Using Optical Tweezers for the Generation of Hybrid Spheroids

Optical tweezers allows the physical trapping and manipulating of objects of micron and nanometer scale non-invasively using only focused laser beam. This tool is now successfully applied to various biological structures, including cleaving cells. Our lab aims to investigate the direct cell-cell interaction at the single cell level and further treat this lateral arrangement and structuring using the promising techniques such as optical tweezers.

First, the current experimental challenge is the lack of the protocol for construction of hybrid spheroids using optical tweezers for the whole process. Second is the stabilization on new reform in hybrid spheroids and retrieving them for subsequent experimental processing. In this protocol, we demonstrate step by step the construction of a hybrid lymphoma spheroid with the use of optical tweezers.

The main advantage of this technique is the opportunity to study the direct interactions between lymphoma cells and the three-dimensional microenvironment in realtime, which is more biologically relevant than traditional cell culture. Importantly, the technique presented here can be easily adapted to leukemia cell lines and other hematopathological malignancies. We plan to use our methods to study the minute changes in single cell adhesion induced by cancer drugs.

We intend to use a patient's tumor lineage for this purpose. Begin by preparing the micro-mold. Rinse micro-mold 3D petri dish in deionized water, and place it under a UV lamb for 30 minutes to perform sterilization.

First, prepare 2%agarose solution by dissolving the agarose powder in 0.9%sodium chloride solution using a microwave. Avoid creating bubbles while mixing or pipetting agarose. Cool agarose to approximately 70 degrees Celsius.

Next, prepare hydrogels by filling previously sterilized micro-molds with 500 microliters of agarose. Remove any bubbles that may be trapped in the small features of a micro-mold by pipetting. After 15 minutes, when the agarose has solidified, carefully remove the gels from micro-molds to a sterile six-well plate.

Before proceeding to the next step, check under a microscope, confirm the gel have been prepared correctly. Add phosphate buffer saline completely covering the micro-molds before placing the plate under a UV lamp for 30 minutes to perform sterilization. Before using the agarose-based devices to produce spheroids, equilibrate gel by completely covering them in RPMI medium and incubate for a minimum of 15 minutes.

Culture the HS-5 mesenchymal stromal cell line in RPMI complete culture medium in standard conditions, ensuring a changing of medium every 48 hours. When the cells reach 90%of confluence, remove the culture media. Wash cells with phosphate buffer saline and detach them from the bottom of culture flask using trypsin.

Stop trypsin by adding two milliliters of culture medium, then transfer them into a 15 milliliter conical tube. Centrifuge cells at 300 times gravity for seven minutes. Remove supernatant and resuspend cells in one milliliter of fresh culture medium.

Count cells in automatic counter using tryp and blue solution. Dilute them to 200, 000 cells per milliliter. Remove culture medium from agarose-based devices.

Carefully seed 190 microliters of cell suspension into the cell seeding chamber of agarose micro-mold. Incubate cells for 30 minutes at 37 degrees Celsius until the cells settle at the bottom of the gel. Slowly add four milliliters of additional medium to the outside of the gels, covering them entirely.

Remember to not add the medium directly onto the gel, but into the culture dish, avoiding damaging the freshly formed spheroids. Incubate for 72 hours at 37 degrees Celsius until uniforms spheroids are formed. Hematological cell line grows free-floating in the culture media in flasks designed for suspension culture.

To maintain optimal cell properties, it is recommended to start preparing the sample no earlier than 30 minutes before the procedure of acid billing distributes spheroids using the optical tweezers. Keep cells in RPMI 1640 medium under standard culture conditions. Maintain cultures between 300 and 500, 000 cells per milliliter by passing every two to three days.

For manipulation in optical tweezers, choose cells in the logarithmic phase. Collect the cells and transfer all the suspension to a 15 milliliter tube. Centrifuge the cells at 300 times gravity for seven minutes.

Remove the supernatant and count cells as previously described. Dilute the suspension to reach a final cell density of 10, 000 cells per milliliter. Keep cells at 37 degrees Celsius until optical manipulation.

The protocol we present here was developed specifically for the custom-built optical tweezers. However, commercial systems are becoming increasingly accessible. Regardless of what type of equipment is available in your laboratory, the basic steps of system preparation and general principles of working with cells remain the same.

