GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration

Abstract

Background: CD133⁺ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133⁺ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics.

Methods: CD133⁺ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133⁺ cells was evaluated and compared to manually isolated CD133⁺ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction.

Results: We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 10⁶ viable CD133⁺ cells with a mean log₁₀ depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo.

Conclusions: Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133⁺ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

Keywords: Adult hematopoietic stem cells; Advanced therapy medicinal product (ATMP); CD133+ cells; Cardiovascular regeneration; Clinical translation; Good Manufacturing Practice (GMP); Prodigy; Stem cell transplantation.

Fig. 1

Characterization of automatically generated fractions. Cell product (CP), non-target cell bag (NTCB), waste bag (WB) and bone marrow (BM) were analyzed in respect of their CD45⁺ cells/μl (a), CD133⁺CD34⁺ cells/μl (b) and viability of CD45⁺ cells (c) using flow cytometry measurement in accordance with ISHAGE guidelines. Additionally, total numbers of thrombocytes (d) and erythrocytes (e) were assessed using a Sysmex device. All data are presented as mean ± SEM. CP, BM (n = 6); NTCB, WB (n = 3). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. CP

Fig. 2

Stability of the automatically generated cell product (CP) over a storage time of 2.5 h and 24 h post manufacturing process. For the first 2.5 h, CP was stored under room temperature (RT) conditions. After 2.5 h CP was stored at 2–8 °C. Samples of CP were taken at the respective storage time and CD45⁺ cells/μl (a), CD133⁺CD34⁺ cells/μl (b) and viability of CD45⁺ cells (c) were measured by flow cytometry in accordance with ISHAGE guidelines. All data are presented as a mean ± SEM. n = 3. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. CP

Fig. 3

Hematopoietic and endothelial differentiation capacity of manually and automatically isolated CD133⁺ stem cells. Hematopoietic colony-forming unit (CFU-H) and colony-forming unit endothelial cells (CFU-EC) assays were performed by seeding 1 × 10³ cells directly after the isolation procedure. Hematopoietic CFUs (CFU-E, BFU-E, CFU-GEMM, CFU-GM) (a) and endothelial CFUs (adherent, non-adherent) (b) were counted after 14 days of incubation. All numbers of counted CFUs are presented in the table (c). All data are presented as a mean ± SEM. n = 3. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Fig. 4

Comparison of the cardiac regeneration potential of manually and automatically isolated CD133⁺ stem cells. 1 × 10⁵ CD133⁺ stem cells were intramyocardially transplanted into SCID bg mice after myocardial infarction (MI). Ejection fraction (EF) (a), velocity of pressure rise (dPdt_max) (b), end-diastolic volume (EDV) (c), and end-systolic volume (ESV) (d) were assessed by pressure-volume (PV) loop measurements 3 weeks after cell transplantation. For control untreated infarction (MIC) and SHAM operation were used. All parameters were measured under baseline and under stress conditions mediated by intravenous dobutamine administration (10 μg/kg/min). Data are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. MIC; ns not significant

Fig. 5

Effects of manually and automatically isolated CD133⁺ stem cells on cardiac remodeling. 1 × 10⁵ CD133⁺ stem cells were intramyocardially transplanted into SCID bg mice after myocardial infarction (MI). For evaluation of histological changes, fibrotic events (a) and decrease of capillary density (b) at infarction border zone and remote area were analyzed 3 weeks after transplantation. Animals with untreated infarction (MIC) and SHAM operation were used as control groups. Data are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. MIC; ns not significant

Fig. 6

Effects of manually and automatically isolated CD133⁺ stem cells on formation of infarction scar. 1 × 10⁵ CD133⁺ stem cells were intramyocardially transplanted into SCID bg mice after myocardial infarction (MI). Three weeks after cell transplantation infarction size (a) and infiltration of the infarcted scar with new blood vessels (b) were analyzed. For control untreated infarction (MIC) and SHAM operation were used. Data are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. MIC; ns not significant