参见作者原研究论文

本实验方案简略版
Jun 2020
Advertisement

本文章节


 

Inkjet 3D Printing of Polymers Resistant to Fungal Attachment
抗真菌附着聚合物的喷墨3D打印   

引用 收藏 提问与回复 分享您的反馈 Cited by

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

  1. Starlab tips (Starlab, catalog numbers: S1111-1706 and S1111-6701)

  2. 90 mm Petri dishes (Fisher Scientific, catalog number: 11308283)

  3. 10 ml and 25 ml pipettes (Greiner Bio-One, catalog numbers: 607180 and 760180)

  4. 50 ml centrifuge tubes (Scientific Laboratory Supplies, catalog number: SLS8110)

  5. 2 ml reaction tubes (Greiner Bio-One, catalog number: 623201)

  6. 12-well and 96-well plates (Greiner Bio-One, catalog numbers: 665102 and 655185)

  7. Nitrogen gas (BOC UN1066 compressed nitrogen)

  8. 5 ml syringe (BD Emerald REF 307731)

  9. PEN film (GTS RPEN-075-0320)

  10. Vials, 22 ml disposable scintillation vials

  11. Ductile tape, Scotch®

  12. Syringe filters, Millex-HA, MF-Millipore Membrane 50pk 0.45 µm

  13. 0.2 μm SartoriusTM MinistartTM syringe filter

  14. Syringe, Braun inkjet 10 ml syringe

  15. BD Discardit II 20 ml syringe

  16. 2,2-Dimethoxy-2-phenylacetophenone (99%, Sigma-Aldrich), stored at 4°C

  17. (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone (AODMBA) (95%, Sigma, catalog number: 376361), stored at 4°C

  18. 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.

  19. PBS (phosphate-buffered saline) (Fisher Scientific, catalog number: 10209252)

    NB: PBS is autoclaved and stored at room temperature for several months.

  20. 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 SartoriusTM MinistartTM syringe filter.

  21. Isopropanol (Fisher Scientific, Laboratory reagent grade ≥99.5%)

  22. 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

  1. 50 ml Erlenmeyer flasks (Fisher Scientific, catalog number: 15499093)

  2. PTFE stirrer 5 mm

  3. FujiFilm Dimatix DMP-2830 material printer

  4. FujiFilm 10pL DMP cartridges DMC-1610/PN2100201146

  5. Inert customized purge box PG-7-0444

  6. UV unit: EPL UV 119-070 high-intensity unit

  7. Stirring stage IKA RCT Basic

  8. Fridge LEC Medical PE207C

  9. Vacuum oven: Fistreem 31L capacity

  10. GilsonTM PIPETMANTM Neo Pipets

  11. Biological safety cabinet with UV unit

  12. Heated incubator IN30 Memmert

  13. Heated orbital shakers: New BrunswickTM Innova® 44 (for flasks) ELMI SkyLine DTS-4 Digital Thermo Shaker (for microplates)

  14. Eppendorf centrifuge 5810

  15. Scientific Industries SITM Vortex-GenieTM 2

  16. Hemocytometer Weber Scientific International Ltd.

  17. Prior Scientific Microscope PX043

  18. Sterile tweezers

  19. BioTek EL800 Microplate Spectrophotometer

  20. Balance (Denver Instrument SI-234)

Software

  1. Fujifilm DMP version 2.0.0.1

  2. GIMP 2.8.14

  3. AutoHotKey 1.1.32.00

Procedure

Development of an ink for inkjet-based 3D printing processes should consider not only viscosity and surface tension as per typical 2D print ink formulations (Derby, 2010) but also the reactivity or solidification speed, which is important and correlates with factors such as photoinitiator concentration and printing environment. We have described these factors in detail in our previous studies (He et al., 2016; Zhang et al., 2019). The protocol below revealed the successful candidate ink formulation developed from a (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone monomer printed in our recent work (Vallieres et al., 2020).

  1. Ink preparation

    1. Place a 5-mm PTFE stirrer into a 22-ml vial.

    2. Place the vial on a balance and tare the weight.

    3. Add 0.1 g 2,2-dimethoxy-2-phenylacetophenone (solid) into the vial.

    4. Wrap the vial in aluminium film before adding liquid monomers.

    5. Place the vial back on the balance and tare the weight.

    6. Add 10 g (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone (liquid) to the vial and close the cap.

    7. Place the vial on the stirring stage (IKA RCT Basic) and mix the contents at 800 rpm for 15 min at room temperature.

    8. Place the vial in a shaded box to help exclude ambient light during nitrogen purging. Open the cap and purge nitrogen gas (flow rate ~10 ml/min) into the mixture for 15 min through a needle, to allow the ink to become saturated with nitrogen. This step helps to minimize the oxygen inhibition effect during printing.

