Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use

Dexamethasone scheduling in MNC culture. The supplement was added in standard medium until the detection of MSC colonies (n = 16 CB units) or alternatively added for the first week of MNC culture only (n = 34). (DOCX 120 kb

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrowederived mesenchymal stromal cells

Abstract Background aims. A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Methods. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. Results. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. Conclusions. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS

P09.01 Adoptive cell therapy of hematological malignancies using cytokine-induced killer cells retargeted with monoclonal antibodies

Background Cytokine-Induced Killer (CIK) cells are a population of effector cells that represents a promising tool for adoptive cell therapy. They are easily expandable ex-vivo, safe, and exert cytotoxicity against a broad range of tumor histotypes.1 We recently reported that they have a relevant expression of FcγRIIIa (CD16a), which can be exploited in combination with clinical-grade monoclonal antibodies (mAbs) to redirect their cytotoxicity in an antigen-specific manner, to improve their antitumor activity.2 Indeed, the engagement of CD16a on CIK cells leads to a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian cancer both in vitro and in vivo. Based on this observation, we investigated whether CIK cells can be specifically retargeted against B-cell malignancies by combination with anti-CD20 mAbs, namely Rituximab® (RTX) and Obinutuzumab® (OBI). Materials and Methods CIK cells were obtained from peripheral blood mononuclear cells of healthy donors, and stimulated in vitro with IFN-γ, CD3 mAb and IL-2 for 14 days; fresh IL-2 was provided every 3–4 days. CIK cell phenotype was analyzed by multicolor flow cytometry; cytotoxic activity was assessed by calcein AM-release assay against B-cell lines, primary samples and patient-derived xenografts (PDX) obtained from B-cell lymphoma patients after written informed consent. Results The combination with both RTX and OBI significantly increased specific CIK cells lysis against several CD20-expressing lymphoma B cell lines, primary tumors from B-cell lymphoma patients and an established PDX, compared to the combination with a control mAb (cetuximab, CTX). NK-depletion demonstrated that the mAb-mediated cytotoxicity is accountable to the CIK cells fraction within the bulk population since no difference in the lytic activity was detectd in the absence of NK cells. In addition, these results are further supported by in vivo preliminary experiments where the treatment with CIK cells in combination with OBI extensively reduced the growth of PDX and increased mice survival, compared to CIK cells or OBI administered alone. Conclusions Here we proved that CIK cells can be retargeted with clinical-grade mAbs against CD20-expressing lymphomas. These data indicate that the combination of CIK cells with mAbs can represent a novel approach for the treatment of haematological malignancies. References Franceschetti M, Pievani A, Borleri G, Vago L, Fleischhauer K, Golay J, et al. Cytokine-induced killer cells are terminally differentiated activated CD8 cytotoxic T-EMRA lymphocytes. Exp Hematol 2009;37:616–28. Cappuzzello E, Tosi A, Zanovello P, Sommaggio R, Rosato A. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies. Oncoimmunology 2016 Aug;5(8):e1199311. The research leading to these results has received funding from Fondazione AIRC under IG 2018 - ID. 21354 project - P.I. Rosato Antonio Disclosure Information A. Dalla Pieta: None. E. Cappuzzello: None. P. Palmerini: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. O. Perbellini: None. M. Tisi: None. C. Visco: None. M. Ruggeri: None. A. Rosato: None

P09.13 Optimization of a GMP-grade large-scale expansion protocol for cytokine-induced killer cells using gas-permeable static culture flasks

