Cryopreservation of functional neutrophils by vitrification

摘要中性粒细胞是对感染性病原体的第一反应细胞，在宿主中起着至关重要的作用。 生死存亡。这些吞噬的粒细胞迅速迁移到感染部位，中和细菌和 真菌入侵者。在几种原发免疫缺陷(如慢性免疫缺陷)中观察到中性粒细胞功能缺陷 肉芽肿性疾病、白细胞粘连障碍、Chédiak-Higashi综合征等) 可观察到黏附、迁移、吞噬或氧化死亡，通常导致严重或复发的真菌和 细菌感染。尽管已经开发了许多方法来评估中性粒细胞的功能，但这些方法 不是常规临床护理的一部分，必须由专门的研究实验室进行。因为中性粒细胞 迅速恶化，理想情况下应该在收集后分析~2-4，严重限制了它们的功能 分析。这种快速的恶化阻止了样本在实验室或诊所之间的运输， 实验时间表，并要求每天从新鲜采集的外周血液中分离中性粒细胞 标本。这种短暂的体外保质期使中性粒细胞用于粒细胞的使用更加复杂。 中性粒细胞减少患者的输血。这个问题的一个潜在解决方案是中性粒细胞的发展。 冷冻保存方法，因为目前还没有办法在低温保存下维持功能性中性粒细胞。 因此，这项提议的总体目标是开发一种冷冻保存功能活跃的中性粒细胞的方法。 基于文献回顾和初步数据，我们假设玻璃化冷冻将导致更好的复苏 功能正常的中性粒细胞。玻璃化冷冻是一种无冰的冷冻保存方法，在这种方法中，细胞中含有高水平的 冷冻保护剂(CPA，例如二甲基亚砜)的浓度和通过玻璃的快速冷却 过渡。其结果是形成了无定形的玻璃态，而不是结晶冰。我们期待着 玻璃化冷冻的主要挑战将是中性粒细胞的渗透敏感性，这使负载足够高变得更加复杂。 玻璃化所需的CPA浓度。我们将克服这一挑战，使用以下工具自动执行该过程 注射器泵，以最大限度地减少CPA负荷期间中性粒细胞的体积变化。然后，我们将对两者进行优化 中性粒细胞玻璃化的生化和相变方面。 在目标1中，我们将对CPA的生化特性进行表征，并优化加载方法以确定优先顺序 毒性最低的玻璃化CPA鸡尾酒。作为概念的证明，我们将使用玻璃化的CPA负载的中性粒细胞 先前报道的微毛细血管。解冻后的中性粒细胞将在一系列复杂的功能中进行测试 化验。在目标2中，我们将通过调整CPA来优化玻璃化的相变参数 以玻璃化官能团为目标的样品的浓度、冷却/复温速率和热质量 中性粒细胞。作为这项工作的结果，我们预计将开发一个强大的玻璃化协议，使 功能中性粒细胞的低温储存。

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.