    9. Collect the ink with a 10-ml syringe in a dark room and purge through a syringe filter into a fresh vial. The filter will help to remove any solid contamination and avoid print head clogging during printing.

    10. Cap the vial and wrap in aluminum foil to exclude ambient light that could trigger polymerization and cause ink solidification.

    11. Store the ink for at least 12 h at 4°C before use.


  2. Filling the ink cartridge

    1. Carry out all the following steps in a dark room.

    2. Carefully collect 3 ml ink using a 5-ml syringe, taking care to avoid the generation of bubbles at this stage.

    3. Fix the flat-head needle that comes with the cartridge to the end of the syringe and carefully transfer the sampled 3 ml ink into the cartridge bag.

    4. Wrap the cartridge in ductile tape to avoid curing triggered by ambient light during printing and assemble the print head.


  3. Printing and post-processing

    1. Clamp the filled print head onto the DMP 2830 printer (Figure 1).



      Figure 1. Interior set up of the Dimatix DMP 2830 printer


    2. Affix the ceramic cleaning pad and purge the cartridge for 2 s to remove residual air from the print head.

    3. Cut a PEN film to A4 size and clean both sides with isopropanol.

    4. Place the film on the printing stage and turn on the vacuum to ensure that the film is firmly held on the stage.

    5. Install the front panel of the glovebox and purge nitrogen until the oxygen level is reduced to 0.2-0.3% (v/v).

    6. Disable the tickle control function, monitor the drop viewer to check for droplet formation, and set the printing waveform as shown below (Table 1).



      Table 1. Key parameters of the jetting and non-jetting waveforms used in (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone ink
      Jetting Waveform
      Level (%) Slew Rate Duration (µs) Maximum Jetting Frequency (kHz) Waveform Width (µs)
      Section 1 0 0.65 3.584 5 11.52
      Section 2 100 0.93 3.712
      Section 3 13 0.6 3.392
      Section 4 27 0.8 0.832
      Non-Jetting Waveform
      Level (%) Slew Rate Duration (µs) Maximum Jetting Frequency (kHz) Waveform Width (µs)
      Section 1 33 1 3.712 5 11.52
      Section 2 33 1 6.976
      Section 3 27 1 0.832


    7. Set the printing temperature to 48°C and the meniscus to 4.0 inches H2O. Adjust the printing voltage of each nozzle such that droplet formation is similar to that shown in Figure 2. The droplet speed needs to be between 7 m/s and 10 m/s. Twelve jets (jets 5-16) were used in our work.



      Figure 2. Exemplar droplet formation for (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone ink captured by the Dimatix instrument drop watcher.

      A full video is attached in the supplementary document.


    8. Set the droplet spacing to 35 µm and the print head angle to 5.8 degrees.

    9. Set the cleaning program to a 0.3 s purge and blot at the beginning of each layer, raising the print head by 12 µm after each layer is finished.

    10. The printing pattern for each layer is a mono bitmap, which is named as number+’.bmp’ starting from 1.bmp; select 1.bmp in the printing program.

    11. Copy the script in the supplement (Supplementary Script) into a notepad and save as an AHK file.

    12. Run the saved script and sequentially input the ‘original height’, ‘number of layers’, and ‘height increment per layer.’ For (R)-α-acryloyloxy-β,β-dimethyl-γ-butyrolactone ink, we used 200 µm, 100 layers, and 11 µm height increment per layer.

    13. Deposit the liquid monomer ink onto the target location using a drop-on-demand inkjet print head. The printed ink is scanned and cured via the attached UV unit as it moves with the print head (He et al., 2017).