Background Cytokine-Induced Killer (CIK) cells are ex vivo expanded T cells with NK cell phenotype. They express both CD3 and CD56 antigens, and exert a potent antitumor activity against a variety of tumors. Several clinical trials demonstrated the safety and the feasibility of CIK cell therapy, with very low side effects and minimal graft-versus-host toxicity. In this study, we developed a GMP-compliant protocol for robust large-scale expansion of CIK cells using G-Rex® gas-permeable static culture flasks. Materials and Methods CIK cells were obtained by stimulating healthy donor PBMCs with GMP-grade IFN-γ, IL-2 and CD3 mAbs, and were cultured in G-Rex6® or G-Rex®6M well plates. CIK cells in G-Rex6® were split only once at day 7 to reduce cell density, whereas the number of CIK cells culterd in G-Rex®6M was not adjusted. In both culture conditions, fresh IL-2 was provided every 3–4 days. We compared these two culture protocols with the culture in standard flasks. Phenotype was analyzed by flow cytometry and cytotoxicity was assessed against several tumor cell lines by calcein-release assay. Results CIK cells cultured in G-Rex6® well plates showed an outstanding cell expansion compared to G-Rex®6M well plates or standard culture flasks, with a 400-fold expansion and a mean of 109 total cells obtained per single well in 14 days, starting from just 2.5 × 106 cells per well. Moreover, the cultures in G-Rex6® were characterized by an higher percentage of CD3+CD56+ cells, as compared to G-Rex®6M or standard culture flasks. Cells cultured in all devices had a comparable expression of NKG2D, NKp30, NKp44, 2B4 receptors. Importantly, CIK cells expanded in G-Rex®6 were as cytotoxic as cells expanded in standard culture flasks. Conversely, CIK cells cultured in G-Rex®6M showed a remarkable reduction of cytotoxicity against tumor cell targets, thus suggesting that cell density during expansion could affect CIK cell activity. Conclusions We propose a GMP-compliant protocol for robust large-scale production of CIK cells. G-Rex® system allows to obtain large amounts of CIK cells highly enriched in the CD3+CD56+ subset and endowed with high cytotoxic activity; this can be accomplished with just a single cell culture split at day 7, which dramatically reduces the culture manipulation as compared to the standard culture flasks. Notably, this strategy can be further and easily scalable to produce CIK cells for clinical immunotherapy applications. Disclosure Information A. Ventura: None. P. Palmerini: None. A. Dalla Pieta: None. R. Sommaggio: None. G. Astori: None. K. Chieregato: None. M. Tisi: None. C. Visco: None. O. Perbellini: None. M. Ruggeri: None. E. Cappuzzello: None. A. Rosato: None

A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokine-induced killer cells: a first-in-human case report

Not available

Definizione di cocktail citochinici per l'espansione, in condizioni FBS-free, di cellule stromali mesenchimali isolate da tessuto adiposo e cordone ombelicale

