RESUMEN
There is a critical need for sorting complex materials, such as pancreatic islets of Langerhans, exocrine acinar tissues, and embryoid bodies. These materials are cell clusters, which have highly heterogeneous physical properties (such as size, shape, morphology, and deformability). Selecting such materials on the basis of specific properties can improve clinical outcomes and help advance biomedical research. In this work, we focused on sorting one such complex material, human stem cell-derived ß cell clusters (SC-ß cell clusters), by size. For this purpose, we developed a microfluidic device in which an image detection system was coupled to an actuation mechanism based on traveling surface acoustic waves (TSAWs). SC-ß cell clusters of varying size (â¼100-500 µm in diameter) were passed through the sorting device. Inside the device, the size of each cluster was estimated from their bright-field images. After size identification, larger clusters, relative to the cutoff size for separation, were selectively actuated using TSAW pulses. As a result of this selective actuation, smaller and larger clusters exited the device from different outlets. At the current sample dilutions, the experimental sorting efficiency ranged between 78% and 90% for a separation cutoff size of 250 µm, yielding sorting throughputs of up to 0.2 SC-ß cell clusters/s using our proof-of-concept design. The biocompatibility of this sorting technique was also established, as no difference in SC-ß cell cluster viability due to TSAW pulse usage was found. We conclude the proof-of-concept sorting work by discussing a few ways to optimize sorting of SC-ß cell clusters for potentially higher sorting efficiency and throughput. This sorting technique can potentially help in achieving a better distribution of islets for clinical islet transplantation (a potential cure for type 1 diabetes). Additionally, the use of this technique for sorting islets can help in characterizing islet biophysical properties by size and selecting suitable islets for improved islet cryopreservation.
RESUMEN
Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24-48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.
