Cryopreservation of functional neutrophils by vitrification

通过玻璃化冷冻保存功能性中性粒细胞

• 批准号: 10288287
• 负责人: Rebecca Sandlin
• 金额: $ 25.2万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-20 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288287
• 关键词: Address; Adhesions; Area; Bacterial Infections; Biochemical; Biological Assay; Blood; Blood specimen; Cells; Cellular Assay; Chemotaxis; Chronic Granulomatous Disease; Clinic; Collection; Complex; Consumption; Cryopreservation; Cryopreserved Cell; Cryoprotective Agents; Data; Defect; Deterioration; Development; Device Designs; Devices; Dimensions; Dimethyl Sulfoxide; Disease; Evaluation; Exhibits; Glass; Goals; Human; Ice; Immune; Infection; Invaded; Laboratories; Laboratory Research; Lead; Leukocytes; Life; Logistics; Methods; Mycoses; Neutropenia; Patients; Phagocytes; Phagocytosis; Phase Transition; Play; Predisposition; Procedures; Process; Property; Propylene Glycols; Protocols documentation; Public Health; Pump; Recovery of Function; Recurrence; Reporting; Research; Research Personnel; Respiratory Burst; Review Literature; Rewarming; Role; Sampling; Ships; Site; Solid; Specimen; Syringes; System; Testing; Therapeutic; Time; TimeLine; Toxic effect; Transfusion; Work; base; chediak-higashi syndrome; chemotherapy; clinical care; congenital immunodeficiency; cryogenics; crystallinity; design; experimental study; first responder; functional outcomes; granulocyte; improved; migration; mortality; neutrophil; pathogen; peripheral blood; preservation; prevent; process optimization; programs; prototype; side effect; success

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.

摘要中性粒细胞是对感染性病原体的第一反应者，在宿主中起着关键作用 生存这些吞噬性粒细胞迅速移向感染部位，中和细菌和 真菌入侵者在几种原发性免疫缺陷（例如慢性免疫缺陷）中观察到神经元功能缺陷。 肉芽肿病、白细胞粘附障碍、Chédiak-Higashi综合征等）， 观察到粘附、迁移、吞噬作用或氧化杀伤，通常导致严重或复发性真菌感染， 细菌感染虽然已经开发了许多测定来评估中性粒细胞功能，但是这些测定是不可行的。 不是常规临床护理的一部分，必须由专业研究实验室进行。由于中性粒细胞 它们会迅速恶化，理想情况下应在采集后约2-4天进行分析，严重限制其功能 分析.这种快速恶化阻止了实验室或诊所之间的样本运输， 实验时间线，需要每天从新鲜采集的外周血中分离中性粒细胞 标本这种短的离体保质期进一步使中性粒细胞用于粒细胞免疫目的的使用复杂化。 在贫血患者中输血。解决这个问题的一个潜在的办法是发展中性粒细胞 冷冻保存方法，因为仍然没有在冷冻保存下保持功能性中性粒细胞的方法。 因此，本提案的总体目标是开发一种冷冻保存功能活性中性粒细胞的方法。 基于文献回顾和初步数据，我们假设玻璃化冷冻将导致提高恢复 功能性中性粒细胞玻璃化是一种“无冰”冷冻保存方法，其中细胞负载高浓度的 浓度的低温保护剂（CPA，例如二甲基亚砜），并通过玻璃快速冷却 过渡其结果是形成一种无定形的玻璃态，而不是结晶冰。我们预计 玻璃化冷冻的主要挑战是中性粒细胞的渗透敏感性，这使得足够高的装载复杂化 玻璃化所需的CPA浓度。我们将克服这一挑战，使用 注射泵，以最大限度地减少CPA加载期间中性粒细胞体积的变化。然后我们将优化两者 中性粒细胞玻璃化的生物化学和相变方面。 在目标1中，我们将表征CPA的生化特性，并优化加载方法，以优先考虑 最低毒性玻璃化CPA鸡尾酒。作为概念验证，我们将使用以下方法玻璃化CPA负载的中性粒细胞： 以前报道的微毛细管。解冻的中性粒细胞将在一系列复杂的功能测试中进行测试。 分析。在目标2中，我们将通过调整CPA来优化玻璃化的相变参数 样品的浓度、冷却/复温速率和热质量，目标是玻璃化功能 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方案， 功能性中性粒细胞的低温保存