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. 2014 May 14;24(18):2648-2657.
doi: 10.1002/adfm.201303891.

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

Victoria L Workman  1 , Liku B Tezera  2 , Paul T Elkington  2 , Suwan N Jayasinghe  1
Affiliations

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

Victoria L Workman et al. Adv Funct Mater. .

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.

Keywords: 3D cell culture; bio-electrospraying; biological models; cell encapsulation; cellular kinetics.

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Figures

Figure 1
Figure 1
a) Effect of voltage, flow rate and nozzle size on diameter of microspheres. b) Lowest (critical) voltage needed to produce smallest microspheres is related to inner diameter of the nozzle used. Alginate concentration was 1.5% Manugel DMB alginate solvated in water. Data points are mean of three independent replicates where 30 microspheres were measured for each replicate. Error bars represent standard deviation.
Figure 2
Figure 2
Observed outcomes when electrospraying a) 3% Manugel DMB alginate and b) 1.5% Manugel DMB alginate using 0.6 mm inner diameter nozzle for various combinations of flow rate and applied voltage. Unstable is used here to indicate unstable jet formation resulting in alginate solution collecting on the ring electrode. c) Teardrop-shaped microspheres and d) tadpole-shaped microspheres observed when electrospraying 3% Manugel DMB. Scale bar 1000 μm.
Figure 3
Figure 3
Effect of solvent on microsphere diameter. Alternative solvents were used to produce 1.5% Manugel DMB alginate solutions, which were electrosprayed using 0.4 mm i.d. nozzle, 10 ml hr-1 flow rate and ∼7 kV applied voltage. Values are mean of 30 microspheres measured and error bars represent standard deviation. Bars labelled with the same letter are not significantly different.
Figure 4
Figure 4
Use of HBSS compared with water does not affect the diameter of microspheres produced. Water a) or HBSS b) was used to produce 1.5% Manugel DMB alginate solutions, which were electrosprayed using 0.6 mm i.d. nozzle, various flow rates and applied voltages. Values are mean of 30 microspheres measured and error bars represent standard deviation.
Figure 5
Figure 5
Effect of alginate type (black bars) and addition of collagen (white bars) on the diameter of produced microspheres. a) Addition of collagen produced microspheres of significantly larger diameter than alginate alone (*p < 0.005). Alginate type b) and addition of collagen c) affected size of microspheres produced. Alginate concentration was 1.5% Manugel DMB, final collagen concentration was 1 mg ml-1, BES conditions were 0.4 mm inner diameter nozzle, 10 ml h-1 alginate solution flow rate, ∼7 kV applied voltage. Values are from two replicate experiments where mean of 30 microspheres were measured for each replicate and error bars represent standard deviation. b and c: Bars labelled with the same letter are not significantly different.
Figure 6
Figure 6
Effect of varying parameters on cell proliferation a), c), e) and cell viability b), d), f) of encapsulated THP-1 cells. Parameters varied were: a), b) voltage applied; no voltage (non-BES) or ∼7 kV applied voltage (BES), c), d) alginate type; food-grade, chemical-grade, MVG or MVM and e), f) initial cell concentration; 10 × 106 cells ml-1 (High), 5 × 106 cells ml-1 (Medium), and 1 × 106 cells ml-1 (Low). Alginate concentration was 1.5%, BES conditions were 0.4 mm inner diameter nozzle, 10 ml h-1 alginate solution flow rate, ∼7 kV applied voltage (unless otherwise stated). Cell counts were carried out using trypan blue. Viability was measured using PI staining and flow cytometry. Error bars represent standard deviation of two measurements.
Figure 7
Figure 7
Fluorescent images showing encapsulated live (green) and dead (red) cells at different time points after BES. Cells encapsulated in 1.5% food-grade alginate are shown a) one day, b) 4 days, and c) 7 days post-encapsulation. Cells encapsulated at d) 1 × 106 cells ml-1 (Low), e) 5 × 106 cells ml-1 (Medium), and f) 10 × 106 cells ml-1 (High). Images were taken 7 days post-encapsulation. Green fluorescence is emitted from intracellular esterase-converted calcein in live cells, whereas red fluorescence is emitted from ethidium homodimer present in the nuclei of dead cells. BES conditions were 0.4 mm inner diameter nozzle, 10 ml h-1 alginate solution flow rate, ∼7 kV applied voltage. Scale bar 500 μm.
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