Non-Invasive Detachment of Vorticella from Calcium Alginate Membrane

Non-invasive detachment of Vorticella from a substrate is essential for fabricating a microsystem based on Vorticella. Here, we dry sodium alginate solution and obtain sodium alginate films with fewer uncrosslinked sol regions. Calcium ions cross-link the alginate polymers of the membrane and form a calcium alginate gel. We first culture V. convallaria on the gel membrane. By dissolving the membrane with a chelating agent, we detatch cells non-invasively from the substrate and obtain cells with intact stalks, which can then be placed in a microsystem without waiting for the stalks to grow. The cells can be patterned by lift-off of the calcium alginate membrane. The proposed method provides new processing capabilities and allows for the design and fabrication of a system based on an actuator of Vorticella. [DOI: 10.1380/ejssnt.2013.25]


I. INTRODUCTION
The contactile filament of Vorticella (i.e., the stalk) is a promising actuator for the power source of a microsystem.A previously reported method of assembling Vorticella in a microsystem is limited in that cells without intact stalks are initially introduced into the microsystem, after which the stalks are subsequently grown [1][2][3].The ability to place a cell with its stalk into a microsystem would offer a new design principle for systems based on an actuator of Vorticella.Detached stalks of Vorticella enables the elimination of waiting time until an individual Vorticella cell anchors its body to a substrate by an adhesive pad and grows its stalk.Typically it takes several hours.The chemical composition of the adhesive pad is not yet fully known [4], and a non-invasive method for detaching Vorticella has not been established, although mechanical detachment methods have been demonstrated [5,6].One such method involves shaking a flask containing Vorticella, which loosens the attached cells and prevents them from adhering to the surface [5].Another involves detaching Vorticella with a scraper [6].Although these mechanical methods successfully detach Vorticella from the substrate, the stalks are not preserved.To detatch cells while preserving the stalks, a less invasive method is preferable to mechanical methods.
Phase-transition gels are prospective substrates for non-invasive detachment [7].A gel on which cells are cultured is dissolved to detatch the cells from the surface.Chemical transition gels are especially promising because they are applicable under a broad range of conditions.Hara et al. developed a method for detaching a mono- * Corresponding author: nagai@me.tut.ac.jp layer of cultured cells from a calcium alginate gel on a porous membrane in a culture dish [8,9].In this method, a calcium alginate gel is formed by cross-linking the alginate polymer chains with calcium ions and is dissolved by removing the calcium ions.The porous membrane facilitates the formation of a thin layer of calcium alginate gel.However, the gel may contain uncrosslinked sol regions that reduce the mechanical strength.
To apply the method to Vorticella, we modify the method developed by Hara et al.A process of drying the alginate solution is added for reducing uncrosslinked sol regions and increasing the mechanical strength.We detatch cells non-invasively from the membrane and obtain cells with intact stalks by dissolving the calcium alginate membrane.

A. Detachment from calcium alginate membrane
Figure 1 shows a schematic of the non-invasive method for detaching Vorticella.Sodium alginate powder (visosity 80-120 cp at 10 g/L and 20 • C, Wako-pure chemical, 194-13321) was dissolved in purified water and adjusted to 10 g/L.A glass Petri dish with an inner diameter of 110 mm was washed with ethanol and pure water and dried at 120 • C on a hot plate.A 4-mL volume of the solution was poured on the dish.The substrate was coated with the solution.The solution was then dried at 120 • C on the hot plate to yield a thin sodium alginate membrane.A solution of CaCl 2 (100 mM) was added to the dish, which was left to stand for 1 min to cross-link the alginic acid with Ca 2+ .After cross-linking, the dish was washed with pure water three times.A suspension of Vorticella convallaria (V.convallaria) was poured onto the dish coated with the alginic substrate.The suspension was prepared according to the method of Vacchiano et al. [5].V. convallaria was cultured in a 1-L flask containing culture medium.An extract of 2.0 g/L of wheat grass powder (Pines International, Inc.) was filtered and used for the medium.The flask was shaken at 100 rpm for 24 h to suspend the cells.The suspension was poured and left to stand for 6-8 h at 20 • C to allow the cells to adhere to the surface.The substrate to which V. convallaria adhered was washed with spring water (Morinomizu, Coca-Cola Japan, pH 7.1, Na 2.02 mg, Ca 0.67 mg, K 0.16 mg, and Mg 0.27 mg in 100 mL) four times.Eight minutes of treatment with 10 mL of 20 mM EDTA solution chelated Ca 2+ and dissolved the membrane.

B. Detachment with scraper
V. convallaria was detached with a scraper to compare the invasiveness of detachment methods.A substrate was prepared without calcium alginate formation.A glass Petri dish was washed and dried at 120 • C. V. convallaria was cultured on a glass substrate at 20 • C. The cells were washed with spring water, and then detached from the surface with a cell scraper.

