Breaking a Stereotype: Suspension Cells and Beads May Not Require Centrifugation

Centrifugation of cells and beads during buffer exchange, commonly known as washing, is a routine procedure in life science and diagnostics laboratories. The traditional protocol requires scientists to wash away the existing solution in a container before introducing a new one to minimize interference. For decades, it has been firmly believed that these suspension cells and beads, which are slightly denser than the solution they float in, require centrifugation to form a pellet—a physically pressed lump of cells and beads. Without this step, particles of cells and beads would be aspirated along with the existing solution, leaving fewer particles than initially present and potentially leading to biased results or, in the worst-case scenario, insufficient particles left for analysis.

Centrifugation serves two primary purposes in buffer exchange: it brings particles to the bottom of the container and forms a pellet. These phenomena help reduce particle loss when removing the existing solution by pipetting or flicking. However, no matter how carefully aspiration is performed, an average of 10% of particles are lost during each centrifugation/aspiration step (Jin, L., Wang, ZS., Cao, Y. et al. Establishment and optimization of a high-throughput mimic perfusion model in ambr® 15. Biotechnol Lett 43, 423–433 (2021)). Once the existing solution is removed, a new solution, typically a wash buffer, is added, followed by resuspension of the particles to mix them evenly. This sequence is repeated 2-3 times depending on the degree of dilution required and the acceptable level of cell loss.

While centrifugation is a useful technique in washing, its automation requires a high cost and expertise beyond a typical labs and scientists. Therefore, a workflow involving centrifugation remains manual and reliant on an operator’s skill. Manual operation presents numerous challenges for the industry, including issues with reproducibility, productivity, traceability, and a shortage of trained personnel. In an era where AI, machine learning, and automation are becoming increasingly prevalent, the persistence of manual workflows of cell and bead preparation for analysis, largely due to the complexities of automating centrifugation, is a significant drawback.

These challenges associated with centrifugation present an opportunity for technologists, engineers, and scientists who aim to bring automation to the highly manual workflows. The first step towards automation was to eliminate centrifugation from the workflow altogether or, if not entirely, reduce it to the bare minimum, thus minimizing the challenges associated with manual handling. Initially, the idea of eliminating centrifugation might seem daunting, if not impossible, as it appeared to defy the law of nature —cells and beads in suspension are easily disturbed by any force within a solution. It seemed unfeasible to replace the solution surrounding the particles without significant loss.

However, upon closer examination, the task of removing centrifugation did not appear to break any law of nature. The two primary roles of centrifugation could be replaced by simple principles of nature and instrumentation that most labs already possess. The sedimentation of particles could be achieved by gravity, and the formation of a pellet might not be necessary if the aspiration of the existing solution and the dispensing of a new solution could be performed without disturbing the cells. In theory, retaining particles during dispensing and aspiration is entirely feasible by controlling the flow velocity near the bottom of a container, keeping it close to zero as proven by our laminar wash technology. This means that while the fluid is displaced, it is not strong enough to lift cells from the surface, thus preventing them from being aspirated along with the fluid. While such control is impossible by hand, it is readily achievable by robotic pipette, which is now ubiquitous in labs.

After years of trial and error, Curiox’s team finally discovered how to achieve this delicate balance and control of fluid dynamics, demonstrating buffer exchange comparable to the manual centrifugation method. The team focused on two primary indicators of successful washing: dilution factor and cell retention, comparing these metrics with those of the centrifugation workflow. The washing process performed by a pre-programmed robotic pipette was shown to achieve dilution and retention levels comparable to or even higher than the centrifugation method. Curiox named this new technology C-Free™. When C-Free technology is integrated with a robotic pipette for conventional containers such as microtiter plates and tubes, it is branded as Pluto technology or the Curiox C-FREE™ Pluto System.

With its extremely precise control over buffer exchange, Pluto technology allows higher particle retention than centrifugation methods. During the washing process, particles are typically lost when the solution is aspirated. Pluto technology, however, employs a robotic pipette with proprietary control, minimizing any disruption to the particles. In contrast, conventional centrifugation methods involve simple flicking or aspiration.

The development of Pluto technology opens the door to automating the last remaining manual workflows in biomedical analysis—cell and bead sample preparation. Like many automated workflows in biomedical fields that utilize a simple liquid handling station with a pipette head and gripper, cell and bead analysis can now benefit from the same simple, inexpensive, and compact automation without the need to integrate a centrifuge and associated instruments. This also means that many existing liquid handling workstations, whether active or idle in labs, can integrate a simple pipette control code and execute workflows that traditionally required centrifugation.

We are at the threshold of embracing the same automation that has revolutionized many other areas, now extending to the analysis of cells and beads. It is exciting to anticipate how this automation will improve the reproducibility and reliability of analyses and free up countless hours for talented scientists, allowing them to focus more on science than on manual labor of endless pipetting and centrifugation. I eagerly await this transformation.