Human mesenchymal stromal cells (hMSCs) are multipotent cells isolated from several tissues that possess self-renewal capacity, long term viability and are capable to differentiate into several cell lineages (osteoblasts, chondrocytes, adipocytes, etc.). They interact with hematopoietic stem cells (HSC) and exert immunoregulatory function. These characteristics make MSCs excellent candidates for regenerative medicine and gene therapy. To date, bone marrow (BM) has been the main source for the isolation of hMSCs,but in the last decade some drawbacks arose: 1) the cells low number that requires an ex vivo expansion; 2) the BM MSCs senescence with increasing of donor age; 3) the invasive procedure. This has led many researchers to investigate alternative MSCs sources and new expansion protocols to bypass FBS/human platelet rich plasma (hPRP) limitations: 1) the risk of prion diseases and immunological reactions associated with the use of FBS; 2) the decreased proliferative effect of hPRP after high g rate centrifugation to purify it, its undefined composition and its full clinical impact that remains to be investigated. In addition, its use is limited by the amount of whole blood necessary to obtain a sufficient quantity of autologous hPRP for cell expansion. In this study we focused on: 1) adipose tissue derived-MSCs (AT MSCs) because of the high number of cells that can be obtained and the easy enzyme-based procedures; 2) umbilical cord derived-MSCs (UC MSCs) which possess greater and faster expansion capability, higher frequency of colony forming unit fibroblast (CFU-F) than BM MSCs. For the first time, we evaluated, measuring bromo-deoxyuridine incorporation in neosynthesized DNA, the effects on AT and UC MSCs expansion of a pool of seven human recombinant growth factors: epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor I (IGF I), platelet-derived Growth factor-bb (PDGFbb) and transforming growth factor β1 (TGFβ1). We defined two cytokines cocktails based on EGF-bFGF-PDGFbb for AT MSCs, and EGF-PDGFbb for UC MSCs expansion in a FBS-free medium supplemented with hPPP (3%), which allows a normal MSCs expansion with a minimal apoptosis and supports GFs proliferative effect. These mixtures permitted us, starting from about 100-150 cm3 of AT and 30 cm of UC, to obtain after 21 days of culture a sufficient number of cells for clinical applications, for example in graft versus host disease (GvHD). Furthermore, in our expansion conditions, we required about 80 ml of hPPP obtainable from 150-200 ml of whole blood, as opposed to 350 ml of hPRP which requires 1000-1200 ml of whole blood. The mitogenic response decreases when MEK signaling is inhibited by U0126, a MEK1/2 inhibitor. This result suggests that MEK 1/2 pathway is required to mediate a maximum mitogenic response of cells to GFs. Our cocktails do not influence surface markers expression. AT and UC MSCs highly expressed CD 105, CD 90, and CD 44, whereas cells expressing CD 31, CD 34, CD 117 and CD 45 were lost with progressive passages, in line with the parameters proposed for MSCs by International Society for Cellular Therapy. High levels of the enzyme aldehyde dehydrogenase (ALDH) have proven to be a novel marker for the identification and isolation of hematopoietic stem cells (HSCs). Co-staining experiments revealed that >80% of ALDH+ umbilical cord blood cells were CD 34+. Based on our studies, ALDH combined with other surface markers (CD 45 and CD 105) can not be used exclusively to define MSCs derived from AT and UC in a manner similar to HSCs because of its low level (less than 50%). Differentiation capacity into adipogenic and osteogenic lineage, tested at the end of P2, was confirmed only for AT MSCs. The exposure to EGF-bFGF-PDGFbb increased adipogenic differentiation as well as osteogenic one respect to cultures in FBS and hPPP, likely because these GFs commit MSCs to specific mesodermal differentiation. This could represent a highly promising approach in tissue engineering for breast soft tissue reconstruction after mastectomy, tissue and subdermal defects after trauma or burn injury. AT MSCs were reported to have a slightly inferior potential for osteogenesis, with our GFs cocktail this “problem” could be overcome, a normal osteogenesis could take place and it could guarantee a large amount of osteogenic precursors for scaffold population. UC MSCs, probably due to their ontogenetic age, did not show mineralization and the formation of lipid vacuoles, but they were more immunocompetent than AT ones in the presence of GFs. UC MSCs immunosuppressive effect, on T cells stimulated with phytohemagglutinin, was confirmed in all tested supplements and was significant when MSCs:T cell ratio was 1:10. AT MSCs immunomodulation was detectable at 1:5 ratio in the presence of GFs cocktail and it was less marked than in FBS, likely because GFs, inducing cell maturation, reduce immunomodulatory