    14. Collect the printed samples and place in a vacuum oven (room temperature, -300 mmHg) for 24 h (Figure 3).



      Figure 3. Voice prosthesis valve-flap samples collected after vacuum drying


    15. Wash the samples by sequential immersion in isopropanol and distilled water, and dry in air.

    16. Transfer the samples to a 12-well plate and irradiate with UV-C for 20 min to sterilize the samples.


  4. Growing the fungal cultures, preparation of inocula, and biofilm formation (use aseptic technique; a biological safety cabinet may be required depending on the fungal species)

    Day 1 – Streak out strains
    1. Inoculate YPD agar medium with C. albicans cells from a stock kept at -80°C (for the stock, freeze C. albicans cells at -80°C in YPD broth with 20% glycerol) and incubate for 2 days in a static incubator at 37°C. We used the C. albicans strain CAF2-yCherry; however, the method can be applied to other C. albicans strains.


    Day 3 – Set up overnight cultures
    1. Inoculate YPD broth medium (typically 10 ml medium in a 50-ml sterile Erlenmeyer flask) with a colony of C. albicans from a 2-day-old culture on YPD agar. We recommend assaying a minimum of three independent colonies. Incubate overnight in an orbital shaker (150 rpm) at 37°C.


    Day 4 – Harvest cells and inoculate printed samples
    1. Harvest cells from the overnight liquid cultures by centrifugation (3,000 × g for 3 min).

    2. Remove the supernatant and wash once in 20 ml PBS and once in 20 ml RPMI 1640 using a 25-ml pipette.

    3. Resuspend the final cell pellets in 10 ml RPMI 1640 using a 10-ml pipette.

    4. From the resulting cell suspensions, prepare 1:100 and/or 1:1,000 dilutions in RPMI 1640 in 2-ml reaction tubes, vortex, and count using a hemocytometer under a brightfield microscope with 40× objective.

    5. Calculate the inoculum required to prepare 40 ml cell suspension at a final concentration of 1 × 106 ml-1 cells in RPMI 1640.

    6. Transfer 1 ml adjusted cell suspension to each well of the 12-well plate (from Step C16).

    7. Cover the plate with its lid, seal with tape, and incubate statically for 2 h at 37°C to allow the cells to adhere to the surface.

    8. Use sterile tweezers to transfer the printed samples to a fresh plate and wash three times with 2 ml PBS to remove non-adhered cells.

      NB: Printed samples must be transferred carefully to fresh plates using tweezers to avoid mechanically disrupting cell attachment. The transfer of samples to fresh plates (here and in Step E2) is important since free cells can also adhere to the surface of the wells, which would add to the crystal violet signal intended to reflect biofilm formation on the printed sample, impairing the analysis.

    9. Add 1 ml fresh RPMI 1640 and incubate for 46 h at 37°C with orbital shaking at 100 rpm.


  5. Fungal biofilm assessment (use aseptic technique and a biological safety cabinet)

    Staining with crystal violet offers an approach for assessing the biomass of the biofilm. Alternatively, biofilm metabolic activity can be assayed; we detect such activity of 24 h-biofilms by measuring the reduction of XTT (tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrzolium-5-carboxanilide). However, crystal violet is less expensive than XTT and allows for the easy visual detection of biofilms on the surface of samples (e.g., Figure 4C in Vallieres et al., 2020).

    1. After 48 h, C. albicans should display mature biofilms. Aspirate the medium carefully so as not to touch or disrupt the biofilm.

    2. Gently transfer the printed samples carrying biofilm to a fresh plate.

      NB: As above, samples must be carefully transferred to fresh plates using tweezers to avoid mechanically disrupting the biofilm (mechanical disruption during sample transfer could lead to the biofilm detaching from the sample surface and interfering in the final assessment. Therefore, multiple specimens (n>3) are required in each test to ensure that biofilm resistance is not a result of operational error. A control group (Vallieres et al., 2020) is also needed to ensure that the biofilm formation is as expected).

    3. Stain the biofilms with 2 ml 0.5% (w/v) crystal violet for 1 min at room temperature.

    4. Wash three times with 2 ml PBS to remove non-adhered biofilm and excess stain.

    5. For quantitation, add 1 ml ethanol to dissolve the crystal violet.

    6. Transfer 100 μl reaction to a 96-well plate. Dilutions of the reaction may be needed if the concentration of crystal violet is too high. We recommend the preparation of a standard curve to estimate the level of saturation of crystal violet absorption before the assay. To do so, measure the absorbance over a range of crystal violet (in ethanol) concentrations.

