The isolation and sorting of specific cell populations from their complex native environment plays a critical role in both chemical and life sciences, including regenerative medicine, tissue engineering, and cancer immunotherapy applications. In such workflows, the key objective is to obtain a highly pure and viable population of cells for downstream applications and analysis. Existing cell sorting methods including Fluorescent-Activated Cell Sorting (FACS) and Magnetic-Activated Cell Sorting (MACS) fail to address many of the challenges associated with cell sorting required for biomedical applications, such as scalability, speed, safety and simplicity. To address the limitations of exiting cell separation techniques, this NSF STTR Phase I project developed an innovative Affinity Chromatographic Cell Sorting (AFFICS) system without residual affinity tags (immunofluorescent or magnetic tags), showing a cost-effective, scalable and efficient multi-marker positive selection technique to separate two or more cell types directly from dense heterogeneous cell environments such as human peripheral blood (buffy coat).

The broader impact/commercial potential of this NSF STTR project will be the development of a cell sorting platform to allow scalable, high-purity isolation of cells for development and manufacturing of cell based therapeutics for cancer, such as CAR-T therapies. Purification of specific cell populations from complex materials such as blood is a critical component of cell therapy manufacturing. However, since most protocols for clinical-grade cell purification rely on binding of magnetic particles to cells, promising cell therapies face the risk of transplanting residual and potentially toxic magnetic particles to human subjects. The steps currently required to eliminate this risk add complexity, time and cost to cell therapy manufacturing, limiting the wide-scale usefulness of promising new cell therapies. This project developed a novel cell separation technology, free of magnetic particles, that can be readily scaled to levels required to enable clinical-scale manufacturing of highly pure and highly viable cell-based therapeutics. Our innovation represents an attractive and disruptive solution for isolating large quantities of healthy, uncontaminated cells. In addition, this cost-effective technology provided solutions in meeting specific clinical needs with a strong emphasis on scalable engineering design, which will exceed the current capabilities of widely utilized MACS or FACS.

Based on a proprietary phase-change hydrogel technology, this STTR Phase I project successfully designed and developed a microsphere droplet formation process for the conjugated hydrogel microparticles that can be used for target cell labeling via specific surface markers. During this project, we for the first time demonstrated this effective AFFICS technique, produced various AFFICS hydrogels, and manufactured AFFICS particles with various types and sizes, developing a complete AFFICS manufacturing process incorporating hydrogel preparation, particle formation and protein-hydrogel particle conjugation. 1) An affinity chromatographic system with functionalized dissolvable hydrogel microparticles was designed and developed for surface marker-based cell isolation from complex cell suspensions, leading to cell capture and target cell populations with >95% purity and viability. By this system, high cell numbers can be isolated rapidly via positive selection by labeling the target cells, resulting in the fastest and most efficient way to isolate a cell subset with high purity and yield. 2) Successful sequential cell separation was performed for efficient simultaneous separation of two and more cell subpopulations while maintaining cell phenotype and viability, indicating highly sensitive labeling and enrichment of target cells based on multiple surface markers. 3) The release mechanism was determined and target cells can then be detached and released from the hydrogel microparticles in a short time point of < 1 min, providing a fast, simple and gentle system for separation of affinity tag-free cell subpopulations. This STTR Phase I project demonstrated a disruptive alternative to current cell separation techniques and proposed the first method to perform multi-target cell sorting without residual affinity tags (immunofluorescent or magnetic tags) at different scales. The AFFICS system represents a significant new bioprocessing platform for cellular therapy development and manufacturing immunotherapies, built around releasable cell purification technology.

Last Modified: 07/24/2017
Modified by: Guokui Qin