A Therapeutic Cell Distillery: Light-Controlled Fractionation of Stem Cells for Next-Generation Biomanufacturing Processes

PI：Menegatti，Stefano Proposal：1743404在开发强大的治疗细胞产品(TCP)制造工艺方面的主要挑战是确保生产的细胞具有所需的治疗效力。目前的许多分离和浓缩过程都是基于使用“亲和标记”的分选技术，“亲和标记”是一种吸附剂，通过锁定细胞的表面标记来结合细胞。在分拣完成后移除这些标签的过程通常与低得率和细胞损伤有关。本项目的研究目标是开发一种创新的基于亲和标签的纯化技术，通过控制光照强度和照射时间来控制不同细胞群的结合和顺序释放，将复杂的细胞混合物分离成具有不同生物活性和独特治疗价值的细胞组分。使用“亲和标签”确保回收的细胞是高度纯净的，而使用光作为生物温和的手段来控制细胞结合，确保回收的细胞具有生物活性。针对的细胞产品包括干细胞和祖细胞，它们可以产生所有其他血细胞，可以通过静脉输注的方式给骨髓或免疫系统受损或缺陷的患者注射。建议的技术是灵活的，可以很容易地适应研究和处理方案，从用于基础细胞生物学和组织工程研究，一直到生产临床相关数量的细胞。由北卡罗来纳州立大学化学工程系、生物医学工程系和生物制造培训和教育中心(BTEC)组成的跨学科团队将把研究与劳动力发展计划和与生物技术公司的接触结合起来。活动包括为学生以及工业和联邦(FDA和BARDA)员工提供关于蛋白质和细胞疗法cGMP处理的培训机会，将结果纳入现有BTEC关于生物分离的课程，以及创建针对生物技术专业人员的特别半学期和短期课程，介绍细胞产品的分级和纯化。该项目还计划与NIIMBL的利益相关者建立联系，NIIMBL是一家NIST研究所，旨在创新美国的生物制造技术，以开发联合研究项目，旨在将该项目的细胞蒸馏厂大规模转化为三聚氰胺生产。该项目建议开发一种基于光控制亲和力的技术，将干细胞群体分离成高纯度和生物活性的、具有独特治疗价值的组份。这个“细胞酿酒厂”利用微流控平台中集成的具有光控结合活性的标记特异性多肽配体，根据细胞表面标记的表达和密度来控制细胞的结合和释放。这些配体被设计成选择性地瞄准细胞标记，并在暴露于特定波长的可见光/近红外光时在开启和关闭结合模式之间进行可逆切换。器件上的配基密度和光剂量的优化确保了高纯度和活细胞组分的回收。该项目将通过(1)从多能造血祖细胞(HPC)中分离出造血干细胞(HSCs)，然后(2)将HSCs分离成长期和短期HSC，以及(3)HPC分离成多能和血统承诺的HPC来展示这项技术。为此，该项目将开发VCAM-1、CD38和Flt-3标记物的光响应性配体(目标1)；将细胞结合与配体密度和光照度相关联(目标2)；构建和表征一个微流体“细胞蒸馏厂”，用于分离从商品全骨髓抽提物或脐带血单个核细胞组分获得的造血干细胞和造血干细胞(目标3)。这项研究的结果有可能改变治疗细胞产品(TCP)的操作和分离的科学和技术，在再生医学和治疗具有挑战性的恶性肿瘤方面开辟新的途径。

PI: Menegatti, StefanoProposal: 1743404A major challenge in developing robust manufacturing processes for therapeutic cell products (TCPs) is being assured that the cells produced have the desired therapeutic potency. Many of the current separation and enrichment processes are based on sorting technologies using "affinity tags," which are adsorbents that bind cells specifically by targeting their surface markers. The process of removing these tags after sorting is completed is often associated with low yield and cell damage. The research goal of this project is to develop an innovative "affinity tag" based purification technology for separating complex mixtures of cells into cell fractions with different bioactivity and unique therapeutic value using light at controlled intensity and exposure time to control the binding and sequential release of different cell populations. The use of the "affinity tags" ensures that the recovered cells are highly pure, while the use of light as a biologically gentle mean to control cell binding ensures that the recovered cells are bioactive. The cell products targeted include stem and progenitor blood cells, which give rise to all the other blood cells, and can be administered by intravenous infusion into patients whose bone marrow or immune system is damaged or defective. The proposed technology is flexible, and can be easily adapted towards both research and processing scenarios, from use in basic cell biology and tissue engineering research, all the way to the production of clinically relevant amounts of cells. The interdisciplinary team encompassing the departments of Chemical Engineering, Biomedical Engineering, and the Biomanufacturing Training and Education Center (BTEC) at North Carolina State University will integrate research with a plan for workforce development and engagement with biotech companies. Activities include BTEC training opportunities for students and industrial and federal (FDA and BARDA) employees on cGMP processing of protein and cell therapeutics, incorporation of outcomes into existing BTEC courses on bioseparations, and creation of ad hoc half-semester and short courses targeted to biotech professionals on fractionation and purification of cell-based products. The project also plans to connect with stakeholders of NIIMBL, a NIST institute designed to innovate biomanufacturing in the United States, to develop joint research projects aimed at the large-scale translation of the project's cell distillery for TCP production. The project proposes to develop a technology based on light-controlled affinity to separate stem cell populations into highly pure and bioactive fractions with distinct therapeutic value. This "cell distillery" employs marker-specific peptide ligands with light-controlled binding activity integrated in a microfluidic platform, to control cell binding and release based on the expression and density of cell surface markers. The ligands are designed to target cell markers selectively, and switch reversibly between ON and OFF binding modes upon exposure to visible/near IR light at specific wavelengths. The optimization of ligand density on the devices and light dosage ensures the recovery of highly pure and viable cell fractions. The project will demonstrate the technology by (1) separating hematopoietic stem cells (HSCs) from multipotent hematopoietic progenitor cells (HPCs), followed by the fractionation of (2) HSCs into long-term and short-term HSCs, and (3) HPCs into multipotent and lineage-committed HPCs. To this end, the project will develop light-responsive ligands for VCAM-1, CD38, and Flt-3 markers (Objective 1); correlate cell binding to ligand density and light irradiance (Objective 2); construct and characterize a microfluidic "cell distillery" for fractionating HSCs and HPCs obtained from commercial whole bone marrow aspirate or cord blood mononuclear cell fractions (Objective 3). The outcomes of this research have the potential to transform the science and technology of manipulation and isolation of therapeutic cell products (TCPs), opening new avenues in regenerative medicine and treatment of challenging malignancies.