    7. Measure the absorbance at 600 nm using the BioTek EL800 microtiter plate reader.

Data analysis

From the resulting colorimetric readings after subtracting the corresponding values for the negative control (ethanol only, i.e., no crystal violet), calculate the mean (of at least three biological replicates) and SEM, and plot the data. Alternatively, results for the 3D-printed samples can be presented as a percentage of the control [in Vallières et al., 2020, the positive control was a commercial prosthesis valve flap provided by Atos Medical (raw material Silastic Q7-4735, Dow Corning)].

Notes

Crystal violet can be incompatible with some materials e.g., polyethylene glycol diacrylate (PEG575DA). Printed PEG575DA samples are completely stained with crystal violet even in the absence of C. albicans. For those materials, we recommend the assessment of fungal biofilm formation using the XTT reduction assay mentioned above.

Recipes

  1. YPD medium

    2% bacteriological peptone

    1% yeast extract

    2% D-glucose anhydrous

    NB: Where necessary, the medium is solidified with 2% (w/v) agar (Sigma, catalog number: A7002). YPD medium is autoclaved and can subsequently be stored at room temperature for several months.

Acknowledgments

This work was supported by the Biotechnology and Biological Sciences Research Council (grant number BB/P02369X/1) and the Engineering and Physical Sciences Research Council (grant numbers EP/N006615/1 and EP/N024818/1).

Competing interests

The authors declare no competing interests.

References

  1. Cavalheiro, M. and Teixeira, M. C. (2018). Candida Biofilms: Threats, Challenges, and Promising Strategies. Front Med (Lausanne) 5: 28.
  2. Derby, B. (2010). Inkjet printing of functional and structural materials: fluid property requirements, feature stability, and resolution. Annu Rev Mater Res 40: 395-414.
  3. He, Y., Wildman, R. D., Tuck, C. J., Christie, S. D., and Edmondson, S. (2016). An Investigation of the Behavior of Solvent based Polycaprolactone ink for Material Jetting. Sci Rep 6: 20852.
  4. He, Y., Tuck, C. J., Prina, E., Kilsby, S., Christie, S. D., Edmondson, S, Hague R.J.M., Rose F.R.A.J., Wildman, R. D. (2017). A new photocrosslinkable polycaprolactone-based ink for three‐dimensional inkjet printing. J Biomedical Mater Res B App Biomater 105(6): 1645-1657.
  5. 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.
  6. Zhang, F., Saleh, E., Vaithilingam, J., Li, Y., Tuck, C. J., Hague, R. J., Wildman R.D., He, Y. (2019). Reactive material jetting of polyimide insulators for complex circuit board design. Addit Manuf 25: 477-484.

简介

[摘要]喷墨3D打印是一种附加制造方法,允许用户生产一小批定制设备,用于对比研究与商业产品。在这里,我们描述了使用商业2D墨水开发系统(Dimatix材料打印)来制造小批量的3D医疗或其他设备,使用最近被鉴定的真菌抗附着材料。

这种印刷设备可以抵抗由于真菌病原体白色念珠菌的定植而困扰商业医疗产品的问题。通过依次引入产品CAD模型的横截面位图并使用自动单击脚本提升打印头高度,我们能够使用2D墨水开发系统创建复杂的自支撑几何图形。使用这一方案,研究人员只需从几克原料中提取一小批样品,就可以对其进行表征。此外,我们还描述了真菌抗附着的制造标本的测试。与大多数商业AM系统相比,它至少需要几百克的油墨进行印刷试验,我们的协议非常适合材料研究中的小规模生产。关键词:添加剂制造、喷墨、3D打印、白色念珠菌、真菌生物膜



