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Isolation of clonal cell populations is a crucial aspect of cell biology during engineering of specific cell strains with both genotypic and phenotypic variations. The use of cloning rings is the most established method, but requires anchoring chemicals or material that can often interfere with quantitative clonal-cell isolation and causes physical damage to the cells. Here we report a non-toxic, cell culture–compatible method that uses agarose for embedding the cloning rings during isolation of cell clones from monolayer cultures, with enhanced cell-viability and reproducibility during downstream applications. The method is simple and rapid, minimizing the chances for desiccation or cross-contamination during colony-lifts.
Stable transfection of mammalian cells with recombinant DNA constructs is a routine procedure in many cell biology laboratories. Chemical-, magnetic particle–, or virus-based methods are usually employed to introduce the exogenous DNA. These DNA constructs also harbor a cDNA cassette, which imparts specific resistance to a cytotoxic drug on the transfected cells to facilitate selection. Prolonged culture (in general, 4–8 weeks) of transfected cell-populations in the presence of drug selects for cell clones that are stably transfected with the recombinant DNA.
In case of adherent cells, stably transfected cell clones appear as macroscopic colonies (~1 mm in diameter) on the culture plates, which are then individually lifted and propagated for evaluation of their genotypic and phenotypic characters. These so-called colony-lifts are routinely conducted by two methods: (i) a ring-based method where a ceramic or plastic hollow cylinder is placed around an individual cell colony to physically isolate it from rest, where the bottom of ring is greased with silicone to create a watertight seal for subsequent manipulations to release the cell-clone from the cell culture plate (1–3), and (ii) a filter-disk–based method, where trypsin-soaked sterile filter circles (2–6 mm diameter) are placed on top of each colony to release and lift the clone from the culture plate for further manipulations (4). Each method has its advantages and drawbacks. With cloning rings, excess grease can cover the clonal cell-colony (as well as other colonies in the vicinity), essentially preventing further enzymatic manipulations to release cells from plate. In addition, due to the hydrophobic nature of grease, the cloning rings may not adhere to the wet culture-dish surface and give rise to leaks during subsequent handling steps; or, the rings tend to “drift” across the plate during subsequent handling steps, resulting in both loss and cross-contamination between cell clones. With filter disks, the key difficulties are visualizing the extent of enzymatic release of cell-clone from the culture dish, and subsequent enumeration of cells after transfer to a new culture vessel, since the cells firmly adhere to the opaque filter disk.
Due to these drawbacks, many researchers have resorted to simply placing a drop of trypsin on each cell clone, followed by aspiration with a micropipette. Despite its simplicity, the method poses a greater likelihood of cross-contamination between clones as physical barriers between individual clones do not exist.
Maintaining the advantages of the cloning ring method while addressing its drawbacks, we have made simple, yet effective changes to the procedure that result in efficient recovery of clonal cell isolates with excellent viability. In brief, we have utilized low melting-point (LMP) agarose for both anchoring cloning cylinders to the culture plate, and for creating a physical barrier against leakage during subsequent enzymatic manipulations. One percent (w/v) LMP agarose (Cat. no. A9414; Sigma-Aldrich Corp., St. Louis, MO, USA, or Cat. no. 15517–014; Invitrogen Corp., Carlsbad, CA, USA) was prepared in phosphate-buffered saline (PBS, pH 7.4) by heating the agarose-PBS suspension to 70°C with stirring. The solution was immediately sterile-filtered via passage through a 0.22-μm filter unit (Millipore-Millex GV 33 mm; Millipore Corp, Danvers, MA, USA) attached to a 10-mL luer-lock syringe, and the filtrate placed in a 37°C water bath until use. Cotton-plugged 5.75-in Pasteur pipettes (Cat. no. 13–678–8A; Fisher Scientific, Hanover Park, IL, USA) with 90° bent tips were formed by heating the pipettes 1.5 cm from the opening with a Bunsen burner (Cat. no. 03–917Q; Fisher Scientific), followed by physically bending the tip against the metal barrel of the burner. These were subsequently sterilized by autoclaving. Sterile tapered cloning cylinders (small, 4.7-mm internal diameter, 8 mm tall; polystyrene) were obtained from Fisher Scientific (Scienceware/Bel-Art Products, Pequannock, NJ). To transfer cell clones after the procedure, a 24-well plate was prepared with 1 mL of fresh complete Dulbecco’s modified Eagle medium (DMEM; containing 10% fetal bovine serum) medium per well.
