抗真菌附着聚合物的喷墨3D打印

Yinfeng  He; Cindy  Vallieres; Morgan R. Alexander; Ricky D. Wildman; Simon  V. Avery

doi:10.21769/BioProtoc.4016

Improve Research Reproducibility A Bio-protocol resource

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Peer-reviewed

Inkjet 3D Printing of Polymers Resistant to Fungal Attachment

抗真菌附着聚合物的喷墨3D打印

YH Yinfeng He ^* email

CV Cindy Vallieres ^*

MA Morgan R. Alexander

RW Ricky D. Wildman

SA Simon V. Avery email

(*contributed equally to this work) 发布: 2021年05月05日第11卷第9期 DOI: 10.21769/BioProtoc.4016 浏览次数: 3974

评审: Jörg ToepelMarie Marie MueheAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Science Advances, featuring study using the protocol.

Jun 2020

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

Abstract

Inkjet 3D printing is an additive manufacturing method that allows the user to produce a small batch of customized devices for comparative study versus commercial products. Here, we describe the use of a commercial 2D ink development system (Dimatix material printing) to manufacture small batches of 3D medical or other devices using a recently characterized fungal anti-attachment material. Such printed devices may resist problems that beset commercial medical products due to colonization by the fungal pathogen Candida albicans. By sequentially introducing the cross-section bitmaps of the product’s CAD model and elevating the print head height using the auto-clicking script, we were able to create complex self-support geometries with the 2D ink development system. The use of this protocol allows researchers to produce a small batch of specimens for characterization from only a few grams of raw material. Additionally, we describe the testing of manufactured specimens for fungal anti-attachment. In comparison with most commercial AM systems, which require at least a few hundred grams of ink for printing trials, our protocol is well suited for smaller-scale production in material studies.

Keywords: Additive manufacturing (增材制造)

Inkjet (喷墨)

3D printing (3D打印)

Candida albicans (白色念珠菌)

Fungal biofilm (真菌生物膜)

Background

Additive manufacturing (AM) or 3D printing is a convenient tool for preparing small batches of products such as biomedical or electronic devices; however, commercial AM systems normally require at least a few hundred grams of material for the smallest batch of product, which remains a challenge for research focused on materials exploration. The Dimatix material printer is a commercial ink formulation development instrument designed for 2D printing, which runs with disposable cartridges that only require 3-5 grams of ink for printing. Its printing unit also allows height adjustment up to 25 mm. With customized scripts, we were able to use this 2D printer to simulate an inkjet-based 3D printing process and prepare experimental specimens from only a few grams of raw material. This protocol has been used in our fungal anti-attachment material screening study (Vallieres et al., 2020), in which a batch of six specimens was produced using only 4 grams of ink made from the candidate monomer discovered in the materials screening. Biofilm formation by fungi such as Candida albicans is regarded as an important source of nosocomial systemic infections, with mortality rates up to 50% (Cavalheiro and Teixeira, 2018). The resistance of the printed materials to C. albicans colonization can be tested by measuring fungal biofilm formation using crystal violet, which binds to negatively charged surface molecules and polysaccharides in the extracellular matrix. The candidate anti-attachment methacrylate polymer, (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone) (AODMBA), was successfully printed into fungal-resistant voice prosthesis components, demonstrating the capability to use inkjet 3D printing to manufacture bespoke medical devices resistant to fungal attachment (Vallieres et al., 2020). In this paper, we detail the protocol for creating small batches of test specimens and subsequent testing against C. albicans. The preparation of inkjet-printable ink formulations and other key parameters for successful printing are described, as well as the scripts enabling the production of self-support 3D geometries. With the help of this protocol, material screening and specimen preparation can be simplified and accelerated for the discovery of printable materials for medical applications (e.g., in-dwelling devices resistant to fungal biofilms, as described here) or non-medical applications (e.g., identification of photoreactive materials for printing electronics).

