Automated Bioprinting

Category: Blog,From Academia
Dec 29, 2020

There is a growing trend of automated biology in recent life science research and development. In this issue of “From Academia”, we included two publications focusing on the eventual realization of automated bioprinting. The first article focus on a new 3D printing method that can produce self-supporting elastomeric microfluidics without sacrificial ink, therefore, reduces contamination while maintaining desired mechanical properties. The author suggests this methodology will allow automatable fabrication. The second article reports an automated one-step bioprinting technique for the production of COC-microbeads of reproducible size and shape and demonstrates that this 3D in vitro maturation (IVM) improves oocyte nuclear maturation and cytoplasmic parameters, biomarkers of oocyte bioenergetic and developmental potential. “From Academia” features recent, relevant, close to commercialization academic publications in the space of healthcare 3D printing, 3D bioprinting, and related emerging technologies.

Email: Rance Tino ([email protected]) if you want to pen an Expert Corner blog for us or want to share relevant academic publications with us.

3D printed self-supporting elastomeric structures for multifunctional microfluidics

– Authored by Ruitao Su, Jiaxuan Wen, Qun Su, Michael S. Wiederoder, Steven J. Koester, Joshua R. Uzarski Michael C. McAlpine. Science Advances, October 2020 

Automated Bioprinting --3D printed microfluidic valve, pump, and spherical microfluidic network. (A) Schematic displaying the configuration of the 3D printed microfluidic valve. (B) Photos displaying the open and closed states of the 3D printed microfluidic valve. The valve was closed with a pressure of 100 kPa. Scale bar, 3 mm, University of Minnesota. (C) Closing pressure test of 3D printed microfluidic valve under varying flow pressures. (D) Flow rate test of a microfluidic pump. (E) 3D printed spherical converging and serpentine microfluidic channels with integrated valves. (F) Filament stacking schemes of the spherical microfluidic channels. (a) to (c) demonstrate the designed and printed profiles of three channel cross sections. Spacer filaments were added to prevent the collapse of asymmetric channels that were distal to the sphere centre. Copyright. Science Advances

Three-point bending at 40% deformation on the 5R9S design. (A) Three-point bending at 40% deformation on the 5R9S design when the probe is centred on top of the ring. (B) Three-point bending at 40% deformation on the 5R9S design when the probe is centred between two rings. (C) Difference in luminal diameter at 40% deformation during three-point bending on the 5R9S design. Copyright. Materials Science and Engineering: C
3D printed microfluidic valve, pump, and spherical microfluidic network. (A) Schematic displaying the configuration of the 3D printed microfluidic valve. (B) Photos displaying the open and closed states of the 3D printed microfluidic valve. The valve was closed with a pressure of 100 kPa. Scale bar, 3 mm, University of Minnesota. (C) Closing pressure test of 3D printed microfluidic valve under varying flow pressures. (D) Flow rate test of a microfluidic pump. (E) 3D printed spherical converging and serpentine microfluidic channels with integrated valves. (F) Filament stacking schemes of the spherical microfluidic channels. (a) to (c) demonstrate the designed and printed profiles of three-channel cross-sections. Spacer filaments were added to prevent the collapse of asymmetric channels that were distal to the sphere centre. Copyright. Science Advances

Abstract: 

Microfluidic devices fabricated via soft lithography have demonstrated compelling applications such as lab-on-a-chip diagnostics, DNA microarrays, and cell-based assays. These technologies could be further developed by directly integrating microfluidics with electronic sensors and curvilinear substrates as well as improved automation for higher throughput. Current additive manufacturing methods, such as stereolithography and multi-jet printing, tend to contaminate substrates with uncured resins or supporting materials during printing.

Here, we present a printing methodology based on precisely extruding viscoelastic inks into self-supporting microchannels and chambers without requiring sacrificial materials. We demonstrate that, in the submillimeter regime, the yield strength of the as-extruded silicone ink is sufficient to prevent creep within a certain angular range. Printing toolpaths are specifically designed to realize leakage-free connections between channels and chambers, T-shaped intersections, and overlapping channels. The self-supporting microfluidic structures enable the automatable fabrication of multifunctional devices, including multi-material mixers, microfluidic-integrated sensors, automation components, and 3D microfluidics.

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

– Authored by Antonella Mastrorocco, Ludovica Cacopardo, Nicola Antonio Martino, Diana Fanelli, Francesco Camillo, Elena Ciani, Bernard A. J. Roelen,Arti Ahluwalia, Maria Elena Dell’Aquila. PLOS ONE. 11 September 2020

Automated Bioprinting--Spherical hydrogel generator Sphyga set up and microbead preparation procedure. (A) Extrusion module. (B) Mechanical framework with coaxial design for holding the syringe (B1) and to direct microdroplets into the beaker (B2-3). (C) Ungelled sodium alginate biopolymer fibers followed by calcium-induced alginate gelification. (D) Sphyga set up under a laminar flow hood with dedicated software. (E) Freshly generated alginate microbeads (white arrows) in 100 mM CaCl2 solution. (F) Photomicrographs showing representative images of microbeads obtained with 1% or 2% alginate and needles of different size. Scale bar represents 300 μm. Copyright. PLOS ONE
Spherical hydrogel generator Sphyga set up and microbead preparation procedure. (A) Extrusion module. (B) Mechanical framework with coaxial design for holding the syringe (B1) and to direct microdroplets into the beaker (B2-3). (C) Ungelled sodium alginate biopolymer fibers followed by calcium-induced alginate gelification. (D) Sphyga set up under a laminar flow hood with dedicated software. (E) Freshly generated alginate microbeads (white arrows) in 100 mM CaCl2 solution. (F) Photomicrographs showing representative images of microbeads obtained with 1% or 2% alginate and needles of different size. Scale bar represents 300 μm. Copyright. PLOS ONE

Abstract: 

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited.

In this study, a bioprinting-based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape, and stability under culture conditions.

COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status, and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6).

The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6, and ATP8 and down-regulation of KHDC3, NLRP5 expression was observed in 3D IVM.

In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

(Notes: Cumulus cells directly surround oocyte to form cumulus oocyte complex (COC). Since oocytes have less glycolytic activity, the energy sources, such as piruvate or amino acid, are transferred from cumulus cells to oocyte via gap junctional communications, which are required for oocyte growth.)

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