6976 photoinitiator (triarylsulfonium hexafluoroantimonate salts in propylene
Co-fabrication of freestanding 1002F:chitosan films
Freestanding 1002F:chitosan hybrid films were fabricated and micropatterned using the strategy depicted in Fig. 1A and described in detail in the Methods section. Briefly, dry chitosan films were prepared on glass by spin coating and baking. After an air-plasma treatment, 1002F photoresist was spin-coated atop the dry chitosan films and micropatterned via UV photolithography. The resulting hybrid films, featuring an array of microwells patterned into the 1002F film and bottomed by a layer of
Conclusions
This microfabricated cellular analysis platform will enable high-throughput study of cellular heterogeneity in response to user-defined and dynamically controlled stimulations. This platform features an array of microscale wells, formed via the co-fabrication of chitosan with an epoxy photoresist to form a freestanding array of microwells bound by a semi-permeable hydrogel layer. Through a variety of methods, we have described numerous material properties of this biocompatible hybrid platform.
Acknowledgments
The authors are grateful to Ms. Lauren Westerhold for fabrication assistance and Mr. Joseph Balowski for helpful discussions, as well as to Dr. Carrie Donley (CHANL) for expertise with XPS analysis and Dr. Wallace Ambrose (CHANL) for SEM imaging assistance. This work was supported by a grant from the National Institutes of Health (CA177993).
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