Hematopoietic Cell Processing and Preservation

Post Thaw Processing of Stem Cells:
Hematopoietic cell grafts (both stem cells and mature cells) have become the standard of care for a wide range of hematologic diseases. New studies suggest that both hematopoietic and non-hematopoietic cells found in the bone marrow may be clinically useful for the treatment of myocardial infarctions and a host of other medical conditions. Tens of thousands of bone marrow (BM) and peripheral blood stem cells (PBSCs) and cord blood (CB) units are routinely cryopreserved, thawed and infused each year. Traditional methods of stem cell cryopreservation use dimethylsulfoxide (DMSO) as a cryoprotective agent and cell grafts are routinely thawed at the patient bedside and infused directly. The clinical toxicity resulting from the infusion of DMSO into humans is well documented. Removal of DMSO using centrifugation or automated cell washers typically results in a 25-30% cell loss. Therein lies the conundrum: direct infusion of cryopreserved hematopoietic stem cells results in significant adverse reactions but washing cells to remove DMSO results in significant cell losses that, in turn, adversely affect transplant outcome.

This project develops a new paradigm for processing of post thaw cell suspensions: the use of microfluidic devices. Microfluidic devices can be used to wash suspension effectively while minimizing cell losses. Liquid flows through microscale devices are characterized by low velocity, laminar flow. In general, the cell motion can be controlled much more precisely and 'gently' than is possible in macroscopic flows such as within a centrifuge. Also, the laminar nature of microfluidic flow will permit diffusion-based extraction without the use of a membrane to separate fluid streams. Finally, the small cross-stream dimension in a microfluidic device allows for relatively rapid diffusion rates and therefore efficient extraction of a contaminant substance. These characteristics, combined with the ability to scale up the device, provide an important platform from which to evaluate processing of cells in concentrations and volumes used clinically.

Mesenchymal Stem Cell Preservation:
Bone marrow is a heterogeneous mixture of differentiated and stem cells of hematopoietic, mesenchymal and endothelial origin. The mesenchymal fraction of the cells has been studied for a variety of applications including cardiac repair and to support hematopoietic stem cell transplantation. Recent studies have shown that stromal cells present in the bone marrow can be differentiated into cardiac myoblasts, endothelial cells, hepatocytes and neural cells. These studies suggest that further clinical studies using the non-hematopoietic cells present in the bone marrow may be forthcoming.

In spite of the tremendous potential for these mesenchymal stem cells (MSCs), studies of their freezing behavior are limited. Until recently, bone marrow was the only source for stromal cells. Previous studies by several investigators suggested that MSCs were not present in cord blood. Recent studies show otherwise. The studies to date have not studies optimal conditions for MSC preservation (composition of cryopreservation media, cooling rate, etc.) and have not developed methods of MSC preservation appropriate for human therapeutic applications. The overall objective of this investigation is to develop methods of MSC preservation appropriate for human therapeutic applications. This work will rely heavily upon the use of microfluidic to introduce freezing solutions and the use of sugars to effectively preserve cells with minimal toxicity.