To lift cell clones, spent medium was aspirated from the culture dishes followed by washing the dishes once in PBS (10 mL per 100-mm diameter dish). The plates were tilted for about 10 s and the residual PBS aspirated out. (is step is necessary, as residual PBS can dilute the agarose during subsequent steps.) Positions of the clones were marked (circled) on the underside of dishes with a permanent marker. The plates were placed in the tissue culture hood and cloning cylinders carefully placed around clones with a sterile curved 4.75-in jewelers’ forcep (Cat. no. 08–953-F; Fisher Scientific). LMP agarose (37°C) was then slowly dispensed dropwise around the outside of the cloning cylinders with a 1 mL pipet (approximately 0.5 mL for a 60 mm diameter tissue culture dish, and 1.0 mL for a 100 mm tissue culture dish, respectively). The agarose was allowed to set at room temperature (20–25°C) for 1–2 min, embedding the cloning cylinders in a thin layer of agarose. Forty microliters of 0.05% trypsin-EDTA (Cat. no. 25300; Invitrogen Corp.) were dispensed into the cylinders with a micropipette. The culture dish was closed and then placed in a 37°C incubator. Detachment and separation of cells within each cylinder were monitored periodically under a low-power microscope (Olympus IX51 inverted microscope, 4× objective, 10× ocular; Olympus America, Inc., Center Valley, PA, USA) (~5 min at 37°C). Then, media from an individual well in the prepared 24-well plate was dispensed into each cloning cylinder using the bent Pasteur pipettes and the solution pipetted in and out several times to detach and resuspend the cells in fresh medium, followed by aspiration and dispensing the contents into the corresponding well in the 24-well plate. Due to the rapidity of the process, and the fact that any agarose that leaks into cloning cylinder wells is both non-toxic and permeable to trypsin-EDTA treatment (and easily dissociates during pipetting), the aspirated cells rapidly establish themselves in the transferred plates and begin to proliferate without any lag period. Photographs of each step of the above procedure are provided in the Supplementary Material.
The above method was compared and contrasted with established protocols, such as anchoring cloning cylinders with grease (1), applying a thin layer of moisture-compatible superglue/dental cement (Cat no. 454; Loctite Corp., Rocky Hill, CT), or using the filter disk based clone-lift method (4). To prevent variations in cell number between colony-lifts during such a study, we used 100-mm tissue culture plates containing adherent U87-MG glioma cells (90% confluent). The plate was demarcated into quadrants and each treated with a different protocol to lift cells enclosed by the cloning cylinders (or covered by the cloning disks). In brief, high-vacuum grease (Dow Corning Corp., Midland, MI, USA) was sterilized according to established methods (1) and used to coat the contact surface of cloning cylinders. Alternatively, the contact surfaces of cloning cylinders were wetted with Loctite 454 cement. Cloning disks (Cat. no. Z374431, Sigma-Aldrich Corp.) were wetted in 0.05% trypsin-EDTA and placed over adherent cells according to established methods (4).
Visualization of trypsin-EDTA–treated areas within cloning cylinders from each method indicated that agarose-based embedding was the most successful technique (100% detachment of cells), followed by that with vacuum grease (where non-detached clumps of cells were evident, likely due to overlay by residual vacuum grease) and filter disks (Figure 1). Although the use of dental cement provided the strongest and fastest bonding of cloning rings to the culture dish surface under the moist conditions, a film of glue (likely due to its hydrophilic nature) spread rapidly over the cells via capillary action resulting in an opaque coating over the cells and forming a partial barrier to cell detachment with trypsin-EDTA (see Supplementary Material). Thus, we did not tabulate the studies with dental glue, as cell detachment was suboptimal.
To enumerate cell viability by each method, the detached cells (in the case of cloning rings) or the cell-containing filter disks were dispensed into individual wells of a 24-well plate, with each well containing 1 mL DMEM medium supplemented with 10% fetal bovine serum. The cells were allowed to establish in the wells for 48 h, followed by another 24 h in 1 mL of phenolred–free DMEM. Subsequently, cell viability was evaluated by 3-(4,5-dimethylthiazol-2--yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described (5). In brief, spent medium was aspirated from each well and fresh phenol-red–free DMEM supplemented with 0.5 mg/mL MTT added, followed by incubation at 37°C for 90 min. The spent medium was removed, and the MTT-formazan dissolved in 0.4 mL DMSO. Forty microliters of glycine buffer (5) were added to normalize pH across each well (in the case of filter disk study, the disks were kept in the wells throughout the procedure). One-hundred-microliter aliquots from each well were placed in a 96-well plate and the absorbance measured at 570 nm. The results, presented in Figure 2, indicate that the agarose-based method is not only quantitative, but also has less variability than the established vacuum grease–based method. In contrast, much fewer cells could be recovered from the filter disks.
In this report, we have demonstrated a much simpler and far less technically tedious method for anchoring cloning cylinders for clone isolation, which can be easily mastered by even a novice. Once a cylinder is positioned over a cell clone, the method only requires dispensing of small aliquots of melted agarose against the outer wall to both anchor and seal the unit. Since the cylinder contact surface is not precoated with grease or glue, the cylinders even can be repositioned around clones if necessary, prior to embedding with agarose. Furthermore, once a solution of agarose is prepared, it can be stored at 4°C, re-melted by boiling or heating at 70°C and reused as many times as necessary. Although we used a filter-based method to sterilize LMP agarose, steam sterilization is possible to prepare larger volumes of the same if necessary. With the vacuum grease method, it is not only necessary to steam sterilize the grease in glass vessels (Petri dishes), but also post-use cleanup requires the use of strong solvents such a 2-butanone (methyl-ethyl ketone; MEK) to clean the glassware, contaminated surfaces in the tissue culture hood, or other non-consumables. These steps are completely eliminated with the current method.
S.P.M is supported by the National Cancer Institute/National Institutes of Health (NCI/NIH; grant no. CA 116257), the Fund for Medical Research and Education (FMRE), Wayne State University, and a gift from the Marvin E. Klein, M.D., Charitable Trust. A.E.S. is supported by NCI/NIH (grant no. KO8 101954) and the Case Western Reserve University School of Medicine. We thank Tom Owok, B.F.A., for help with digital file conversions in preparing figures for the manuscript. This paper is subject to the NIH Public Access Policy.