[背景]增材制造(AM)或3D打印是一种方便的工具,用于制备小批量产品,如生物医学或电子设备;然而,商业AM系统通常需要至少几百克的材料来生产最小的一批产品,这对于专注于材料探索的研究来说仍然是一个挑战。Dimatix材料打印机是一种为2D打印设计的商业墨水配方开发工具,使用一次性墨盒运行,打印只需要3-5克墨水。它的印刷单元还允许高度调整高达25毫米。通过定制的脚本,我们能够使用这个2D打印机来模拟基于喷墨的3D打印过程,并仅从几克原料中制备实验样本。该方案已用于我们的真菌抗附着材料筛选研究(Vallieres et al.,2020),其中仅使用材料筛选中发现的候选单体制成的4克墨水制备了一批6个样本。白念珠菌等真菌形成的生物膜被视为医院内系统感染的重要来源,死亡率高达50%(Cavalheiro和Teixeira,2018)。印刷品对C的抵抗力。白色念珠菌的定植可以通过使用结晶紫测量真菌生物膜的形成来测试,结晶紫与带负电荷的表面分子和细胞外基质中的多糖结合。候选抗附着甲基丙烯酸酯聚合物(R)-α-丙烯酰氧基-β,β-二甲基-γ-丁内酯(AODMBA)被成功印刷到抗真菌语音假体组件中,证明了使用喷墨3D打印技术制造抗真菌附着的定制医疗器械的能力(Valliers等人,2020)。在本文中,我们详细介绍了创建小批量的测试样本和随后对C。白色念珠菌。描述了喷墨打印墨水配方的制备和成功打印的其他关键参数,以及能够产生自支撑三维几何图形的脚本。在本协议的帮助下,可以简化和加速材料筛选和样品制备,以发现可打印材料用于医疗应用(例如,如本文所述的抗真菌生物膜的居住设备)或非医疗应用(例如。,印刷电子产品用光活性材料的鉴定。

关键字:增材制造, 喷墨, 3D打印, 白色念珠菌, 真菌生物膜

材料和试剂

1.    星实验室提示(星实验室,目录号:S1111-1706和S1111-6701)<

2.    90 mm培养皿(Fisher Scientific,目录号:11308283)<

3.    10 ml和25 ml移液管(Greiner Bio One,目录号:607180和760180)<

4.    50 ml离心管(科学实验室用品,目录号:SLS8110)<

5.    2 ml反应管(Greiner Bio One,目录号:623201)<

6.    12孔板和96孔板(Greiner Bio One,目录号:665102和655185)<

7.    氮气(BOC UN1066压缩氮气)<

8.    5毫升注射器(BD翡翠参考号307731)<

9.    笔膜(GTS RPEN-075-0320)<

10.  小瓶,22毫升一次性闪烁小瓶<

11.  韧性胶带,苏格兰威士忌®<

12.  注射器过滤器,Millex HA,MF微孔膜50pk 0.45µ米<

13.  0.2μm SartoriusTM MinistartTM注射器过滤器<

14.  注射器,布劳恩喷墨10毫升注射器<

15.  BD Discardit II 20毫升注射器<

16.  2,2-二甲氧基-2-苯乙酮(99%,Sigma-Aldrich),储存于4°C级<

17.  (右)-α-丙烯酰氧基-β,β-二甲基-γ-丁内酯(AODMBA)(95%,西格玛,目录号:376361),储存于4°C级<

18.  RPMI 1640,含20 mM HEPES和L-谷氨酰胺,不含碳酸氢钠(Sigma,目录号:R7388)<

注:RPMI 1640可存储在4°好几个月了。<

19.  PBS(磷酸盐缓冲盐水)(Fisher Scientific,目录号:10209252)NB:PBS在室温下高压灭菌并储存数月。<

20.  结晶紫(Sigma,目录号:C3886)<

注:结晶紫每天都是新鲜的,用BD Discardit II 20ml注射器和0.2ml注射器过滤消毒μm SartoriusTM MinistartTM注射器过滤器。<

21.  异丙醇(Fisher Scientific,实验室试剂级≥99.5%)<

22.  YPD培养基(见配方)<

细菌蛋白胨(Oxoid,目录号:LP0037)<

酵母提取物(Oxoid,目录号:LP0021)<

D-无水葡萄糖(Fisher Scientific,目录号:G/0500/61)

设备


1.    50 ml锥形烧瓶(Fisher Scientific,目录号:15499093)<

2.    聚四氟乙烯搅拌器5 mm<

3.    富士胶片DMP-2830材料打印机<

4.    FujiFilm 10pL DMP墨盒DMC-1610/PN2100201146<

5.    惰性气体净化箱PG-7-0444<

6.    紫外线装置:EPL UV 119-070高强度装置<

7.    搅拌阶段IKA RCT基本<

8.    冰箱LEC Medical PE207C<

9.    真空烤箱:Fistreem 31L容量<

10.  GilsonTM移液管Neo移液管<

11.  带紫外线装置的生物安全柜<

12.  30分钟加热培养箱<

13.  加热轨道振动筛:新不伦瑞克TM Innova® 44(用于烧瓶)ELMI SkyLine DTS-4数字热振动筛(用于微孔板)<

14.  Eppendorf离心机5810<

15.  科学工业<

16.  韦伯科学国际有限公司。<

17.  先前的科学显微镜PX043<

18.  无菌镊子<

19.  BioTek EL800微孔板分光光度计<

20.  天平(丹佛仪器SI-234)

