Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications

Abstract

We present (1) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and (2) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 μl each within 3 min at a pitch of 2.3 mm within an array of 48 × 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 × 3 HUVEC spheroids with intermediate distances of 500 μm embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 h of incubation indicating a good viability of the deposited spheroids.

Bioprinting of high cell‐density constructs leads to controlled lumen formation with self‐assembly of endothelial cells

“…Once produced, cell numbers within spheroids could however be considered constant. With the printing method used, the spheroids could even be sorted by means of size selection criteria (Gutzweiler et al, ). Major issue of the spheroid bioink was the random distribution of spheroids in the liquid suspension, though the concentration was adjustable.…”

“…This suspension was used for automated printing of droplet arrays (1 μl) on flat culture plates, which were subsequently used for hanging drop formation of spheroids (250 cells/spheroid) overnight. The automated spheroid production procedure is described in detail in (Gutzweiler et al, 2017). After 24 hr of incubation, spheroids were harvested by flushing with ECGM, washed and concentrated by centrifugation (800 rpm for 10 min), and finally resuspended in ECGM containing fibrinogen (10 mg/ml) to obtain the ready to use bioink (3,000 spheroids/ml).…”

“…If none or multiple spheroids were detected in the ROI, a vacuum suction system was deflecting the ejected droplets in flight, whereas only droplets containing single spheroids were passing the vacuum shutter system and were deposited onto the substrates. A more detailed description of the single spheroid printing process, in analogy to the single-cell printing technology, can be found in (Gross et al, 2013;Gutzweiler et al, 2017).…”

Aspiration-assisted bioprinting for precise positioning of biologics

“…Despite these advantages, only a few methods including extrusionbased bioprinting (2,11,13,14), droplet-based bioprinting (15), Kenzan (16), and biogripper approaches (4,17) have been demon-strated for 3D bioprinting of spheroids made of cells, such as but not limited to human articular or nasal chondrocytes, human umbilical vein endothelial cells (HUVECs), human umbilical vein smooth muscle cells, etc. Extrusion-based bioprinting technique dispenses similar-sized spheroids suspended in a hydrogel ink through a glass nozzle.…”

“…Although extrusion-based bioprinting of cellular aggregates in the form of strands can generate scalable tissues (3,5), their use in high-precision applications, such as organ-on-a-chip platforms or microphysiological systems, is quite challenging. In droplet-based bioprinting, a single spheroid is loaded into a droplet during bioprinting, which enables the positioning of spheroids in 2D (15). Kenzan method uses an array of needles on which spheroids are skewered by a robotic arm; however, this method requires same-size spheroids since bioprinted spheroids have to fit properly in the needle array and smaller spheroids would be prone to fragment during insertion on the needle (16).…”