Cell Separation System for Fully Automated Clinical Scale Separation of CD133+ Cells From Bone Marrow Aspirates.
Authors: Essl Mike , Stuth Juliane , Huppert Volker , Balshüsemann Dirk , Bauer Petra , Miltenyi Stefan 
Published: October 2019

Abstract
Abstract 1188
An increasing number of clinical trials are enrolling patients in studies designed to examine the safety and efficacy of autologous stem cells for cardiac repair. Recent reports suggest that patients receiving CD133+ bone marrow cells after myocardial infarction, or as a treatment for ischemic cardiomyopathy, may benefit from an increase in global left ventricular function. Today the clinical scale enrichment of CD133+ cells has to be performed as a complex procedure involving numerous manual handling steps as well as a semi-automated magnetic separation process.
We have developed a fully automated clinical scale process to purify CD133+ cells out of human bone marrow aspirates. The whole process was performed in a closed system, containing appropriate adaptors and tubing material, suitable for sterile connection of the bone marrow sample and required solutions, respectively.
For the whole separation process, the total processing time was reduced from about 4.5 h (previous process) to 2.5 h. In this context, erythrocyte reduction, generation of autologous plasma, labeling time as well as the conditions for immunomagnetic separation of the CD133+ cells and the automatic monitoring of the whole process by a newly developed camera were optimized.
To determine the reproducibility and stability of the process, CD133+ cells were separated from bone marrow aspirates with an initial volume of about 60 mL (n=10). The intitial frequency of CD133+ cells amounted to 0.34% (range: 0.11% to 0.66%) and the number of isolated CD133+ cells was 7.9×105 (range: 3.7×105 to 1.9×106). The yield was 47% (range: 23.9% to 50.9%) and the average viability of the separated CD133+ cells achieved 90% (range: 69.9% to 96.9%).
The separation process typically achieved a >3.0 log depletion of CD133 negative cells, i.e. 99.9% of CD133 negative cells were removed. The log depletion of different cell types were: 4.0 for CD3+ cells (i.e. 99.99% removal), 3.1 for CD19+ cells, 3.4 for CD56+ cells, 3.2 for CD14+ cells, and 3.7 for CD15+ cells (n=3, respectively).
After separation the CD133+ cells were automatically resuspended in 6 mL of clinical grade isotonic NaCl solution. For storage or transport of the cells, the NaCl solution could be automatically supplemented with 10% autologous plasma, generated out of the bone marrow sample during the separation process.
The described cell separation system provides a safe and easy way to purify CD133+ cells from bone marrow aspirates within 2.5 h without any intermediate manual steps. The cell preparation in a closed sterile system facilitates a fast and robust enrichment of CD133+ cells. After separation the CD133+ cells are available in small volume and can be formulated for further use e.g. according to requirements for use in regenerative medicine.

Disclosures:
Essl: Miltenyi Biotec GmbH: Employment. Stuth:Miltenyi Biotec GmbH: Employment. Huppert:Miltenyi Biotec GmbH: Employment. Balshüsemann:Miltenyi Biotec GmbH: Employment. Bauer:Miltenyi Biotec GmbH: Employment. Miltenyi:Miltenyi Biotec GmbH: Employment.