软件


1.    富士胶片DMP 2.0.0.1版<

2.    GIMP 2.8.14<

3.    自动热键1.1.32.00

程序


根据典型的2D打印墨水配方(Derby,2010),用于喷墨3D打印工艺的墨水的开发不仅应考虑粘度和表面张力,还应考虑反应性或固化速度,这一点很重要,并与光引发剂浓度和打印环境等因素相关。我们在之前的研究中详细描述了这些因素(He等人,2016;Zhang等人,2019年)。下面的方案揭示了从a(R)开发成功的候选油墨配方-α-丙烯酰氧基-β,β-二甲基-γ-我们最近的工作中印刷了丁内酯单体(Vallieres等人,2020)。

答。油墨制备<

1.    将5 mm聚四氟乙烯搅拌器放入22 ml小瓶中。<

2.    将小瓶放在天平上并用皮重称量。<

3.    向小瓶中添加0.1 g 2,2-二甲氧基-2-苯乙酮(固体)。<

4.    在添加液体单体之前,用铝膜包裹小瓶。<

5.    将小瓶放回天平上,并用皮重称量。<

6.    添加10 g(R)-α-丙烯酰氧基-β,β-二甲基-γ-将丁内酯(液体)倒入小瓶中并盖上瓶盖。<

7.    将小瓶置于搅拌阶段(IKA RCT Basic),并在室温下以800 rpm混合内容物15分钟。<

8.    在氮气吹扫过程中,将小瓶放在有阴影的盒子中,以帮助排除环境光。打开瓶盖,用针头将氮气(流速约10毫升/分钟)吹入混合物中15分钟,使油墨充满氮气。这一步有助于减少印刷过程中的氧气抑制效果。<

9.    在暗室中用10毫升注射器收集墨水,并通过注射器过滤器吹扫到一个新的小瓶中。过滤器将有助于清除任何固体污染物,并避免打印过程中打印头堵塞。<

10.  盖住小瓶并用铝箔包裹,以排除可能引发聚合并导致墨水凝固的环境光。<

11.  在4小时内储存油墨至少12小时°C使用前。

B。加注墨盒<

1.    在暗室中执行以下所有步骤。<

2.    使用5毫升注射器小心收集3毫升墨水,注意避免在此阶段产生气泡。<

3.    将墨盒附带的平头针固定在注射器末端,并小心地将取样的3毫升墨水转移到墨盒袋中。<

4.    用韧性胶带包裹墨盒,以避免打印过程中环境光引发固化,然后组装打印头。

C。印刷和后处理<

1.    将装满的打印头夹在DMP 2830打印机上(图1)。



图1。Dimatix DMP 2830打印机的内部设置


2.    贴上陶瓷清洁垫并吹扫墨盒2秒钟,以清除打印头中的残留空气。<

3.    将笔膜剪成A4大小,并用异丙醇清洁两侧。<

4.    将胶卷放在印刷台上,打开真空,确保胶卷牢固地固定在印刷台上。<

5.    安装杂物箱的前面板并吹扫氮气,直到氧气液位降至<

0.2-0.3%(v/v)。<

6.    禁用挠痒痒控制功能,监视水滴查看器以检查水滴的形成,并设置如下所示的打印波形(表1)。<

       

表1。(R)中使用的喷射和非喷射波形的关键参数-α丙烯酰氧基-β,β-二甲基-γ-丁内酯油墨
喷射波形









水平(%)<

回转率<

持续时间(µ秒)<

最大喷射量<

频率(kHz)<

波形宽度<

(µ秒)<

第1节<

0<

0.65<

3.584<

5<

11.52<

第2节<

100<

0.93<

3.712<

第3节<

13<

0.6<

3.392<

第4节<

27<

0.8<

0.832<

非喷射波形









水平(%)<

回转率<

持续时间(µ秒)<

最大喷射量<

频率(kHz)<

波形宽度<

(µ秒)<

第1节<

33<

1<

3.712<

5<

11.52<

第2节<

33<

1<

6.976<

第3节<

27<

1<

0.832

7.    将打印温度设置为48°C和弯月面到4.0英寸H2O。调整每个喷嘴的打印电压,使液滴形成类似于图2所示。液滴速度需要在7 m/s和10 m/s之间。我们的工作中使用了12架喷气机(喷气机5-16)。



