Cryopreservation of functional neutrophils by vitrification

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ABSTRACT Neutrophils are among the first responders to infectious pathogens and play a critical role in host survival. These phagocytic granulocytes migrate rapidly towards sites of infection, neutralizing bacterial and fungal invaders. Neutrophil functional defects are observed in several primary immunodeficiencies (e.g. Chronic Granulomatous Disease, Leukocyte Adhesion Disorder, Chédiak-Higashi Syndrome, etc) where inadequate adhesion, migration, phagocytosis or oxidative killing is observed, often leading to severe or recurrent fungal and bacterial infections. Though many assays have been developed to assess neutrophil function, these assays are not part of routine clinical care and must be performed by specialized research laboratories. Since neutrophils deteriorate rapidly, they should ideally be analyzed ~2-4 after collection, severely limiting their functional analysis. This rapid deterioration prevents shipment of samples between laboratories or clinics, strains experimental timelines and requires daily isolation of neutrophils from freshly collected peripheral blood specimens. This short ex vivo shelf-life further complicates the use of neutrophils for the purpose of granulocyte transfusion among neutropenic patients. A potential solution to this issue is the development of neutrophil cryopreservation methods, as there is still no method to maintain functional neutrophils under cryogenic storage. As such, the overall goal of this proposal is to develop a method to cryopreserve functionally active neutrophils. Based on literature review and preliminary data, we hypothesize that vitrification will lead to improved recovery of functional neutrophils. Vitrification is an `ice-free' method of cryopreservation where cells are loaded with high concentrations of cryoprotective agents (CPAs, e.g. dimethyl sulfoxide) and rapidly cooled through the glass transition. The result is formation of an amorphous glassy state as opposed to crystalline ice. We anticipate the major challenge to vitrification will be neutrophil osmotic sensitivity, which complicates loading sufficiently high concentrations of CPAs necessary to vitrify. We will overcome this challenge automating the procedure using syringe pumps to minimize neutrophil volumetric changes during CPA loading. We will then optimize both biochemical and phase transition aspects of neutrophil vitrification. In Aim 1, we will characterize the biochemical properties of CPAs and optimize loading methods to prioritize minimum toxicity vitrification CPA cocktails. As proof of concept, we will vitrify the CPA-loaded neutrophils using previously reported microcapillaries. Thawed neutrophils will then be tested in a range of sophisticated functional assays. In Aim 2, we will optimize the phase-transition parameters of vitrification by tuning the CPA concentration, cooling/rewarming rates and thermal mass of the sample with a goal of vitrifying functional neutrophils. As a result of this work, we anticipate that a robust vitrification protocol will be developed, enabling the cryogenic storage of functional neutrophils.