Materials and Reagents

Starlab tips (Starlab, catalog numbers: S1111-1706 and S1111-6701)
90 mm Petri dishes (Fisher Scientific, catalog number: 11308283)
10 ml and 25 ml pipettes (Greiner Bio-One, catalog numbers: 607180 and 760180)
50 ml centrifuge tubes (Scientific Laboratory Supplies, catalog number: SLS8110)
2 ml reaction tubes (Greiner Bio-One, catalog number: 623201)
12-well and 96-well plates (Greiner Bio-One, catalog numbers: 665102 and 655185)
Nitrogen gas (BOC UN1066 compressed nitrogen)
5 ml syringe (BD Emerald REF 307731)
PEN film (GTS RPEN-075-0320)
Vials, 22 ml disposable scintillation vials
Ductile tape, Scotch^®
Syringe filters, Millex-HA, MF-Millipore Membrane 50pk 0.45 µm
0.2 μm Sartorius^TM Ministart^TM syringe filter
Syringe, Braun inkjet 10 ml syringe
BD Discardit II 20 ml syringe
2,2-Dimethoxy-2-phenylacetophenone (99%, Sigma-Aldrich), stored at 4°C
(R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone (AODMBA) (95%, Sigma, catalog number: 376361), stored at 4°C
RPMI 1640 with 20 mM HEPES and L-glutamine, without sodium bicarbonate (Sigma, catalog number: R7388)
NB: RPMI 1640 can be stored at 4°C for several months.
PBS (phosphate-buffered saline) (Fisher Scientific, catalog number: 10209252)
NB: PBS is autoclaved and stored at room temperature for several months.
Crystal violet (Sigma, catalog number: C3886)
NB: Crystal violet is made fresh each day and filter-sterilized using a BD Discardit II 20 ml syringe and a 0.2 μm Sartorius^TM Ministart^TM syringe filter.
Isopropanol (Fisher Scientific, Laboratory reagent grade ≥99.5%)
YPD medium (see Recipes)
Bacteriological peptone (Oxoid, catalog number: LP0037)
Yeast extract (Oxoid, catalog number: LP0021)
D-glucose anhydrous (Fisher Scientific, catalog number: G/0500/61)

Equipment

50 ml Erlenmeyer flasks (Fisher Scientific, catalog number: 15499093)
PTFE stirrer 5 mm
FujiFilm Dimatix DMP-2830 material printer
FujiFilm 10pL DMP cartridges DMC-1610/PN2100201146
Inert customized purge box PG-7-0444
UV unit: EPL UV 119-070 high-intensity unit
Stirring stage IKA RCT Basic
Fridge LEC Medical PE207C
Vacuum oven: Fistreem 31L capacity
Gilson^TM PIPETMAN^TM Neo Pipets
Biological safety cabinet with UV unit
Heated incubator IN30 Memmert
Heated orbital shakers: New Brunswick^TM Innova^® 44 (for flasks) ELMI SkyLine DTS-4 Digital Thermo Shaker (for microplates)
Eppendorf centrifuge 5810
Scientific Industries SI^TM Vortex-Genie^TM 2
Hemocytometer Weber Scientific International Ltd.
Prior Scientific Microscope PX043
Sterile tweezers
BioTek EL800 Microplate Spectrophotometer
Balance (Denver Instrument SI-234)

Software

Fujifilm DMP version 2.0.0.1
GIMP 2.8.14
AutoHotKey 1.1.32.00

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

He, Y., Vallieres, C., Alexander, M. R., Wildman, R. D. and Avery, S. V. (2021). Inkjet 3D Printing of Polymers Resistant to Fungal Attachment. Bio-protocol 11(9): e4016. DOI: 10.21769/BioProtoc.4016.
Vallieres, C., Hook, A. L., He, Y., Crucitti, V. C., Figueredo, G., Davies, C. R., Burroughs L., Winkler D.A., Wildman R.D., Irvine D.J., Alexander, M. R., Avery S.V., (2020). Discovery of (meth) acrylate polymers that resist colonization by fungi associated with pathogenesis and biodeterioration. Science Adv 6: eaba6574.