Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures
Introduction
Cells in tissues and organs exist in a three-dimensional (3D) environment surrounded by other cell types. Homotypic and heterotypic cell–cell interactions, as well as individual cellular shapes, play important roles in cellular behaviors such as migration, proliferation, and differentiation. However, most tissue culture techniques lack such morphological and architectural characteristics as cells are typically cultured as single cell types spread out on a two-dimensional (2D) flat surface [1]. The ability to recreate in vivo-like microenvironments may lead to advances in diverse fields ranging from tissue engineering to fundamental studies of cell biology [2], [3], [4].
Co-cultures of two or more cell types have been used to make more biomimetic environments. These approaches have already demonstrated the importance of heterotypic cell–cell interactions on regulating cell behaviors [5]. Standard co-culture methods that mix two or more cell types, however, cannot be used to easily control the degree of homotypic and heterotypic cell–cell interactions. Micropatterned co-cultures are used to enhance microenvironmental control through spatial localization of multiple cell types relative to each other [6]. In this approach, cells have been patterned on various substrates using photolithography [7], [8], microcontact printing [9], inkjet printing [10], and microfluidics [11]. Although micropatterned co-cultures have been used to study the effects of cell–cell interactions on various cell functions, one potential disadvantage is that cells are generally patterned on a flat surface and form outspread 2D monolayers. While some cell types such as fibroblasts and endothelial cells actively grow and retain metabolism in 2D monolayer cultures, many cells such as hepatocytes and pancreatic cells frequently lose their organ-specific functions in 2D monolayer cultures and require 3D culture conditions to maintain such functions [12], [13], [14]. Thus, co-cultures that are suited for each cell type, such as the combination of 3D and 2D cultures, may be of benefit to enhance efficacy of co-cultures and lead to more advanced tissue engineered constructs.
Spheroid culture, in which cells form 3D multicellular aggregates, has been used to culture cells in 3D environments. For example, hepatocytes forming spheroids have cuboidal cell shapes, reconstruct bile canaliculi, and express intercellular adhesion molecules that are required for cellular communications [15], [16], [17]. Hepatocyte spheroids also exhibit liver-specific functions such as albumin secretion, urea synthesis, and drug metabolism for an extended period of time [15], [18], [19], [20]. Recently, micropatterns of spheroids have been generated using microscale technologies such as micromolding and microfabrication. In these approaches, geometric features such as microwells have typically been fabricated with non-cell adhesive polymers such as poly (ethylene glycol) (PEG). For example, photocrosslinkable PEG hydrogels have been used to form microwells using micromolding techniques [21]. Alternatively, microwells fabricated from other materials can be modified using chemical modification, electrostatic force or physical adsorption with PEG to make these surface cell repellent [17], [22]. Such non-adhesive PEG microwells create regions of low shear stress for cell immobilization and subsequent spheroid formation while preventing random cell adhesions on substrate surface [21]. Although these systems allow for 3D cell aggregations of a single cell type, they may not be suitable for co-cultures of additional cell types in a spatially controlled manner because of the non-adhesive property of the polymers.
With the goal of generating spatially controlled 3D co-culture systems, here we used photocrosslinkable chitosan. Chitosan is a hydrophilic and non-toxic polysaccharide [23]. Because of its biocompatibility and similarities to naturally occurring glycosaminoglycans, chitosan is useful for various biomedical applications in tissue engineering [24], [25], drug delivery [26], wound healing [27], and surgical adhesives [28]. In this study, we describe micromolding process using photocrosslinkable chitosan and show that the cellular attachment properties are significantly changed from cell repulsive to adhesive. These properties facilitate the formation of spheroids inside the microwells and the subsequent adhesion of a second cell type. This spatially controlled spheroid co-culture system may be useful for fabricating biomimetic cellular microenvironment.
Section snippets
Materials
Cell lines were purchased from American Type Culture Collection. Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Co. Chitosan glutamate (Protasan UP G113; Mw: <200 kDa; degree of deacetylation: 75–90%) was purchased from Novamatrix (Norway). 4-azidobenzoic acid was purchased from TCI America (Portland, OR, USA). All other chemicals were purchased from Sigma, unless otherwise indicated.
Photocrosslinkable chitosan synthesis
Photocrosslinkable chitosan (Fig. 1) was synthesized using a
Cell attachment on a chitosan hydrogel flat surface
Most synthetic and natural hydrogels such as PEG and dextran are known to prevent cellular adhesion for a long period of time, because most cells do not have receptors to hydrogel polymers [31]. Furthermore, due to the hydrophilic nature of hydrogels, extracellular matrix proteins, such as laminin, fibronectin, and vitronectin, typically do not readily absorb to the hydrogel surfaces. These attributes have been exploited in the application of post-operative adhesion barriers and in the design
Conclusion
We demonstrated a novel approach to prepare spheroid microarrays and co-cultures using micromolding technology with a photocrosslinkable chitosan hydrogel. The synthesized photocrosslinkable chitosan was compatible with the micromolding processes. Chitosan surface changed significantly from cell-repulsive to cell-adhesive, which facilitated the formation of spheroids inside the microwells and the subsequent adhesion of a second cell type. This spatially controlled spheroid co-culture system
Acknowledgements
This research has been supported by NIH (NIH Grant # HL60435), Draper laboratory (DL-H-550154) and Institute of Soldier Nanotechnology (DAAD-19-02-D-002) to Dr. Langer. Dr. Fukuda would like to acknowledge the support by a Grant-in-Aid for JSPS fellows, 16-4754.
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Present address: Institute of Materials Science, University of Tsukuba 3F528, 1-1-1 Tennoudai, Tsukuba, Ibaraki, Japan 305-8573.