Introduction of DMSO into cell suspensions using a two-stream microfluidic device.

Abstract

Cryoprotective agents (CPAs) are used during cryopreservation of cells in order to protect them from freezing injuries and increase the viability post thaw. Dimethyl sulphoxide (DMSO) is the most commonly used and in this investigation we aim to understand the feasibility of using a two stream microfluidic device to introduce a 10% (v/v) solution of DMSO into a cell suspension. DMSO will change the osmolarity of the solution and if preservation solutions containing DMSO are not added appropriately, cells may respond adversely. We have developed a microfluidic device that can be used to add cryopreservation solutions in a gradual fashion and avoid cell damage. The two-stream microfluidic channel is operated in two different flow configurations, one with a DMSO rich heavy donor stream on the top of the cell stream and the other with the vice-versa arrangement, for a range of Reynolds numbers (0.7 < Re < 10) The effects of different initial donor stream concentrations (1% - 15%) and flow rate fractions (0.23 – 0.77), on the transport of CPA molecules from one stream to another, is experimentally modeled for these configurations. For the former flow configuration, convective motion is produced (along the depth of channel) due to the influence of gravity resulting in high outlet cell stream concentrations even for very low residence times within the channel. This behavior is directly contrasting to the observations in the alternate configuration where-in mass transport is dominated by diffusion. Prior research on laminar flows in micro channels typically neglect effects of gravity and even if accounted, assumes the lack of other significant physical effects of viscosity or diffusion. The extent of mixing of two miscible fluids of different densities (DMSO and PBS) is characterized using Atwood number (At) as a modeling parameter. We have determined a range of At (1.5x10-3 < At < 3x10-3) where in transition occurs from gravity driven advection to molecular diffusion based mixing in the channel, for low residence times in the channel (Re > 2) and the highest effectiveness of mixing for a flow rate fraction of q f = 0.77. The ability to recover cells flowing through the device is critical so we monitored cell recovery and cell motion for specific operating conditions of Pe = 2000, 4000, an initial donor stream concentration of 15% (v/v) and a flow rate fraction q f = 0.5. It was determined from the study that the effects of gravity is very important and needs to be accounted in both the flow configurations in the channel. It has been found that it is most beneficial to operate the device at flow conditions when equilibrium DMSO concentration is achieved at the outlet of the device. This equilibrium is attained for a faster cell stream processing rate in the flow configuration with the heavier donor stream on the top, keeping all other operating conditions to be exactly the same. The recovery of the cells from the device has been found out to be appreciably high for the tested operating conditions mentioned above, if both the streams are collected at the outlet.