potential. Whereas, UC MSCs, having relatively primitive nature, seem to be less influenced by cytokines than mature MSCs. We did not measure a significant immunosuppression when AT/UC MSCs and lymphocytes were separated by transwell. This suggests that the suppressive factor(s) are not constitutively secreted by MSCs and probably a pre-activation mediated by cell contact is required. Recently, a number of clinical trials used cytokine induced killer (CIK) cells in an attempt to improve the effectiveness of HSC transplantation. CIK cells are a population of immune-effector cells CD 3+ CD 56+, expanded in vitro by exposure to interferon γ, monoclonal anti CD3 and interleukin 2. They show cytotoxicity against a broad range of malignant cell targets, but not against normal CD 34+ stem cells. In view of relevant role of both MSCs and CIK cells, we explored the interactions between the two cell types. We found that CIK cells lysed UC MSCs in dose dependent manner, while UC cells killed CIK ones and suppressed their cytotoxicity against K562, a tumour target. These effects required cell to cell contact. Our results should be taken into account in evaluating novel protocols of adoptive immunotherapy. They suggest to administer, at first, mesenchymal cells to optimize engraftment and to prevent GvHD, and then CIK cells to mediate graft versus leukemia. In conclusion, our cocktails guarantee the expansion of an extremely homogeneous population of MSCs and a sufficient number of cells for clinical applications: committed AT MSCs in reconstructive medicine, and UC MSCs in hematologic context in combination with CIK cells.Le cellule stromali mesenchimali umane (hMSC) sono cellule multipotenti, isolate da numerosi tessuti, con capacità di autoreplicarsi e di differenziarsi in più linee cellulari (osteoblasti, condrociti, adipociti, ecc.). Interagiscono con le cellule staminali ematopoietiche (HSC) e hanno proprietà immunomodulanti. Queste caratteristiche hanno reso le MSC eccellenti candidate per la medicina rigenerativa e la terapia genica. Fino ad oggi il midollo osseo (BM) ha rappresentato la fonte principale di MSC, ma nell’ultimo decennio sono state sollevate delle riserve: 1) la bassa frequenza che richiede un’espansione ex vivo; 2) la comparsa di senescenza con il progredire dell’età del donatore; 3) la procedura di prelievo invasiva. Questo ha spinto i ricercatori ad investigare, da una parte, fonti alternative e, dall’altra, nuovi protocolli di espansione per bypassare gli inconvenienti associati all’uso del siero fetale bovino (FBS) e di plasma umano arricchito di piastrine (hPRP). Le principali limitazioni sono legate, per l’FBS, al rischio di trasmissione di malattie prioniche e di reazioni immunitarie causate dalle proteine xenogeniche; per hPRP, alla sua composizione variabile, ad un effetto clinico poco conosciuto e, soprattutto, all’alta quantità di sangue intero richiesto per ottenere un volume di hPRP autologo sufficiente per l’espansione ex vivo delle MSC. In più, la centrifugazione ad alte g, nel tentativo di rimuovere le membrane delle piastrine, decresce l’effetto proliferativo di hPRP. In questo studio sono state prese in esame due fonti alternative: 1) il tessuto adiposo (AT), da dove le MSC sono ottenibili in alto numero e con una facile procedura enzimatica; 2) il cordone ombelicale (UC), dal quale sono isolabili, come riportato in letteratura, MSC caratterizzate da frequenza e capacità replicativa maggiori rispetto alle BM MSC. Per la prima volta è stato valutato, misurando l’incorporazione della bromo-deossiuridina nel DNA neosintetizzato, l’effetto sulla proliferazione di AT e UC MSC di un pool di sette fattori di crescita (GF) umani ricombinanti: epidermal growth factor (EGF), basic-fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), insulin-like growth factor I (IGF I), platelet-derived growth factor-bb (PDGFbb) e transforming growth factor β1 (TGFβ1). Sono stati così definiti due cocktail a base di EGF-bFG-PDGFbb per le AT MSC e EGF-PDGFbb per espansione delle UC MSC in un mezzo FBS-free supplementato con plasma umano povero di piastrina (hPPP, 3%), che supporta la crescita delle mesenchimali minimizzando l’apoptosi e consente l’azione dei GF. Queste combinazioni di citochine sono state in grado di fornire, dopo 21 giorni di coltura, un numero sufficiente di cellule per un eventuale trattamento, ad esempio, della graft versus host disease (GvHD), partendo da 100-150 cm3 di AT e 20-30 cm di UC. Inoltre, nelle condizioni di coltura definite, sono serviti circa 80 ml di hPPP ottenibili da 150-200 ml di sangue intero, invece dei 350 ml di hPRP ricavabili da 1000-1200 ml di sangue. La risposta mitogenica riscontrata sembra coinvolgere due protein-kinasi specifiche, MEK1 e MEK2, come evidenziato in presenza di un loro inibitore specifico (U0126). Questo suggerisce la priorità di questa via nel mediare la risposta mitogenica