Cell patterning is an intuitive and powerful technology for individual studies. Our research aims to present a versatile, user-friendly, and inexpensive cell patterning method, which could be replicated in most biomedical laboratories. Compared to other methods, our protocol doesn't require specialized skills and equipment.
Also, it advances parallel to characters of ink-free, label-free, substrate-independent, and the ability for high-throughput patterning. The protocol requires only two necessities, magnets and the gadolinium-based contrast agents, which are both easy to get at low prices. The magnetic-based cell patterning method provides convenient solutions for the first step of a variety of experiments, including cell migration, biomimetic multicellular co-culture, organoid assembly, biomaterial studies, and more.
To begin, select flat rectangular magnets to assemble the magnet sets for strip patterns. Cut two millimeter-thick silicone plates into rectangles of the dimensions 2 by 8 by 30 millimeters. Then assemble the rectangular magnets and silicone plates layer by layer.
For dot array patterns, assemble the miniature cylindrical magnets, ensuring each magnet set consists of 36 cylindrical magnets arranged in a six by six grid. To prepare a cell culture device, employ a puncher to create a one by one centimeter rectangular hole in the two millimeter-thick silicone plate. Trim the silicone plate around the hole to craft a round mold with the hole at the center.
For sterilization, place the molds in the ultrasonic cleaner. Sterilize the molds with pure water for 10 minutes. Then replace the water with 75%ethanol for 10 minutes.
Dry the molds in a sterilizer box at 65 degrees Celsius for 60 minutes. Next, attach the sterilized silicone mold to a 25-millimeter round glass cell slide and gently press the mold so that the silicone adheres to the glass. Place the magnet sets on a 6-well plate and position the cell culture device on top of the magnet set.
To prepare 30-millimolar Gd-DTPA injection solution, mix 30 microliters of Gd-DTPA with 470 microliters of complete cell culture medium. To create the cell patterns, centrifuge the HUVEC suspension at 200G for five minutes, and re-suspend the pellet in Gd-DTPA-containing medium. After cell counting, adjust the cell density to approximately two times ten to the five cells per milliliter using Gd-DTPA-containing medium.
Gently mix the cell suspension and add 200 microliters to the cavity of the cell culture mold to create a brim-full liquid surface. Incubate the plate for three to six hours until the cells adhere to the substrate. After incubation, replace the Gd-DTPA-containing medium with a complete culture medium and remove the cell culture device from the magnet set.
To prepare a cell culture device for striped cell patterning, replace the thick silicone plates from the device with thin plates. After patterning the first type of cells as demonstrated previously, move the cell culture device one millimeter from the magnets below to the right. Apply the same patterning procedure to the second type of cells, and move the third cell culture device one millimeter from the magnets below to the right.
Pattern the third type of cells as demonstrated. After patterning all types of cells, wash the glass slides twice with 200 microliters of PBS. Finally, replace the PBS with a complete cell culture medium.
Viability tests conducted on the HUVECs treated with Gd-DTPA for 12 hours showed no significant decrease in cell viability, except for 50 millimolar Gd-DTPA, which resulted in a statistical difference, but preserved a living rate of approximately 90%In multi-type cell patterning, a tripartite cell pattern of side-by-side stripes and concentric dot arrays was generated.