图2。(R)液滴形成示例-α-丙烯酰氧基-β,β-二甲基-γ-丁内酯墨水由Dimatix仪器滴落观察者捕获。补充文件中附有完整的视频。

8.    将液滴间距设置为35µm和打印头的角度为5.8度。<

9.    将清洁程序设置为在每层开始时进行0.3秒的清洗和吸干,将打印头升高12µ每层完成后m。<

10.  每一层的打印图案是一个单位图,从1.bmp开始命名为number+“.bmp”;在打印程序中选择1.bmp。<

11.  将补充(补充脚本)中的脚本复制到记事本中并另存为AHK文件。<

12.  运行保存的脚本,依次输入(R)的“原始高度”、“层数”和“每层高度增量”-α-丙烯酰氧基-β,β-二甲基-γ-丁内酯油墨,我们用200µm、 100层,11层µm每层高度增量。<

13.  使用点滴式喷墨打印头将液体单体墨水沉积到目标位置。印刷油墨随打印头移动时,通过连接的UV单元进行扫描和固化(He等人,2017)。<

14.  收集印刷样品并放置在真空烘箱(室温,-300 mmHg)中24小时(图3)。



图3。真空干燥后采集的人工语音瓣样本


15.  将样品依次浸入异丙醇和蒸馏水中清洗,并在空气中干燥。<

16.  将样品转移到12孔板上,用UV-C照射20分钟,使样品灭菌。

D。真菌培养物的生长、接种物的制备和生物膜的形成(使用无菌技术;根据真菌种类,可能需要一个生物安全柜)<

第1天-去除菌株
1用C。白念珠菌细胞从一个股票保持在-80°C(对于股票,冻结C。白念珠菌细胞-80°在YPD肉汤中加入20%甘油),在37℃的静态培养箱中培养2天°C。我们用了C。白色念珠菌CAF2-yCherry株;但该方法也适用于其它C。白色念珠菌菌株。

第3天-建立隔夜培养
2用C。在YPD琼脂上培养2天的白色念珠菌。我们建议至少检测三个独立的菌落。在37转/分的轨道摇床(150转/分)中培养过夜°C。

第4天-收获细胞并接种打印样本
3.    通过离心(3000)从过夜液体培养物中收获细胞× g,3分钟)。<

4.    去除上清液,用25毫升移液管在20毫升PBS和20毫升RPMI 1640中清洗一次。<

5.    使用10 mL移液管将最终细胞颗粒重新悬浮在10 mL RPMI 1640中。<

6.    从得到的细胞悬浮液中,在2mL反应管中制备1:100和/或1:1000的RPMI 1640稀释液,旋涡,并在brightfield显微镜下用40× 目标。<

7.    计算制备最终浓度为1的40 mL细胞悬浮液所需的接种量× RPMI 1640中的106 ml-1细胞。<

8.    将1 ml调整后的细胞悬液转移到12孔板的每个孔中(从步骤C16开始)。<

9.    盖上盖子,用胶带密封,在37℃静置2小时°使细胞粘附在表面。<

10.  用无菌镊子将打印好的样品转移到一个新的盘子里,用清水冲洗三次<

2毫升PBS去除不粘附的细胞。<

注:打印的样品必须小心地转移到新鲜的平板上,使用镊子,以避免机械破坏细胞附着。将样品转移到新鲜平板(此处和步骤E2中)很重要,因为游离细胞也可以粘附在微孔表面,这将增加结晶紫信号,以反映打印样品上生物膜的形成,从而影响分析。<

11.  加入1毫升新鲜RPMI 1640,在37℃下培养46小时°以100转/分的速度进行轨道震动。

E。真菌生物膜评估(使用无菌技术和生物安全柜)<

结晶紫染色提供了一种评估生物膜生物量的方法。或者,可以测定生物膜的代谢活性;我们通过测量XTT(四唑盐,2,3-双[2-甲氧基-4-硝基-5-磺苯基]-2H-四唑-5-羧基苯胺)的还原来检测24小时生物膜的这种活性。然而,结晶紫的价格比XTT便宜,可以方便地目视检测样品表面的生物膜(例如,Valliers等人,2020年的图4C)。<

