Controlled Generation of Microspheres Incorporating Extracellular Matrix Fibrils for Three‐Dimensional Cell Culture

Abstract

A growing body of evidence suggests that studying cell biology in classical two-dimensional formats, such as cell culture plasticware, results in misleading, non-physiological findings. For example, some aspects of cancer biology cannot be observed in 2D, but require 3D culture methods to recapitulate observations in vivo. Therefore, we developed a microsphere-based model to permit 3D cell culture incorporating physiological extracellular matrix components. Bio-electrospraying was chosen as it is the most advanced method to produce microspheres, with THP-1 cells as a model cell line. Bio-electrospraying parameters, such as nozzle size, polymer flow rate, and voltage, were systematically optimized to allow stable production of size controlled microspheres containing extracellular matrix material and human cells. We investigated the effect of bio-electrospraying parameters, alginate type and cell concentration on cell viability using trypan blue and propidium iodide staining. Bio-electrospraying had no effect on cell viability nor the ability of cells to proliferate. Cell viability was similarly minimally affected by encapsulation in all types of alginate tested (MVM, MVG, chemical- and food-grade). Cell density of 5 × 106 cells ml-1 within microspheres was the optimum for cell survival and proliferation. The stable generation of microspheres incorporating cells and extracellular matrix for use in a 3D cell culture will benefit study of many diverse diseases and permit investigation of cellular biology within a 3D matrix.

A comprehensive review on droplet-based bioprinting: Past, present and future

“…Dripping mode is observed at low voltages and flow rates, Although process parameters do not significantly affect the cell viability (see Table 2) and genomic expression immediately after bioprinting, applied voltage, cell concentration and bioink constituents can collectively affect the long-term post-bioprinting cell viability [71]. The applied voltage determines the ejected droplets size [43,71] whereas bioink constituents (such as hydrogels including but not limited to crosslinked alginate, collagen and fibrin) and concentration used for cell encapsulation alter the diffusion of media. Hence, the droplet size and bioink constituents affect media transport to the encapsulated cells.…”

“…The volume of ejected droplets is mediated by several operating parameters such as the bioink material characteristics, the printhead geometry (orifice diameter) and its actuation voltage pulse characteristics [23,32,36,43,46,71,82,83]. For example, the width of each M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13 coalesced line in Fig.…”

“…Consequently, bioink droplets are ejected when the electrostatic stresses overcome the surface tension at the orifice under a sufficiently high voltage depending on the process parameters. The strength of the electric field (applied voltage), the bioink flow rate and the bioink properties (including cell type and concentration [69]) determine the jetting mode [42,70] and cell viability [71]. Dripping mode is observed at low voltages and flow rates, Although process parameters do not significantly affect the cell viability (see Table 2) and genomic expression immediately after bioprinting, applied voltage, cell concentration and bioink constituents can collectively affect the long-term post-bioprinting cell viability [71].…”

“…The strength of the electric field (applied voltage), the bioink flow rate and the bioink properties (including cell type and concentration [69]) determine the jetting mode [42,70] and cell viability [71]. Dripping mode is observed at low voltages and flow rates, Although process parameters do not significantly affect the cell viability (see Table 2) and genomic expression immediately after bioprinting, applied voltage, cell concentration and bioink constituents can collectively affect the long-term post-bioprinting cell viability [71]. The applied voltage determines the ejected droplets size [43,71] whereas bioink constituents (such as hydrogels including but not limited to crosslinked alginate, collagen and fibrin) and concentration used for cell encapsulation alter the diffusion of media.…”

Hybrid Cross‐Linking Characteristics of Hydrogel Control Stem Cell Fate

“…Our molecular engineering strategy to control cellular organization is different from current approaches which usually involve variation of polymer fraction [19] and cross-linking density [20] in hydrogels or three-dimensional scaffolding, e.g., cell laden electrospun fibers, [21,22] microsphere-based cellular aggregates. [23][24][25] …”