© 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...

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

The Croatian Cardiovascular Tissue Bank (CTB) was established in June 2011. Activities managed by CTB are processing of heart valves and blood vessels, as well as quality control, storage, medical release and distribution of allografts. The aim of this report is to present CTB's vascular tissue activities and retrospectively evaluate the outcomes of their use in the University Hospital Centre Zagreb. Between June 2011 and July 2021, 90 vascular allografts (VAs) from 55 donors after brain death were referred to CTB. Only 54% of VAs met the tissue quality requirements while 46% of tissues were discarded. The most frequent reasons for discard were unacceptable morphology and initial microbiological contamination. Altogether 42 VAs were released for transplantation and 37 of them were used in 27 surgical procedures. The most common indication for surgery was prosthetic graft or stent infection. According to the anatomic position of vascular reconstruction, patients were divided in the aortic and peripheral reconstruction group. A total of 23 patients were treated. In the aortic reconstruction group 58% of patients did not experience any graft-related complications. In the group of patients who underwent peripheral reconstruction significant incidence of reinfection was observed highlighting it as a major graft-related complication. Despite the small patient groups and limited duration of follow-up, presented clinical outcomes provide valuable information on the efficacy of vascular allografts. Additional clinical results collected on a larger patient groups and comparison to other reconstructive treatment options are necessary.

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