Cyto-3D-print to attach mitotic cells
The Cyto-3D-print is an adapter that adds cytospin capability to a standard centrifuge. Like standard cytospinning, Cyto-3D-print increases the surface attachment of mitotic cells while giving a higher degree of adaptability to other slide chambers than available commercial devices. The use of Cyto-3D-print is cost effective, safe, and applicable to many slide designs. It is durable enough for repeated use and made of biodegradable materials for environment-friendly disposal.
KeywordsCytospin Mitosis 3D printing Centrifugation
Cytospin is a cytology method that is specifically designed to concentrate and attach cells for eventual immunochemistry evaluation (Brown et al. 1996; Campaner et al. 2005; Choi and Anderson 1979; Martinazzi 1971; Shanholtzer et al. 1982; Watson 1966). Cytofunnel devices and specialized cytocentrifuges are sometimes used to concentrate cell suspension onto slide surfaces (Nassar et al. 2003; Piaton et al. 2004). In our studies on mitotic cells, we encountered difficulty in cell attachment even with adherent HeLa cells. During mitosis, cells gain a defined geometry and sufficient space for a mitotic spindle with proper orientation and correct chromosome segregation (Brown et al. 1996; Fischer-Friedrich et al. 2014). Adherent cells round up, forming spheres, this is a sign of healthy dividing cells, but the change in attached surface area leads to frequent loss of cells during experiments (Fischer-Friedrich et al. 2014). Cytospinning is employed and increases the number of mitotic cells that remain on the slide surface. Centrifuge adapters are available for standard six-well plates but not for microslide chambers (25 × 75 mm) or 35-mm slide plates. So we developed Cyto-3D-print inserts made of polylactic acid (PLA) plastic, that fit into existing centrifuge buckets for cytospinning these smaller slide chambers. Our aim in this study is to prove that Cyto-3D-print provides a rapid, safe and cost effective way to attach mitotic cells and suspension cell lines to slide surfaces.
Materials and methods
Mammalian cell culture
HeLa cells (ATCC, Manassas, VA, CCL-2) were grown in DMEM (Corning, Corning, NY, USA, 10-017-CV) with 10 % Fetal Bovine Serum (GE Healthcare Life Sciences, HyClone Laboratorie, Logan, Utah, USA, SH30071.03), 1 % Penicillin and Streptomycin (Life Technologies, Grand Island NY, USA, 15240-062). These monolayer cells were sub-cultured every 3–4 days.
Thymidine and nocodazole were purchased from Millipore (Billerica, MA, USA; Calbiochem #6060 and #487928) For immunofluorescence preparation, we utilized poly-l-lysine (Sigma, St. Louis, MO, USA; P4832), 10 % formaldehyde (Polysciences, Warrington, PA, USA; 04018) and BSA (Sigma, A3059) in Lab-Tek microslide chambers (Thermo Scientific, USA, Nunc 155361).
MAb414 (Covance, Princeton, NJ, USA; MMS-120P) and anti-lamin B1 (Santa Cruz Biotechnology polyclonal gAb, Santa Cruz, CA, USA; 6216) antibodies were used for immunofluorescence with DAPI (Sigma, 471224) to visualize DNA.
Synchronizing HeLa cells
Cells were synchronized by double thymidine and nocodazole block (Glavy et al. 2007; Kaur et al. 2010). A 2 mM concentration of thymidine was added to the medium for 18 h in which HeLa cells were grown in culture. Introducing thymidine prohibits synthesis of DNA by a negative feedback mechanism. Cells in this population go through the cell cycle and will be halted at interphase. Cells were washed 3X with phosphate buffered saline pH 7.4 (PBS) and released into the medium without thymidine for the next 8 h. Cells were then treated again with 2 mM thymidine for 18 h and released into the medium with 0.02 μg/mL nocodazole for 12 h. Cells were collected by mitotic-shake off.
Evaluation of cell attachment using Cytospin with the Cyto-3D-print
Both poly-l-lysine and cytospinning
Both poly-l-lysine and cytospinning
The first and second columns were coated with poly-l-lysine a few hours before the experiment. Mitotic HeLa cells or suspension cells were first placed on the first and third columns. 10 μL of the cell suspension were placed in a hemocytometer to count how many cells were originally in each well. To ultimately test the function of the Cyto-3D-print, equal numbers of isolated mitotic cells were seeded in each well (approximately 5 × 104 HeLa cells). The cells were then spun in an Eppendorf 5810R Centrifuge for 3 min at 800 rpm (130 g) (Fig. 1). The microslide chamber fits inside the Cyto-3D-print, which fits inside the centrifuge bucket (Fig. 1). The centrifuge was balanced. After, mitotic HeLa cells or suspension cells were then placed in the second and fourth columns. After a 10-min incubation period at 37 °C, 5 % CO2, the cells were washed twice with 250 μL of PBS. Cells were counted and photographed under a light compound microscope. The percentage of cells that remained attached to the surface chamber over the original cell number for each well was calculated.
Mitotic cell suspension was seeded and cyto-spun on pre-treated microslide chambers as described above. Cells were then fixed with 2 % formaldehyde in PBS for 30 min. Chambers were washed three times for 7 min with PBS and permeabilized with 0.2 % Triton X-100 for 5 min. After two additional PBS washings, cells were blocked with 5 % BSA in PBS for 1 h. Cells were washed with PBS three times (7 min each time), and incubated with designated dilutions of primary antibodies in 2 % BSA for 1 h. After PBS washing, cells were incubated with Alexa Fluor 488 or Alexa Fluor 555-conjugated secondary antibodies for 40 min (anti-mouse A21422, anti-goat A21467, Invitrogen, Carlsbad, CA, USA), which were previously diluted 1:100 in 2 % BSA-PBS. Based on the need of specific purposes, the incubation was repeated with a different pair of primary and corresponding secondary antibody. The nuclei in the cells were stained with DAPI. The prepared samples were visualized using a Zeiss LSM 5 Pascal confocal microscope.
Results and discussion
In summary, our Cyto-3D-print offers an inexpensive flexible alternative to expensive metal plate adaptors with additional inserts. The use of microslide chambers has increased in recent years. Their reduced surface area saves on antibodies and reagents compared to larger six-well plates. We have developed Cyto-3D-print to bridge the gap between cytospinning and new slide technology. We hope this work stimulates more ideas to apply 3D printing to lab use.
We thank David Peacock, Sandra Furnbach and Stevens’ office of Innovation and Entrepreneurship for support of this project. We thank Arthur B. Ritter and members of the Warren G. Wells BioRobotics Laboratory. A Shulman Scholarship and Innovation and Entrepreneurship Summer Scholarship supported MRC. National Institute on Aging (NIA) grant 1R21AG047433-01 and Stevens’ Ignition Grant Initiative supported ZL, YZ and JSG. We thank Thomas Cattabiani for critical reading of the manuscript.
MRC, ZL, YZ and JSG designed the experiments. MRC, ZL, and YZ executed the experiments and finalized the figures. MGM printed the 3D insert. JSG wrote the initial draft and JSG, ZL, YZ and MRC revised the final manuscript.
Compliance with ethical standards
Conflict of interest
U.S. Provisional Patent Application No. 62/063,595.
Supplementary material 1 (MOV 166666 kb)
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