© Ferrata Storti Foundation IntroductionSince the first clinical trial of mesenchymal stromal cells (MSC) 9,10 Another important issue regarding the clinical application of MSCs is their culture under serum-free conditions. The majority of clinical studies have used MSCs that were expanded in media supplemented with fetal bovine serum (FBS). 1,[11][12][13][14][15] To avoid the risks associated with the use of FBS, 16 platelet lysate (PL) was proposed as a supplement to tissue culture media for MSCs. 17 Recently, several studies showed that MSCs that were expanded in PL exhibited the same efficacy as MSCs cultured in serum-containing media for the treatment of GvHD. 18-22To date, clinical studies have used MSCs that have been generated from several individual donors. Considering the aforementioned inter-donor heterogeneity and the need for a large number of "off-the-shelf" MSCs, the establishment of MSC banks appears to be an indispensable strategy for providing a continuous supply of MSCs with predictable potency. To our knowledge, there are few established MSC banks worldwide, and these MSC banks were generated by separately isolating, expanding, and freezing MSCs from up to 10 donors in FBS-containing media. [23][24][25][26] In the current study, we report for the first time the establishment of a serum-free and GMP-compliant MSC bank generated from pooled bone marrow mononuclear cells (BM-MNCs) of multiple donors as a novel strategy to circumvent donor-to-donor variability. Clinical-grade MSC endproducts (MEPs) derived from the MSC bank were thoroughly assessed for their proliferation, differentiation, and, in particular, for the allosuppressive potential in vitro. Importantly, 81 MEPs were administered as a rescue therapy to 26 pediatric patients with severe steroid-refractory aGvHD in seven transplantation centers. Safety and efficacy of MEPs was compared to MSCs generated from a single or several individual donors that have been used in the GvHDclinical studies reported thus far. Methods Generation of MSC bank and clinical-grade MEPsBone marrow was collected from 8 healthy volunteers (age 21-45 years old) after written informed consent and after the approval of the local Ethics Committee (n. 275/09). BM-MNCs were enriched from the bone marrow aspirate by using the Sepax II NeatCell process (Biosafe, Eysins, Switzerland) and frozen individually. After thawing and washing these BM-MNCs were pooled. This pool of BM-MNCs from 8 donors was used to generate MSCs over 14 days in culture. After their detachment, passage 1 mesenchymal stromal cells (MSC-P1) were washed and aliquoted into 209 cryovials (each containing 1.5x10 6 MSC-P1). Cryopreserved vials with MSC-P1 were referred to as the MSC bank.To generate clinical-grade MEPs, MSC-P1 aliquots from the MSC bank were thawed and after washing they were expanded in medium containing 10% PL till the end of passage 2. These MSCs were re-suspended in cryomedium (0.9% NaCl containing 5% HSA and 10% DMSO), distributed in cryobags (each containing 1-3x10 6 MS...

All methods are in principle suitable and can be selected based on sample volume, available technology, and desired product specifications beyond RBC depletion and MNC and/or CD34+ cell recovery.

The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

BackgroundRed blood cell (RBC) depletion is a standard graft manipulation technique for ABO-incompatible bone marrow (BM) transplants. The BM processing module for Spectra Optia, “BMC”, was previously introduced. We here report the largest series to date of routine quality data after performing 50 clinical-scale RBC-depletions.MethodsFifty successive RBC-depletions from autologous (n = 5) and allogeneic (n = 45) BM transplants were performed with the Spectra Optia BMC apheresis suite. Product quality was assessed before and after processing for volume, RBC and leukocyte content; RBC-depletion and stem cell (CD34+ cells) recovery was calculated there from. Clinical engraftment data were collected from 26/45 allogeneic recipients.ResultsMedian RBC removal was 98.2% (range 90.8–99.1%), median CD34+ cell recovery was 93.6%, minimum recovery being 72%, total product volume was reduced to 7.5% (range 4.7–23.0%). Products engrafted with expected probability and kinetics. Performance indicators were stable over time.DiscussionSpectra Optia BMC is a robust and efficient technology for RBC-depletion and volume reduction of BM, providing near-complete RBC removal and excellent CD34+ cell recovery.