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Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

互联网2013-11-13

963

实验材料

Name	Company	Catalog Number	Comments
HP DeskJet 500	Hewlett-Packard	C2106A	Discontinued from manufacturer. Purchased refurbished DEC Trader.
HP 26 Black Ink Cartridge	Hewlett-Packard	51626A
Actin from rabbit muscle, Alexa Fluor 488 conjugate, 200 ?g	Invitrogen	A12373
Phosphate Buffered Saline (PBS)	MP Biomedicals	ICN1860454
Dulbeccos Modified Eagles Medium (DMEM)	Thermo Scientific	SH3002201
Fetal Bovine Serum	Sigma-Aldrich	F4135
Amphotericin B	Sigma-Aldrich	A2942	Used at 0.5% in cell culture media
Penicillin-Streptomycin	Sigma-Aldrich	P4333	Used at 0.5% in cell culture media
Unidirectional Flow Clean Bench	Envirco	VLF 797	Optional housing for keeping printer aseptic

实验步骤

1. Converting the HP DeskJet 500

It should be noted that this technique should work with many commercially inkjet printers. However, older printers tend to work better as they use ink cartridges with larger diameter nozzles, which do not clog as easily. In addition, older printers tend to use mechanical paper feed sensors that are easier to bypass. Printers with optical sensors can be tricked but using a small strip of paper on the far edge of the printer during each cycle, but is a bit more difficult than the mechanical system to "trick". The currently commercially available printers that work the best have low resolution (DPI).

Higher resolution printers tend to clog more easily. The resolution of the HP Deskjet 500 is 300 DPI. There are many commercially available printers (HP Deskjet series and others) that have a resolution of 600 DPI. This type of printer can be used with only a small increase in nozzle clogging issues that can be alleviated using careful cleaning (section 3).

1) Remove the top plastic case of the printer by unlocking several plastic clips from the bottom base of the printer and slowly lifting the top off.

2) Unscrew button/display light panel from top of printer, leaving it connected to the printer's motherboard.

3) Clean the inside of the printer, especially the areas where the ink cartridge rests and where printing occurs.

4) Locate the cables supplying power to the paper feed mechanisms and unplug them from the motherboard.

a) In the HP DeskJet 500, these are found just below the paper tray towards the left of the front.

5) Locate paper detection mechanism. Bypass the mechanism by affixing a string or wire loop to serve as a manual pull handle.

a) In the HP DeskJet 500, the paper detection mechanism is a gray plastic lever found above and behind the printing/paper feed mechanism.

6) Create a stage in front of the paper feed mechanism (where the paper would be fed from and deposited) in order to bring the desired printing slides to a level just below the cartridge print head.

a) For our experiments, the foam shipping holder for 15 mL centrifuge tubes was used with several microscope slides taped in place in the printing region to bring the final level of the slides to the desired height.

7) To maintain aseptic technique, the printer can be placed inside a standard biohazard cabinet or tabletop laminar flow hood.

8) It is important to note that modifying a commercially available ink-jet printer will usually void the printer's manufacturer warranty.

2. Converting Stock HP Ink Cartridges (HP 26 Black ink cartridge)

1) Remove the cartridge from its packaging, leaving the protective tape covering the printer contacts and print head for the time being.

2) Stabilize the body of the cartridge (black portion) either with a clamp or vise (simply firmly gripping the cartridge by hand usually worked as well), leaving the green top clear of any obstacle.

3) Using pliers or an adjustable wrench, grasp the green top of the cartridge and twist back and forth several times until it breaks free.

a) This should not take much force, but the top is no longer needed, so it is okay if it breaks when removing it.

4) Using screwdrivers, pry off the clear plastic piece now exposed.

a) Again, this should not take much force, but it will not be needed, so it is okay if it breaks during removal.

5) Empty any remaining from the reservoir.

6) Remove the plastic protective tape covering the printer contacts and print head.

7) Thoroughly flush the reservoirs with water.

a) Use a pipette or syringe to push water through the channels.

b) Water will likely leak from the print head during this process; this is acceptable, and does not cause any damage to the functionality of the cartridge.

8) When the water runs clear, allow the cartridge to dry.

3. Cleaning Ink Cartridge

1) In order to maintain a clean printing environment, the ink cartridge should be cleaned before and after use.

a) This also helps with avoiding crystallization of salts and other biological material in the cartridge that could cause blockages.

