Platelet transfusions are
a highly effective for treating bleeding disorders in patients with low platelet counts and/or functionally defective platelets. Currently, around 10.3 million units of platelets are transfused in the United States annually.1
Since platelets can only be stored at room temperature for 5 days,2
this coupled with the demand for platelets can result in shortages of transfusable platelets.
Megakaryocytes (MKs), which are produced from hematopoietic stem cells (HSCs), are giant cells capable of producing around 104
To produce platelets, MKs undergo a cytoplasmic maturation that involves the formation of proplatelet processes that extend into marrow sinusoids, where shear forces generated from blood flow facilitate the release of the proplatelet processes.4,5
Understanding the molecular pathways and physical mechanisms in vivo
and in vitro
that regulate MK development and platelet release are of significant interest by the hematology field.6,7 Ex vivo
large-scale generation of platelets from MKs is also emerging as a possible solution to platelet shortages.8–10
However, MKs constitute only 0.03%–0.06% of all nucleated cells in the bone marrow and are difficult to isolate in large numbers11
; further, the generation and amplification of MKs from HSCs is time consuming and costly, and can take >10 days.12
Therefore, the ability to cryopreserve MKs after HSCs differentiation and before platelet production could provide crucial flexibility in producing platelets in vitro
on a large scale.
To successfully achieve cryopreservation, one has to prevent possible cryoinjury to the cells, which is most likely to occur at a temperature around −20°C during the freezing and thawing processes. This is a temperature where extracellular ice formation and the rising of solute concentration in the surroundings may induce intracellular ice formation (IIF)13,14
and excessive volume shrinkage15,16
when the cooling rate is too high and low, respectively.17
To develop optimized protocols for freezing, thawing, and removal of cryoprotectant agent (CPA) for different cell types, the cell membrane transport properties at subzero temperatures need to be determined. Specifically, these properties are the membrane permeability coefficient to water (Lp
), the membrane permeability coefficient to CPAs (Ps
), and the activation energies of all permeability coefficients (Ea
). Currently, the membrane transport properties of human MKs have not been determined yet.
In previous works, we proposed that cell membrane permeability could be determined by several techniques by measuring cell volume change under controlled anisotonic conditions, such as microdiffusion,18
and differential scanning calorimetry (DSC).22,23
However, there were difficulties associated with each of these techniques. The dialysis membrane method sometimes introduced complicated mass transfer and concentration gradient with the membrane,18
and the micropipette method can only hold a single or a few cells in one experiment and is limited to cell types with large size and shell.19,20
The microperfusion chamber provides an improved microfluidic tool21
; however, nonuniform flow and induced cell deformation due to its cell blocking geometry may also generate a heterogeneous exchange rate of mass between intra- and extraenvironment. Importantly, very few such data from above direct-observation methods at subzero temperatures are reported. Toner et al. have measured volumes of embryos between 0°C and −20°C at a low cooling rate (−2°C/min) with direct cryomicroscope observations,13
but in our study this method was found to provide inaccurate membrane transport information for MKs as a large cell type due to the presence and growth of extracellular ice, which may squeeze and damage the cell membrane integrity during freezing. The presence of the extracellular ice also makes the image processing for measuring the cell volume difficult. The DSC has been used to derive water permeability and activation energy of mammalian sperm by measuring latent heat change during freezing at subzero temperatures.22
However, the difference of the latent heat curve may not be distinguishable for cell types with high permeability that require a high cooling rate to induce IIF.23
Limited ability on measuring membrane permeability to CPAs and the cost of the DSC also make the popular use of this method difficult.
To overcome above-mentioned difficulties and achieve membrane transport properties measurement at subzero temperatures under a light microscope, we developed a simple cell-adhesive microperfusion system that could replace isotonic medium in the channel with desired solutions in less than a second. Besides, for a microscale problem, diffusion at the boundary between 2 consecutive solutions is negligible. Rapid heat transfer and uniform temperature distribution was ensured due to low heat capacity of solutions in the microfluidic channel. It is also observed that fluids in this microenvironment easily remain supercooled at temperatures below the freezing point.24
Hydrophobic surfaces of the polydimethylsiloxane (PDMS) microchannel further facilitated supercooling of solutions at subzero temperatures.25
Our microperfusion channel maintained the isotonic solution ice-free down to around −23°C. This new system was applied to determining the membrane transport properties of MKs.