1.    48小时后,C。白色念珠菌应显示成熟的生物膜。小心吸入培养基,以免接触或破坏生物膜。<

2.    轻轻地将带有生物膜的印刷样品转移到一个新的平板上。<

注:如上所述,必须使用镊子小心地将样品转移到新鲜平板上,以避免机械破坏生物膜(样品转移过程中的机械破坏可能导致生物膜从样品表面分离并干扰最终评估)。因此,每次试验需要多个样本(n>3),以确保生物膜阻力不是操作错误造成的。还需要一个对照组(Vallieres et al.,2020),以确保生物膜的形成符合预期)。<

3.    在室温下用2ml 0.5%(w/v)结晶紫染色1分钟。<

4.    用2ml PBS冲洗三次,去除不粘附的生物膜和多余的污渍。<

5.    定量时,加入1毫升乙醇溶解结晶紫。<

6.    转移100μ我对96孔板有反应。如果结晶紫的浓度过高,可能需要稀释反应。我们建议在分析前准备一个标准曲线来估计结晶紫吸收的饱和水平。为此,测量结晶紫(乙醇中)浓度范围内的吸光度。<

7.    使用BioTek EL800微量滴定板阅读器测量600 nm处的吸光度。

数据分析


从减去阴性对照(仅乙醇,即无结晶紫)的相应值后得到的比色读数,计算平均值(至少三个生物复制品)和SEM,并绘制数据。或者,3D打印样本的结果可以表示为对照的百分比[以Valli表示]ères等人,2020年,阳性对照是由Atos Medical提供的商用人工瓣膜(原料硅橡胶Q7-4735,道康宁公司)。

笔记


结晶紫可能与某些材料不相容,例如聚乙二醇二丙烯酸酯<

(PEG575DA)。即使在没有C的情况下,印刷的PEG575DA样品也完全被结晶紫染色。白色念珠菌。对于这些材料,我们建议使用上述XTT还原试验评估真菌生物膜的形成。

食谱


1YPD培养基<

2%细菌蛋白胨<

1%酵母抽提物<

2%无水葡萄糖<

注:必要时,用2%(w/v)琼脂(Sigma,目录号:A7002)固化培养基。YPD培养基经过高压灭菌,随后可在室温下储存数月。

致谢


这项工作得到了生物技术和生物科学研究理事会(批准号BB/P02369X/1)和工程和物理科学研究理事会(批准号EP/N006615/1和EP/N024818/1)的支持。

相互竞争的利益相互竞争的利益

作者声明没有相互竞争的利益。

参考文献


1.    卡瓦莱罗,M。特谢拉,M。C(2018). 念珠菌生物膜:威胁,挑战和有前途的策略。前地中海(洛桑)5:28。<

2.    德比,B(2010). 特征稳定性和分辨率。年鉴40:395-414。功能材料和结构材料的喷墨打印:流体性能要求,<

3.    他,Y,怀德曼,R。D.,塔克,C。J.,克里斯蒂,S。D.和Edmondson,S(2016). 溶剂型聚己内酯喷墨材料的性能研究。Sci报告6:20852。调查<

4.    他,Y,塔克,C。J.,Prina,E.,Kilsby,S.,Christie,S。D.,埃德蒙森,S.,黑格R.J.M.,罗斯F.R.A.J.,威尔德曼,R。D(2017). 一种用于三维喷墨打印的新型光交联聚己内酯基油墨。105(6): 1645-1657. 生物医学材料研究杂志<

5.    瓦利耶斯,C.,胡克,A。L.,他,Y.,克鲁西蒂,V。C.,菲格雷多,G.,戴维斯,C。R.,Burroughs L.,Winkler D.A.,Wildman R.D.,Irvine D.J.,Alexander,M。R.,Avery S.V.,(2020年)(甲基)丙烯酸酯聚合物,可抵抗与致病和生物退化有关的真菌的定植。科学杂志6:eaba6574。发现<

6.    Zhang,F.,Saleh,E.,Vaithilingam,J.,Li,Y.,Tuck,C。J.,海牙,R。J.,Wildman R.D.,他,Y(2019). 附加制造商25:477-484。用于复杂电路板设计的聚酰亚胺绝缘体活性物质喷射。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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.
  2. 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.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。