Bioprinting of stem cell expansion lattices

Abstract

Stem cells have great potential in regenerative medicine, with neural progenitor cells (NPCs) being developed as a therapy for many central nervous system diseases and injuries. However, one limitation to the clinical translation of stem cells is the resource-intensive, two-dimensional culture protocols required for biomanufacturing a clinically-relevant number of cells. This challenge can be overcome in an easy-to-produce and cost-effective 3D platform by bioprinting NPCs in a layered lattice structure. Here we demonstrate that alginate biopolymers are an ideal bioink for expansion lattices and do not require chemical modifications for effective NPC expansion. Alginate bioinks that are lightly crosslinked prior to printing can shield printed NPCs from potential mechanical damage caused by printing. NPCs within alginate expansion lattices remain in a stem-like state while undergoing a 2.5-fold expansion. Importantly, we demonstrate the ability to efficiently remove NPCs from printed lattices for future down-stream use as a cell-based therapy. These results demonstrate that 3D bioprinting of alginate expansion lattices is a viable and economical platform for NPC expansion that could be translated to clinical applications.

Introduction

Stem cell-based therapies have shown great potential to benefit patients across a wide range of medical conditions [1]. Stem cells are multipotent cells that have the capacity to regenerate tissue both through differentiation into specific cell lineages and through secretion of regenerative factors [2], [3]. While stem cells have shown promise in regenerative medicine, there are still significant hurdles that must be overcome. One such challenge is the large number of cells required to treat a single patient [4], [5], [6]. To meet this need, three-dimensional (3D) expansion platforms have emerged as an efficient method to manufacture clinically-relevant numbers of stem cells in a significantly smaller footprint than traditional two-dimensional (2D) culture [7]. Because different types of stem cells have different culture requirements [2], [8], the effective and affordable biomanufacturing of distinct stem cell types may require the design and validation of unique expansion strategies.

Towards this goal, we have recently identified matrix remodeling as a requirement for the 3D expansion of adult murine neural progenitor cells (NPCs) [8]. NPCs are multipotent stem cells found in the central nervous system (CNS) that are capable of proliferation and differentiation into the most common lineages present in the CNS [9]. Transplanted NPCs have been identified as a potential therapy for a number of CNS dysfunctions including spinal cord injury, stroke, and amyotrophic lateral sclerosis (ALS), with several clinical trials underway [10], [11]. Recent reports have indicated that clinical-grade NPCs (i.e. those expanded using Current Good Manufacturing Practices) may not have the same regenerative efficacy as research-grade NPCs used in pre-clinical models, which limits their value as a translational therapy without further improvement of NPC expansion protocols [12], [13]. Achieving clinically relevant numbers of high-quality NPCs using 3D expansion culture will require the development of new biomanufacturing platforms.

Manufacturing NPCs in 3D reduces the spatial footprint of expansion when compared to traditional, adherent 2D culture, thereby reducing the energy and reagent costs, increasing the space-efficiency, and potentially improving the ease of handling. NPCs have been expanded in 3D either through culture as neurospheres (i.e. non-adherent, multicellular aggregates) [14], [15] or through encapsulation within bulk hydrogels [8]. However, both neurospheres and bulk gel systems are limited by the insufficient diffusion of oxygen, nutrients, and waste. In neurosphere culture, the slow rate of transport through the cell-dense aggregate results in apoptosis at the core and inhomogeneous cell populations, especially for spheroids >200 µm [16], [17]. In 3D bulk hydrogels, the overall size of the construct is limited, since cells further than 200 µm from a free-surface can suffer from limited diffusion of oxygen and nutrients through the gel [18], [19], [20]. To overcome these transport limitations, we propose using a 3D-printed lattice of a cell-laden hydrogel as an efficient NPC expansion platform. Here, 3D-printing enables the rapid creation of large-scale, cell-laden scaffolds with an open lattice structure to eliminate diffusional limitations in an easy-to-handle hydrogel.

An often unexplored aspect of designing effective platforms for 3D stem cell expansion is extraction of expanded cells following culture. Efficient extraction of stem cells from the expansion platform (i.e. high cell viability while maintaining stemness) is required whether the stem cells are to be used directly for therapeutic applications or other biomanufacturing processes. While others have demonstrated expansion of stem cells in 3D [7], [21], [22], there has been limited characterization of extraction efficiency from scalable expansion platforms. Beyond simply remaining viable, extracted cells must retain their stemness to be clinically relevant; thus, quantitative characterization of cell phenotype after expansion and extraction is a prerequisite for eventual clinical translation.

To fabricate this platform, we first must select an appropriate bioink. An ideal gel-based bioink scaffold for NPC expansion has several requirements. First, the gel must mechanically support the NPCs before and after printing [23], [24], [25]. Second, the gel must result in high cell viability throughout the printing and post-printing process. Third, the bioink must be remodelable by NPCs. We recently reported that NPCs cultured as single-cells within a 3D matrix must be able to remodel the scaffold to initiate cell-cell contacts between neighboring NPCs in order to maintain their stemness and effectively proliferate [8]. Finally, the bioink must enable the efficient extraction of NPCs after they have been expanded for down-stream use in cell-therapies or for further differentiation.

Electrostatically crosslinked alginate is a popular bioink material that can be manipulated to have all of the required functionalities for an ideal NPC expansion scaffold [26], [27]. Alginate is a polysaccharide biopolymer that can be crosslinked through electrostatic bonding with divalent cations to form hydrogels with tunable mechanical stiffness [28]. Furthermore, because electrostatic bonds are reversible, the crosslinked hydrogels exhibit stress relaxation and can be remodeled by encapsulated cells [8], [28]. We hypothesized that alginate bioinks subject to distinct first- and second-stage cation crosslinker concentrations would make capable NPC expansion lattices. First-stage crosslinking with a low cation concentration produces a weak, extrudable gel before printing. This weak gel can prevent cell sedimentation within the ink cartridge and protect the cell membrane from damage during printing. Second-stage crosslinking with a high cation concentration produces a stiffer material post-printing that is remodelable, supports cell-cell contacts, promotes cell proliferation, and permits dissociation for on-demand gel extraction of expanded NPCs. Here, we provide demonstration that 3D-printed, cell-laden alginate lattices are a scalable and effective approach to producing clinically-relevant numbers of NPCs that maintain their multipotent potential.