C. Observation method
Samples of V. convallaria prepared by the two methods were centrifuged at 3,000 g for 10 min and examined on a cover slip.An inverted microscope (IX71, Olympus) was set up for differential interference contrast observation to compare the rate of stalk preservation.Images were taken with a cooled CCD camera (Cascade II, Photometrics).The dissolution of the calcium alginate membrane was observed with the same setup.

III. RESULTS AND DISCUSSION
The alginate solution spread uniformly on a hydrophilic glass Petri dish.The solution height before evaporation was estimated as 0.4 mm by dividing the solution volume by the surface area of the dish.After drying the solution, we obtained a transparent and hard sodium alginate membrane.The estimated thickness of the sodium alginate was about 3 µm, as calculated from the solution concentration of 10 g/L and assuming a density of 1.6 g/cm 3 for sodium alginate.On immersing the dried sodium alginate membrane in a solution of calcium chloride, water dissolved the surface of the membrane and the alginic acid was cross-linked with calcium ions.A calcium alginate membrane without uncrosslinked sol regions was formed on the glass dish.As Young's modulus of calcium alginate is proportional to the concentration of alginic acid [10], our membrane is expected to have high mechanical strength.
V. convallaria adhered to the cross-linked membrane and grew stalks (Fig. 2).Most cells adhered to the surface of the membrane.The mean horizontal length of the stalk was 46 ± 18 µm (n = 17).The membrane did not cause any noticeable swelling during the experiment and remained adhered to the glass for five days.For the control experiment, the calcium alginate membrane was formed without the drying process.The membrane detached from the cover slip and floated after five days of immersion.Insufficiently cross-linked regions in the membrane were mechanically weak and easy to deform or detach from the surface.The diffusion of calcium ions through the calcium alginate is slow.Calcium ions insufficiently reached the inside of the alginate membrane and did not fully cross-link the boundary between the solution and the glass.
Subsequent injection of 20 mM EDTA solution started the dissolution of calcium alginate within 10 s.The dissolved area spread and a new surface gradually appeared.The process of dissolving the calcium alginate membrane with the EDTA solution is presented in Fig. 3.The exposed area increased from ∼10% at 0 s to ∼30% at 19.8 s.V. convallaria began to detach and move away from the surface by using its ciliary motion as the membrane dissolved.Dissolution of the gel was complete within 3 min.http://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/)Cells of V. convallaria with an intact stalk were distributed evenly in the dissolved solution.Centrifuged and concentrated cells were observed for comparing the rate of stalk preservation (Fig. 4).V. convallaria cells that detached from the calcium alginate membrane retained their stalks.The spontaneous contraction and extension of a stalk showed that its motility was preserved.In contrast, V. convallaria cells detatched by a scaper lost their stalks.A stalk with an adhesive pad adhered to a new cover slip, indicating that the pad was functional several hours after secretion.Table I summarizes the rate of stalk preservation.In the case of the scraper, the rate was 1.5% (5/343).In the case of the alginate membrane, the rate was 77.1% (330/428).The dissolution of a sacrificial layer is far less invasive than scraping the cells.

IV. CONCLUSIONS
Vorticella was detached from the surface of calcium alginate, with stalk intact, by dissolving the membrane with EDTA.The mechanical strength and crosslinked portion were improved beyond those of the previous method by adding a proceess of drying the alginate solution.The modified detatchment method may be applicable to other adherent cells.In this study, Ca 2+ is used as a gelling agent.Other divalent cations such as Sr 2+ and Ba 2+ are available for formation of alginate gels and can produce stronger alginate gels than Ca 2+ [11].The proposed method offers new processing capabilities and enables the design and fabrication of a system based on an actuator of Vorticella.Cells can be patterned by lift-off of the calcium alginate membrane.Selective removal or patterning of the membrane can be achieved by combining this method with soft lithography [12].The membrane contains high levels of Ca 2+ and may become a source of contamination, which has the potential to inhibit the precise motion control.Several rinses with chelating agents reduce the concentration of Ca 2+ to sufficiently low level.Cells with intact stalks can be placed in an arbitrary site by cell manipulation [13].

FIG. 2 :
FIG. 2: Micrograph of V. convallaria on an alginate membrane.Approximately 17 cells of V. convallaria adhere to the membrane.Inset shows enlarged view of V. convallaria.Scale bar: 50 µm.

FIG. 3 :
FIG. 3: Time series of micrographs showing dissolution of calcium alginate membrane.Approximately 21 cells of V. convallaria are observed.When the membrane dissolves, the cells of V. convallaria detach from the surface.

TABLE I :
Rate of stalk preservation in Vorticella prepared by two methods: scratching with a scraper and dissolving the calcium alginate membrane.