2) Fully submerge the cartridge in a beaker full of de-ionized water, and sonicate for 15 minutes before and after printing.

3) After sonication, remove the cartridge from the water, and shake out excess water.

4) Spray 70% ethanol into the cartridge to create a more aseptic environment.

a) Ensure that the ethanol has dried before adding the bioink printing solution.

4. Making Cell Suspension - "Bioink"

1) Culture cells until ready to passage.

a) For 3T3 fibroblasts: Seed cells on T-75 flasks at approximately 1.3x10⁴ cells/cm² in Dulbeccos Modified Eagles Medium (DMEM) with 10% Fetal Bovine Serum (FBS). Culture cells for two days in an incubator at 37 °C and 5% CO₂ .

2) Make fluorescent g-actin stock solution at concentration 50 μg/ml in Phosphate Buffer Solution (PBS).

3) Passage cells.

a) For 3T3 fibroblasts: Remove media from flask and rinse twice with PBS. Cover cells with 5 ml 0.25% Trypsin EDTA and incubate for 5 mins.

b) Add 5 ml of fresh medium and pipette the cell suspension into a 15 ml conical centrifuge tube and centrifuge at 1000 rpm for 5 min. Aspirate spent medium.

4) Resuspend the cells in PBS with fluorescent g-actin stock solution to create bioink.

a) Bioink should have a final concentration of g-actin of 10 μg/ml and a cell concentration of 1x10⁵ cell/ml. This concentration has been optimized to limit amount of cells per drop and clogging of the printer head.⁸

b) Note that 250 μl of bioink prints three cover slips with the pattern shown in Figure 2.

1) Power on and let the printer warm up.

2) Place desired printing surface (here, we use 22 mm x 22 mm coverslips) on the center of the stage prepared in step 1.6.

3) Create a printing pattern file.

a) Open Microsoft Word (or any other drawing software), and draw the desired pattern.

b) Pick a desired print pattern for cell printing in the premade file.

4) Load the prepared cartridge with desired cell suspension.

a) Pipette suspension into the small circular well at the bottom of cartridge compartment. Use approximately 100-120 μl of solution. The printed drop size is around 130 picoliters.⁸

5) Print the file with the HP Deskjet 500 Printer.

a) For best results, print smaller patterns multiple times (5), by changing the amount of copies desired in the word processing program.

6) Printer will warm up again, then cartridge will move to the "ready position".

7) When cartridge moves into the ready position (slightly to the left of the ink drip well below and stays there), pull up on the paper feed mechanism wire, and printing should commence.

a) For multiple copies of the printing, the paper feed mechanism will have to be released after every cycle or page printed. The cartridge will then return to the "ready" position, and the paper feed mechanism wire should be raised again.

8) The printer will print onto the coverslip placed on the center of the printing stage in step 5.2 (see Figure 2).

6. Representative Results

The representative results of the entire conversion process of a standard, off-the-shelf HP Deskjet 500, standard HP 26 series cartridges will create a printer with the capability of printing cell solutions for many types of analysis as stated before. The completed printer after conversion is shown in Figure 3, with a printing stage to place the microscope cover slip onto. The printer can be useful in analysis in many fields, including but not limited to: single cell mechanics, tissue engineering, gene transfection, biosensor micropatterning, and direct cell therapies. ^{1-10, 16-22}

In this example, an HP Deskjet 500 printer and HP 26 series ink cartridges were modified for bioprinting. Using this printer setup with a bioink consisting of a fibroblast cell suspension in a g-actin monomer solution, cells were printed onto glass microscope coverslips. Figure 1 illustrates representative results of printed fibroblast cells, which show incorporated fluorescent actin monomers. The results were obtained in a controlled aseptic environment in which the cells were printed into customizable patterns.

The pattern used in this example was created in Microsoft Word (Figure 2). This pattern created a continuous line of the printing solution across the majority of the microscope slide. Figure 4 shows the straight line in which the bioink and cells are mutually deposited. It should be noted that immediately after printing, there is an increase of background fluorescence because the bioink solution, in which the cells are suspended, contains free excess fluorescent actin monomers. This background fluorescence significantly decreases after the addition of growth media on the cells (Figure 1), which washes excess monomer from the substrate.

