Assessment of Bioreactor-Derived Human Platelet Quality, Storage Profile, Safety, and Function

 描述(申请人提供)：血小板生物生成正在开发一种微流控生物反应器，以生产临床规模的人血小板。血小板是血液的“创可贴”，负责血栓的形成和血管修复。低血小板计数是癌症治疗、移植和手术的重要后果，对于癌症治疗、移植和手术，血小板是预防因失控出血而死亡的关键一线治疗。每200-400毫升含有3x1011个血小板的单位目前仅来自人类志愿者捐赠者，必须储存在22oC或以上，以避免不可逆转的与温度相关的激活/聚集。在室温储存期间细菌生长的风险将保质期限制在5天内，其中2天用于细菌筛选，1天用于运输。因此，血液中心通常没有超过1.5天的可供输血的血小板库存，这些库存在紧急情况下会迅速耗尽[1，2]。为了解决这一主要的未得到满足的需求，我们开发了一种血小板生物反应器，它可以再现成人骨髓的关键特征(生理微环境)，以触发不断扩大的新血小板的产生。到目前为止，我们已经证明：(1)从人诱导的多能干细胞(IP-SCs，一种可冷冻保存多年的可补充祖细胞来源)生成功能性巨核细胞和血小板是可行的[3]；(2)我们的生物反应器将触发血小板形成，并提高从人IPSC来源的MK产生血小板的速度和程度，高于已建立的静态培养方法[4，5]。这份直接到第二阶段的SBIR提案概述了3个具体目标，以确定在人源化小鼠血小板减少模型中生物反应器衍生血小板(BdPLT)的质量、安全性和功能，并扩大血小板生产规模以生产人类输血装置：目标1.评估bdPLT的质量、储存情况和体外安全性。我们将在FDA批准的储存条件下评估血小板结构、生物标记物的表达、代谢活性和功能，并通过计数残余白细胞计数和评估病毒、败血症和畸胎瘤风险来确定其安全性。目的2.评价bdPLT在人源化小鼠体内的止血和血栓形成能力，以及对血小板清除率和循环时间的影响。我们将在一种新的小鼠模型中评估bdPLT的体内行为，该模型允许人而不是小鼠的血小板在动脉损伤部位聚集(非GLP试点研究)。胡曼捐献的血小板将作为生理对照。目标3.将生产规模扩大3,000倍，以生产一个人体输血单位(每300毫升3x1011个bdPLT)。我们将优化我们的生物反应器设计，以实现持续介质灌流和并行化，最大限度地提高巨核细胞的带状分布和捕获率，并使整个设备的剪应力暴露均衡。

 DESCRIPTION (provided by applicant): Platelet BioGenesis is developing a microfluidic bioreactor to produce human platelets at clinical scale. Platelets are the 'band-aids' of the bloodstream, responsible for clot formation and blood vessel repair. Low platelet count is a significant consequence of cancer treatment, transplant, and surgery, for which platelets are a critical first-line therapy to prevent mortality due to uncontrolled bleeding. Platelet units comprising 3x1011 platelets per 200-400 mL are at present derived exclusively from human volunteer donors, and must be stored at or above 22oC to avoid irreversible temperature-related activation/aggregation. Risk of bacterial growth during room temperature storage limits shelf life to 5 days, 2 of which are consumed by bacterial screening, and 1 by transport. As a result blood centers typically do not have more than a 1.5-day platelet inventory avail- able for transfusion, which is rapidly depleted in emergencies [1, 2]. To address this major unmet need we have developed a platelet bioreactor that reproduces key features of adult bone marrow (physiological microenvironment) to trigger new platelet production at increasing scale. To date we have shown that: (1) It is feasible to generate functional megakaryocytes and platelets from human induced pluripotent stem cells (iP-SCs, a replenishable source of progenitor cells which can be stored frozen for years)[3] and (2) our bioreactor will trigger platelet formation and improve the rate and extent of platelet production from human iPSC-derived MKs above established static culture approaches[4, 5]. This Direct-to-Phase II SBIR proposal outlines 3 specific aims to determine quality, safety, and function of bioreactor-derived platelets (bdPLTs) in humanized mouse models of thrombocytopenia, and to scale platelet production to yield a human transfusion unit: Aim 1. Assess bdPLT quality, storage profile, and safety in vitro. We will evaluate platelet structure, biomarker expression, metabolic activity, and function under FDA-approved storage conditions, and determine their safety by enumerating residual leukocyte counts, and assessing viral, septic, and teratoma risk. Aim 2. Assess the hemostatic and thrombogenic potential of bdPLT in humanized mice as well as platelet clearance and circulation time. We will evaluate the in vivo behavior of bdPLTs in a novel murine model that permits human but not mouse platelet accrual at sites of arterial injury (non-GLP pilot study). Hu- man donor platelets will serve as a physiological control. Aim 3. Scale production 3,000-fold to yield a human transfusion unit (3x1011 bdPLTs per 300 mL). We will optimize our bioreactor design for continuous media perfusion and parallelization, maximize megakaryocyte zonal distribution and trapping, and equalize shear stress exposure throughout the device.