Turn on the microscope light source. Wear laser-safe protective glasses. Turn on the laser and set the power at 100 megawatts.

Before proceeding, wait 10 minutes, allowing the laser beam to stabilize. In the meantime, start up the computer software. Once the system is ready, add 30 microliters of water to the center of the bottom objective.

Place a 35 millimeter microscopy dish with a glass bottom on the microscope stage. Stabilize the dish with the holders. Raise the objective using the micrometric microscope screw until the bead of water touches the glass bottom of the microcopy dish.

Using the micrometer screw, gently adjust the stage until there's a sharply focused laser beam on the screen. In the software window, you will see an image of the microscope sample. Use the markers visible on the screen to move the optical trap in the desired location.

Add 100 microliters of previously-prepared lymphoma cell suspension. Let them sink to the bottom of the dish. This will take approximately five minutes.

Using the movements of the microscope stage, find a floating lymphoma cell. Allow the optical trap to hit the sample by clicking on the trap cursing. Try to move the cell to the desired location.

A cell to be useful for manipulation should be able to move easily in any given direction. Remove a single stromal spheroid from the agarose mold with a pipette tip and place it on an uncoated glass bottom dish. Add two milliliters of culture medium and incubate for 10 minutes at 37 degrees Celsius.

During this time, the spheroid will gently stick to the glass, which prevents movement during manipulation. Place the dish containing the spheroid on the microscope stage. Add 1000 lymphoma cells to 100 microliters of culture medium.

Let them sink to the bottom of the dish, taking approximately five minutes. Select a single lymphoma cell and trap it using the laser beam. Move the optical trap to deliver the cell into contact with the stromal spheroid surface.

Start by attaching the lymphoma cell to the stromal spheroid for 10 seconds. Next, check if the cell is permanently attached with three repeated attempts of detaching the lymphoma cell using the optical tweezers. If the cellular contact is broken, attach the lymphoma cell with a spheroid surface once more using the optical trap, this time, for a longer period of time, approximately 20 seconds.

Repeat this process with subsequent lymphoma cells until the entire surface of the stromal spheroid is covered by lymphoma cells. By changing the imaging plane using micrometric microscope screw, one can change the position of the optical trap, which enables the movement of trapped objects more profoundly into the sample. For a better representation of the spontaneously-formed spheroid, cover the core of stromal cells with two to three layers of lymphoma cells.

Gently remove the culture medium and directly wash the spheroid with 100 microliters of phosphate buffer solute. Incubate the hybrid spheroids with 100 microliters of PBS containing one micromole of calcium ion and two micromoles of Ethidium Homodimer-1 for 20 minutes at 37 degrees Celsius. Using fluorescence microscopy, observe the spheroids and capture the images.

Here, we successfully reconstructed a human hybrid spheroid consisting of mesenchymal stromal cells and lymphoma cells with the use of optical tweezers. First, the mesenchymal stromal cells spheroids were prepared in an agarose micro-well device, which results in relatively uniform spheroids after 72 hours of incubation. The viability of spheroids was higher than 98%Next, mesenchymal spheroids were used as a core of newly constructed hybrids spheroid with the use of optical tweezers.

Lymphoma cells were individually trapped by optical traps and attached to the surface of the stromal spheroid. The procedure was repeated until the entire surface of the stromal spheroid was covered with lymphoma cells. We established that the RI-1 cells attached to the stromal spheroid in 11.67 seconds with a 3.73 second deviation.

During the process, 65 cells were attached, taking approximately one hour and 12 minutes. The live-dead staining of newly engineered spheroids showed a high viability of cells used for manipulation, at 93%This paper proposes a non-invasive protocol for the formation of lymphoma stromal cell spheroids using optical tweezers. This method not only construct hybrid spheroids de novo, but also allows for precise control over the number of attached cells and the timing of adhesion formation, providing valuable insight for tissue engineering.

Further, this optical tweezers approach facilitates the study of early-stage of high risk spheroid formation, which cannot be achieved using standard bulk techniques. Most importantly, our protocol can be easily applied to other cell types, which will enable this optical tweezers-based approach to become increasingly employed in the field of 3D cell culture.