Microfluidics for cryopreservation

Abstract

Cryopreservation has utility in clinical and scientific research but implementation is highly complex and includes labor-intensive cell-specific protocols for the addition/removal of cryoprotective agents and freeze-thaw cycles. Microfluidic platforms can revolutionize cryopreservation by providing new tools to manipulate and screen cells at micro/nano scales, which are presently difficult or impossible with conventional bulk approaches. This review describes applications of microfluidic tools in cell manipulation, cryoprotective agent exposure, programmed freezing/thawing, vitrification, and in situ assessment in cryopreservation, and discusses achievements and challenges, providing perspectives for future development.

Keywords: Cryopreservation; Cryoprotective agent loading/unloading; Freezing/thawing; Microfluidics; Vitrification.

Figure 1.. Basic theory for cryopreservation.

(A) Cell survival corresponds to cooling rate. (B) Cooling-rate dependent cell fates during freezing. Typical thermal profiles and cell volume responses for slow programmable freezing (C) and vitrification (D). (a) Temperature profiles; (b) Cell volume excursions. (A) adapted from (Mazur, 1984) and (He, 2011) with permission; (B) Reproduced from (Zhao, G. et al., 2014) with permission; (C) and (D) adapted from (Acker, 2008) and (Zhao et al., 2016) with permission.

Figure 2.. PDMS microfluidics for quantitative examination of cell osmotic responses.

Mechanical block- (A) and biological settling- (B) based microfluidic chips for cell perfusion. (a–d) are schematics of the microfluidic perfusion chamber, the microperfusion system, and the typical micrography of cells perfused in the microchannel of the two microfluidic chips, respectively. (A) Reproduced from (Chen et al., 2007; Chen et al., 2008) with permission and courtesy of Prof. Chen; (B) Reproduced from (Tseng et al., 2011) with permission and courtesy of Dr. Shu.

Figure 3.. Schematics of the representative oocyte-specific microfluidic perfusion devices.

(A) A two-layer PDMS device on glass, including a microfluidic network layer (lower layer) and a control layer (upper layer) to handle single oocyte for perfusion. Reproduced from (Heo et al., 2011; Lai et al., 2015) with permission. (B) A two-layer PDMS device, including a microfluidic channel layer that houses the oocytes and delivers CPA solutions, and a holding pipette layer for suction of the oocytes. Reproduced from (Lai et al., 2015a) with permission. (C) A first microfluidic device used for in vitro fertilization of pig oocytes. (D) A modified microfluidic device used for in vitro fertilization of mouse oocytes. (C) and (D): Reproduced from (Swain et al., 2013) with permission. (E) A novel microwell-structured microfluidic device that integrates single oocyte trapping, fertilization and subsequent embryo culture. Reproduced from (Han et al., 2010) with permission.

Figure 4.. Measurement of cell membrane transport properties using hydrodynamic switching.

(A) Cell trapping by two laminar flows with the same velocity. (B) Changing the extracellular solution by adjustment of the velocity ratio of the two flows. (C) Cell volume responses to the change in extracellular osmolality (switching time constant τ = 0.23 s). (D) Relative cell volume corresponds to time during hydrodynamic switching with two different time constants. Reproduced from (Lyu et al., 2014) with permission.

Figure 5.. Improved sandwich-structured microperfusion chamber for quantitative characterization of cell membrane permeability.

(A) Sectional view of microchamber. (B) Transient concentration distributions in the microchannel. (C) Concentration profile of point ‘P’ during osmotic shift. (D) Micrographs for typical cell volume responses. Reproduced from (Liu et al., 2015; Niu et al., 2015; Wang et al., 2016) with permission.

Figure 6.. A MEMS-based Coulter counter for cell counting and sizing using multiple electrodes.

(A) Three-dimensional schematic of the MEMS Coulter counter. (B) A complete fabricated and packaged Coulter counter with PDMS cover. (C) SEM image of the microfluidic channel for mixing and sensing. (D) SEM image of gold electroplated focusing and detection electrodes. (E) Simulation of the mixing of two dyes. (F) Simulation of electric field and its gradient distribution in the focusing electrode pair. (G) Simulated electric field in a vertical electrode pair. (H) Measurement of the impedance changes of yeast cells after mixing with dimethylsulfoxide (Me₂SO) at four different temperatures. Reproduced from (Wu, Y. et al., 2010; Wu et al., 2012) with permission.

Figure 7.. Microfluidics enabled CPA loading/unloading.

(A) Two-stream microfluidic device. (B) three-inlet T-junction microchannel. (C) membrane-based microfluidic device. (D) three-inlet Y-junction microchannel. (E) A sequential logarithmic microfluidic mixer for zebrafish sperm activation. Reproduced from (Bala Chandran et al., 2012; Lusianti and Higgins, 2014; Scherr et al., 2013; Song et al., 2009)(Scherr et al., 2015) with permission.

Figure 8.. Microfluidics for controllable freezing and thawing.

(A) On-chip direct freezing/thawing of mammalian cells. (B) Individual-cell-based microfluidic chip mounted onto a cryostage for programmed freezing/thawing of cells. (C) A microfabricated chip with an incubation microchamber, microfluidic channels, and microheaters for on-chip cell cryopreservation. Reproduced from (Afrimzon et al., 2010; Deutsch et al., 2010; Li et al., 2014; Li et al., 2010) with permission.

Figure 9.. Microfluidics facilitate microencapsulation of cells for low-CPA vitreous cryopreservation (Huang et al., 2015).

(A) A nonplanar microfluidic flow-focusing device for microencapsulation of cells. (B) Vitrification of VSP but not VSQ during cooling with PS, and devitrification of the vitrified VSP during warming with the PS. (C) Viability of mESCs and hADSCs with (W/) or without (W/O) alginate hydrogel encapsulation (Enap) before and after vitrification in VSP. **: p<0.01. VSP: cell culture medium containing 2M PROH and 1.3M trehalose. VSQ: cell culture medium containing 1.5M PROH and 0.5M trehalose. PS: conventional plastic straw. mESCs: mouse embryonic stem cells. hADSCs: human adipose–derived stem cells.