1.

Introduction

Mammalian cells are extensively used in biomedical research to understand the function of the body and to improve the treatment of diseases. During the development of cell lines that are genetically engineered, single cells must be isolated and then cloned from a population. Typically, cells are cultivated on an adherent surface from which specific individual cells selected under the microscope can be studied for criteria such as the cell surface expression of specific proteins detected.¹ A postprocess of removing these cells from the surface allows for analysis of their deoxyribonucleic acid or ribonucleic acid.^{2, 3} Mechanical and enzymatic techniques have been traditionally used for this. However, they are fraught by loss of viability and morphology, removal of surface markers, damage to the membrane, and alterations in physiology. In the last two decades, the technique based on laser-capture microdissection (LCM) followed by laser-pressure catapulting (LPC) has been touted to provide better adherent cell selection and collection.^{4, 5, 6, 7, 8} Nevertheless, in many instances, these processes still result in some degree of cellular injury. In LPC, for instance, initial specimen velocities of 50–60 m/s are typical⁷ and lead to strong shear forces developing in the cell. It has been found that damage from these shear stresses is much higher than that emanating from heat or UV exposure.⁷

A more recent alternative approach applies the use of SU-8 polymer micropallets mounted on a glass surface to separate the living cells.⁹ The top of these micropallets is coated with collagen or fibronectin to facilitate culture. The delivery of a pulsed laser beam then releases the micropallet from the glass surface into the liquid chamber. The advantages claimed for this approach include the following: (i) side-step of photo-UV damage from LCM, (ii) a thicker substrate to withstand the mechanical stresses and heating damage during pallet release, (iii) immersion in liquid media in a chamber throughout, and (iv) an ability to address the coordinate of a specific pallet of interest for release. After the micropallet is removed, the cells are collected in a microplate well placed above the micropallets in a one-to-one corresponding fashion.¹⁰ The advantages claimed with this scheme include reduction in contamination via immediate collection, lowered cell damage from minimal mechanical contact, and the ability to match the micropalletes to the coordinate of their release. Once collected inside the microplate well, the adherent cells are released from the micropallet via a second pulsed laser operation. Although this scheme, overall, promises to have degrees of cell damage lower than LPC, it is a more lengthy process and requires the fabrication of specialty structures.

In this work, we describe an approach that primarily offers reduction in contamination and can be conducted rather quickly in a single step without the fabrication of specialty structures. It is based on the concept of creating fluidics with microscope coverslips that we have developed.^{11, 12}

2.

Method

The method of specific collection of adherent cells in a capillary cell is described in Fig. 1. The system is constructed by trapping a droplet between two glass surfaces, the area of the chamber is defined by the area of the smaller glass piece and the height by the droplet volume.^{13, 14} The lower glass surface is a coverslip that has been treated to permit good cell adhesion, the upper surface is created using a smaller shaped coverslip that is sealed at all edges except one and a small aperture for droplet dispensation [see top view in Fig. 1]. The introduction of the droplet imposes a hydrostatic pressure on the solution in the chamber that produces a movement of the contact line at the free edge of the chamber [depicted as being from A to B in Fig. 1] The contact angle is denoted by θ. The ability of the meniscus to deform in such a manner is caused by a pinning effect at the top sharp edge of the coverslip C, which allows the contact angle there to have a large range. The ensuing effect is a flowrate Js _s through the liquid chamber.¹² This flow rate arises from evaporation that occurs at the free edge of the coverslip; hence, the flow pattern is unusual in that it leads to a single line location rather than a more typical swirling pattern.^{15, 16, 17}

Fig. 1

Schematic of method of specific collection of adherent cells using low laser release in a capillary cell. (a) Top view depicts release of droplet at one end of the liquid chamber that causes adherent cells at the bottom coverslip selectively dislodged by the microbeam laser to flow to the right in (b) the sideview. The cells collect at the proximity of the triple phase contact line in a hydrated state.

A microbeam laser delivered from underneath the bottom coverslip serves to selectively dislodge the adherent cells to enter into the flow. The travels of these cells terminate near the contact line within the liquid medium, ensuring that they remain continuously hydrated. If the cells to be dislodged are located close to the free edge of the liquid chamber, then the amount of cell travel and, thus, contamination is minimized. Essentially, this method enables us to capitalize on the ability to detach an extended choice of cells from their positions in proximity to the edge of the liquid chamber for maximal harvest while keeping contamination to a minimum. Such a scheme has not been reported before to the best of our knowledge. More importantly, it can be accomplished rather easily in the laboratory, wherein the flow is conveniently created by a droplet onto the liquid chamber made using a coverslip and glass slide rather than with a pump and expensive fluidic channels. The surface tension forces at the free edge keep the cells in place somewhat for a later collection process with a receptacle. It is conceivable that the flow generated orthogonal to cell dislodgement may introduce a shear peeling action on the cells and thus reduce the amount of force needed to dislodge them from the surface.

3.

Materials and Methods

4.

Results and Discussion

Figure 2 provides image sequences of a typical adherent cell before and 14 s after the laser beam was delivered to remove it via a direct delivery on the cell. The cell was found to be dislodged from the surface (in an out-of-focus sense), but cell death occurred in this case as evidenced by its color change to blue. Observation of the video footage enabled us to determine that the cell was brought to the edge of liquid chamber with a droplet induced flow. Figure 3 gives image sequences associated with the laser beam delivered 30 μm from the nearest cell at 63% maximum power, wherein no visible effect on the liquid was seen as well as the cell failed to dislodge. Having the laser beam delivered 35 μm from the nearest cell at 61% maximum power produced a bubble growth that cavitated in time to produce an indirect force needed that dislodged the cell. This is evident in the image sequences of Figs. 4, 4, 4. In this case, the cell remained viable.