References：

1. Calvert, P. Materials science. Printing cells. Science. 318 (5848), 208-209 (2007).

2. Mironov, V., Reis, & Derby, B. Review: Bioprinting: A beginning. Tissue Eng. 12 (4), 631-634 (2006).

3. Pepper, M.E., Parzel, C.A., Burg, T., Boland, T., Burg, K.J.L., & Groff, R.E. Design and Implementation of a two-dimensional inkjet bioprinter. Proc. IEEE Eng. Med. Biol. Soc. 3-6, 6001-6005 (2009).

4. Campbell, P. & Weiss, L. Tissue engineering with the aid of inkjet printers. Expert Opin. Biol. Th. 7 (8), 1123-1127 (2007).

5. Boland, T., Xu, T., Damon, B., & Cui, X. Application of inkjet printing to tissue engineering. Biotechnol. J. 1 (9), 910-917 (2006).

6. Mironov, V., Prestwich, G., & Forgacs, G. Bioprinting Living Structures. J. Mater. Chem. 17 (20), 2054-2060 (2007).

7. Xu, T., Rohozinski, J., Zhao, W., Moorefield, E.C., Atala, A., & Yoo, J.J. Inkjet-mediated gene transfection into living cells combined with targeted delivery. Tissue Eng. Part A. 15 (1), 95-101 (2009).

8. Cui, X., Dean, D., Ruggeri, Z., & Boland, T. Cell Damage Evaluation of Thermal Inkjet Printed Chinese Hamster Ovary Cells. Biotechnol. Bioeng. 106 (6), 963-969 (2010).

9. Hamm, A., Krott, N., Breibach, I., Blindt, R., & Bosserhoff, A.K. Efficient transfection method for primary cells. Tissue Eng. 8 (2), 235-245 (2002).

10. Saunders, R. & Derby, B. Bioprinting, Inkjet Deposition. Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology. John Wiley & Sons, 15 April 2010.

11. Prabha, S., Zhou, W., Panyam, J., & Labhasetwar, V. Size-dependency of nanoparticle mediated gene transfection: studies with fractioned nanoparticles. Int. J. Pharm. 244 (1-2), 105-115 (2002).

12. Derby, B. Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures. J. Mater. Chem. 18 (47), 5717-5721 (2008).

13. Apodaca, G. Endocytic Traffic in Polarized Epithelial Cells: Role of Actin and microtubule Cytoskeleton. Traffic. 2 (3), 149-159 (2001).

14. Hotulainen, P., Llano, O., Smirnov, S., Tanhuanpää, K., Faix, G., Rivera, C., & Lappalainen, P. Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J. Cell Biol. 185 (2), 323-339 (2009)

15. Allen, P.G. Actin filament uncapping localizes to ruffling lamellaw and rocketing vesicles. Nat Cell Biol. 5 (11), 972-979 (2003)

16. Cooper, G.M., Miller, E.D., Desesare, G.E., Usas, A., Lensie, E.L., Bykowski, M.R., Huard, J., Weiss, L.E., Losee, J.E., & Campbell, P.G. Inkjet-based biopatterning of bone morphogenetic protein-2 to spatially control calvarial bone formation. Tissue Eng. Part A. 16 (5), 1749-1759 (2010).

17. Deitch, S., Kunkle, C., Cui, X., Boland, T., & Dean, D., Collagen matrix alignment using inkjet printer technology, Mater. Res. Soc. Symp. Proc. 1094 (DD), 7-16 (2008).

18. Ilkhanizadeh, S., Teixeira, A., & Hermanson, O. Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation. Biomaterials. 28 (27), 3936-3943 (2007).

19. Ringeisen, B.R., Orthon, C.M., Barron, J.A., Young, D., & Sparago, B.J. Jet-based methods to print living cells. Biotechnol. J. 1 (9), 930-948 (2006).

20. Cui, X. & Boland, T. Human microvasculature fabrication using thermal inkjet printing technology. Biomaterials. 30 (31), 6221-6227 (2009).

21. Langer, R. & Vacanti, J.P. Tissue engineering. Science. 260 (5110), 920-926 (1993).

22. Okamoto, T., Suzuki, T., & Yamamoto, N. Microarray fabrication with covalent attatchment of DNA using Bubble Jet technology. Nature Biotechnol. 18 (4), 438-441 (2000).

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