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: 10408182
• 负责人: Rebecca Sandlin
• 金额: $ 21万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408182
• 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; rational design; 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 中性粒细胞是感染性病原体的第一反应者，在宿主体内发挥着关键作用。 这些吞噬性粒细胞迅速迁移到感染部位，中和细菌并促进其存活。 在几种原发性免疫缺陷（例如慢性）中观察到中性粒细胞功能缺陷。 肉芽肿病、白细胞粘附障碍、Chédiak-Higashi 综合征等）不足的情况 观察到粘附、迁移、吞噬或氧化杀灭，通常导致严重或复发的真菌和真菌感染。 尽管已经开发了许多测定法来评估中性粒细胞功能，但这些测定法是不可行的。 不属于常规临床护理的一部分，必须由专门的研究实验室进行。 迅速恶化，理想情况下应在收集后进行约 2-4 次分析，严重限制其功能 这种快速恶化阻碍了实验室或诊所之间的样品运输。 实验时间表，需要每天从新鲜采集的外周血中分离中性粒细胞 这种较短的离体保质期使中性粒细胞用于粒细胞的用途进一步复杂化。 中性粒细胞减少症患者的输血这一问题的一个潜在解决方案是中性粒细胞的发育。 冷冻保存方法，因为目前还没有方法在低温储存下维持功能性中性粒细胞。 因此，该提案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。 根据文献综述和初步数据，我们发现玻璃化冷冻将提高恢复率 玻璃化是一种“无冰”冷冻保存方法，其中细胞负载高浓度。 冷冻保护剂（CPA，例如二甲亚砜）浓度并通过玻璃快速冷却 结果是形成非晶态玻璃态，而不是结晶冰。 玻璃化的主要挑战是中性粒细胞渗透敏感性，这使得加载足够高变得复杂 我们将使用自动化程序来克服这一挑战。 注射泵可最大限度地减少 CPA 加载过程中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化和相变方面。 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 最小毒性玻璃化 CPA 鸡尾酒作为概念证明，我们将使用 CPA 负载的中性粒细胞进行玻璃化。 然后将在一系列复杂的功能中测试先前报道的微毛细血管。 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 作为这项工作的结果，我们预计将开发出强大的玻璃化协议，从而实现 功能性中性粒细胞的低温储存。

项目成果

β-Dispersion of blood during sedimentation.

β-沉淀过程中血液的分散。

• 发表时间: 2021-01-29
• 影响因子: 4.6
• 作者: Sabuncu, Ahmet C;Muldur, Sinan;Cetin, Barbaros;Usta, O Berk;Aubry, Nadine
• 通讯作者: Aubry, Nadine

Cryogenic enrichment of Plasmodium falciparum gametocytes from spiked whole blood.

从加标全血中低温富集恶性疟原虫配子细胞。

• 发表时间: 2024-03
• 影响因子: 2.7
• 作者: Nesbitt, Jenny E;Jaskiewicz, Justyna J;Bean, Hailey;Toner, Mehmet;Tessier, Shannon N;Sandlin, Rebecca D
• 通讯作者: Sandlin, Rebecca D

Progressive hypoxia-on-a-chip: An in vitro oxygen gradient model for capturing the effects of hypoxia on primary hepatocytes in health and disease.

芯片上进行性缺氧：一种体外氧梯度模型，用于捕获缺氧对健康和疾病状态下原代肝细胞的影响。

• 发表时间: 2020
• 影响因子: 3.8
• 作者: Kang, Young Bok Abraham;Eo, Jinsu;Bulutoglu, Beyza;Yarmush, Martin L;Usta, O Berk
• 通讯作者: Usta, O Berk

A comparison of hepato-cellular in vitro platforms to study CYP3A4 induction.

研究 CYP3A4 诱导的肝细胞体外平台的比较。

• 发表时间: 2020
• 影响因子: 3.7
• 作者: Bulutoglu, Beyza;Rey;Mert, Safak;Tian, Lipeng;Jang, Yoon;Yarmush, Martin L;Usta, O Berk
• 通讯作者: Usta, O Berk

High-Voltage, Pulsed Electric Fields Eliminate Pseudomonas aeruginosa Stable Infection in a Mouse Burn Model.

高压脉冲电场消除小鼠烧伤模型中铜绿假单胞菌的稳定感染。

• 发表时间: 2021
• 作者: Wu, Mengjie;Rubin, Andrey Ethan;Dai, Tianhong;Schloss, Rene;Usta, Osman Berk;Golberg, Alexander;Yarmush, Martin
• 通讯作者: Yarmush, Martin