Fig. 2

Images sequences (a) before and (b) after 14-s delivery of the laser at 60% power directly hitting the cell (spot indicated by arrow). The cell is seen to experience death as evidenced by its color change to blue.

Fig. 3

Images sequences (a) before and (b) after delivery of the laser at 63% power delivered 30 μm from the nearest cell (spot indicated by arrow).

Fig. 4

Images sequences (a) at the instant, (b) 3 s, and (c) 11 s after laser at 61% power delivered 35 μm from the nearest cell. A bubble is seen to instantaneously form that diminished in size over time. In this case, the cell was dislodged, but no reduction in cell viability was observed.

Figure 5 presents results depicting cell death probability of six cells against the laser power (i.e., six cells per data point) used by direct delivery of laser on the cell as well as inducing a bubble 60 μm away from the cell. Six cells were used per data point in order to keep the total experimentation time from being too extended. The latter approach is clearly able to sustain lower cell death probabilities in relation to an equal amount of laser power used. However, the ability of the cell to survive better is of little consequence if the cell cannot be dislodged sufficiently for the fluid flow to carry it to the collection region of the liquid chamber. Figure 5 gives the average movement of six cells against the laser power used by direct delivery of laser on the cell and inducing a bubble 60 μm away from the cell. In this case, the former approach is able to deliver greater average cell movement in relation to the laser power used. This result is largely expected due to the larger shear forces delivered to the cell⁷ by this means. Again, the ability for cell movement is inconsequential if the cell does not survive the dislodgement process. A comparison of Figs. 5 and 5 together is thus necessary in order to arrive at an apt conclusion. By considering the direct laser delivery approach alone, it would appear that there is no laser power range wherein both cell survival and cell movement are possible. The laser power ranges between 50 and 60% of the maximum with the bubble created 60 μm away, alternatively, offers such a possibility. We have further investigated the effect of creating the bubble at different distances away from the cell by keeping the laser power at 60% of its maximum.

Fig. 5

Plots of (a) comparative cell death probability of six cells against the laser power used by direct delivery of laser on the cell and inducing a bubble 60 μm away from the cell. The latter approach is able to sustain lower cell death probability in relation to the laser power used. Alternatively, (b) comprises plots of comparative average cell movement of six cells against the laser power used by direct delivery of laser on the cell and inducing a bubble 60 μm away from the cell. The former approach is able to deliver greater average cell movement on the basis of dislodgement in relation to the laser power used.

Figure 6 outlines the results obtained. The cell death probability distribution follows a distinct reducing trend with distance. The average cell movement distribution, however, shows that there is an optimal distance away (30–40 μm), where laser delivery produces the best effect for this. We do not fully understand the reason for this but postulate that it may be due to the cell being flexible rather than rigid and the force induced by the bubble having a vertical as well as a horizontal component. When the cell is close to the bubble, the resultant force may be large in magnitude but more vertically directed; resulting in the cell deforming but not being able to move much horizontally. If the cell is far from the bubble, then the resultant force is lower in magnitude despite being more horizontally oriented, again resulting in a smaller cell displacement. The lack of correlation between the two graphs appears to show that mechanical forces may not be the contributing cause for cell death from dislodgement via the bubble approach. This is in contrast to the findings with the direct laser delivery on cell approach.⁷ In fact, a plausible cause for the cell death probability distribution following a reducing trend with laser delivery distance may, in fact, be due to phototoxicity. It should be noted that the distances mentioned refer to the center of the laser beam and must not be taken to mean that the laser beam is confined to an infinitesimally small spot. The specified diameter of the laser beam for sharpest focus at its waist is 1 μm. However, this is used only for dissection purposes. We estimate that for catapulting purposes, the plane containing the cell corresponds to a beam diameter of 10 μm. If we assume a Gaussian profile, the percentage of energy delivered at the cell center is then around 10% of the laser spot center at this particular plane.

Fig. 6

Cell death probability and average cell movement (taken from six cells) determined by inducing a bubble at various distances away from the cell using a constant laser power of 60% of the maximum. The cell death probability distribution follows a distinct reducing trend with distance but not the average cell movement distribution.

Figure 7 presents an image of cells collected at the collection region of the fluidic chamber. It can be seen that the cells remain hydrated throughout. Closer inspection of these harvested cells using trypan blue indicates the presence of both viable and nonviable numbers. We believe that the nonviable cells are a consequence of the cell dislodgement process using the microlaser and not due to the fluid flow and collection process. To check for this, we have conducted tests of collecting nonadherent cells using the liquid chamber. With this, we observed no nonviable cells at the collection region. It is also important to note that the cells were located beyond the coverslip edge, which would enable physical removal if required.

Fig. 7

Image of cells collected at the collection region of the fluidic chamber. It can be seen that the cells remain hydrated throughout and that they can be kept viable. The arrow shows the direction of liquid flow in the chamber.

5.

Conclusion

We demonstrate that the approach of a microbeam laser delivered from underneath the bottom coverslip serves to selectively dislodge the adherent cells to enter into a flow. The transport of these cells terminate near the contact line within the liquid medium, ensuring that they remain continuously hydrated. By dislodging the cells close to the free edge of the liquid chamber, the amount of travel and thus contamination is minimized. The investigation of the modes of dislodgement via the metrics of cell death and movement show that the firing of the laser beam center a distance away from the cell to create a bubble that cavitates with time is more viable. The results obtained also show that phototoxicity appears to play a greater role than mechanical shear stresses in determining cell viability. The flow process was found to cause no death effects on the cells as they traveled to the coverslip edge for collection. More importantly, this technique can be readily performed in the laboratory without expensive fluidic channels and external flow creating sources (e.g., pumps).