A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.
Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15
The process to convert a standard inkjet desktop printer for bioprinting is not particularly difficult. The most challenging step is determining how to bypass the paper feed mechanism, which is dependent on the brand and model of printer used. However, this is relatively simple when the paper feed sensor is mechanical as described here. For models with optical feed sensors, other techniques may need to be employed to trick the printer into thinking it is using paper; for instance, one can run a small piece of paper through the printer while it prints on the microscope slide. Bypassing the paper feed mechanism is likely to be the most difficult step in applying these procedures to different printer models.
When constructing a stage to hold the coverslips for printing, it is important to ensure proper alignment and height. The stage should allow for the coverslip to be placed in the middle of the printing area. In addition, it should place the coverslip at an adequate height to allow clearance for the print cartridge to pass over the slide without disrupting it. The exact height of the stage will depend on the printer model.
To ensure that printed cells do not get washed away, the slides were placed in an incubator immediately after printing for approximately 30 minutes to allow for cell attachment before additional cell growth media was added. Because the cells were printed in a solution that includes large amounts of PBS the cells did not dry out and remained viable. Cells were imaged using fluorescence microscopy to visualize the distribution of the tagged g-actin monomers inside the cell (Figures 1, 5-7). When printing with 3T3 fibroblasts, Figure 5 shows a representative result, with the actin causing much of the cell to fluoresce, but showcasing lines of increased brightness. The incorporation of fluorescently tagged g-actin into the cytoskeleton is useful for the study of cytoskeletal dynamics and cellular mechanics.12-15 A limitation of this technique is that it is only applicable for molecules and proteins that have diameters smaller than approximately 10 nm.
To ensure that cells were actually being processed by the converted printer, DAPI (1:5000 in PBS) was added to the bioink in place of half of the volume of pure PBS. The fluorescent stain DAPI binds to amino acid rich portions of DNA located in the nucleus. Therefore DAPI is a useful stain in fluorescently imaging nuclei of the printed cells. Figure 6 shows a representative image of a cell displaying both Alexa Fluor 488 (actin) and DAPI (nucleus) fluorescence with an overlay image of both. Figure 7 also illustrates how the cells will fluoresce once the g-actin monomers have been incorporated while non-biological material that has been deposited into the sample will not fluoresce because of the absence of g-actin monomers.
One important consideration for bioprinting is the aqueous media being used for the bioink. It was found that using standard cell growth media with serum yielded inconsistent results. This is most likely due to clogging of the print-head nozzles by serum proteins. The use of PBS increased the consistency of printed patterns and the number of cells deposited. One drawback to using PBS is that cells should not be left in the suspension for prolonged periods of time. However, fibroblasts in these examples were able to tolerate the bioink conditions for at least an hour with no change in cell viability. This is consistent with several prior findings, which report that cells printed via thermal inkjet mechanisms have been shown to have high viability rates.2-8
This modified printer setup can be used for applications other than cell printing. 16-22 Matrix proteins, such as collagen or fibronectin, can be easily printed onto substrates with this technique, which can be useful for cell patterning. For instance, collagen type I printed into line patterns will result in aligned collagen substrates that can be used for in vitro cell culture studies.17 In addition to matrix proteins, other molecules, including growth factors, can be reliably localized on substrates for cell studies and potential therapeutic applications.18
A major limitation of the design described in the above steps is that this printer is not capable of printing in more than one dimension. This limits the potential for use in patterned applications such as scaffold printing. To allow for 3D printing, a specialized stage needs to be used. The stage needs to have incremental height adjustments for layer-by-layer deposition of bioink.
Bioprinting has shown promise as an efficient and cost-effective method for tissue engineering, gene transfection, micropatterning and microarray fabrication.1-12, 16-22 The future applications of this type of device are numerous, including: creation of controlled and patterned cellular microenvironments, incorporation of macromolecules into cell cytoplasm, and deposition of cells onto scaffolds and structures that do not occur naturally or efficiently.
The authors have nothing to disclose.
The authors would like to acknowledge Dr. Thomas Boland for the idea of using the HP DeskJet printers for cell printing. Funding for this project from NSF RII-EPS 0903795 and NIH K25 HL0922280.
Name of the reagent | Company | Catalogue 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 μg | 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 |