massima delle citochine. I cocktail, inoltre, non influenzano l’espressione dei marker di superficie caratteristici delle mesenchimali. CD 105, CD 90 e CD 44 sono risultati altamente espressi sia sulle AT che sulle UC MSC, mentre la percentuale di cellule positive al CD 31, CD 34, CD 117 e CD 45 decresceva con il progredire dei passaggi, in linea con i criteri definiti dall’International Society for Cellular Therapy. Le aldeidi deidrogenasi (ALDH), una classe di enzimi ossidativi, sono state di recente proposte come marker per l’identificazione e l’isolamento delle HSC dove sono espresse ad alti livelli (>80% nelle cellule CD 34+ isolate dal sangue della vena del cordone ombelicale). In questo studio si è indagato se le ALDH potessero svolgere un ruolo analogo per le MSC. I risultati ottenuti hanno evidenziato che le ALDH, combinate con CD 45 e CD 105, non possono essere usate come marker identificativi per le MSC derivate da AT e UC a causa della loro bassa espressione (inferiore al 50%). La capacità differenziativa in senso adipogenico ed osteogenico, testato a fine del secondo passaggio (P2), è stata confermata per la AT MSC. L’esposizione a EGF-bFGF-PDGFbb ha incrementato l’adipogenesi e l’osteogenesi rispetto alla coltura in FBS e hPPP, probabilmente le citochine stimolano l’attivazione dei pathway coinvolti nel differenziamento adipogenico ed osteogenico. L’utilizzo di cellule mesenchimali “indirizzate” al differenziamento adipogenico potrebbe rappresentare una promettente risorsa in chirurgia plastica e ricostruttiva per la ricostruzione del seno dopo mastectomia, per la riparazione di difetti tissutali e subdemici conseguenti a traumi o ustioni. Per le AT MSC è stata riferita una potenzialità osteogenica inferiore alle altre MSC adulte. Con il cocktail definito, che predispone le AT MSC anche all’osteogenesi, questo “problema” potrebbe essere superato, garantendo un numero sufficiente di precursori osteogenici per il popolamento di uno scaffold. Le UC MSC, forse a causa della loro età ontogenia, non hanno evidenziato mineralizzazione e la formazione di vacuoli lipidici, ma si sono dimostrate più immunocompetenti delle AT MSC in presenza dei GF. L’effetto immunomodulante, a carico dei linfociti T attivati con fitoemagglutinina, è stato registrato per le UC MSC ad un rapporto MSC: cellule T pari a 1:10 in tutte le condizioni testate. L’immunomodulazione indotta dalle AT MSC è stata invece riscontrata a 1:5 in presenza del cocktail risultando così più blanda di quella in FBS. Probabilmente i fattori di crescita, inducendo la maturazione e predisponendo le cellule al differenziamento, compromettono le potenzialità immunomodulanti delle mesenchimali isolate da AT. Le UC MSC, essendo invece più immature, sono forse meno suscettibili all’azione dei GF rispetto alle MSC adulte. Quando tra mesenchimali e linfociti si è interposta una “barriera” che ne impediva il contatto “fisico”, non è stato rilevato alcun blocco significativo della proliferazione delle cellule T. Si può concludere che il contatto cellulare rappresenti la condizione necessaria per attivare i meccanismi di immunosoppressione come la produzione di fattori solubili. Recentemente, si sono moltiplicati i trial clinici dove si vanno ad infondere cellule CIK (cytokine induced killer) con lo scopo di aumentare l’efficienza del trapianto di HSC. Le cellule CIK sono una popolazione linfocitaria CD 3+ CD 56+, espanse in vitro con interferone γ, anti CD3 e interleuchina 2, e dotate di azione citotossica verso numerosi target tumorali, ma non sulle staminali CD 34+. Visto il ruolo rilevante in campo ematologico sia delle MSC che delle CIK, è stata indagata l’interazione tra le due popolazioni cellulari. Le CIK hanno mostrato un’azione citotossica sulle UC MSC dose dipendente, mentre le mesenchimali hanno determinato la soppressione delle CIK e la riduzione della loro potenzialità citotossica a carico della K562, un target tumorale. Questi risultati dovrebbero essere tenuti presenti nella definizione di nuovi protocolli clinici di immunoterapia. Essi suggeriscono di somministrare prima le mesenchimali al fine di supportare l’attecchimento delle HSC e prevenire la GvHD, e solo in un secondo tempo le CIK ad azione antitumorale. In conclusione, i cocktail definiti garantiscono l’espansione di una popolazione omogenea di mesenchimali e in numero sufficiente per l’uso clinico delle AT MSC “committed” in medicina ricostruttiva, e delle UC MSC in un contesto ematologico in combinazione con le cellule CIK a patto di una somministrazione in due tempi

Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue

Mesenchymal stem cells (MSCs) are non-hematopoietic, adult, fibroblast-like, multipotent cells that are plastic adherent in standard culture conditions. They can be isolated from several tissues, but it is always necessary to expand them for clinical practice

The cytotoxic effects of bendamustine in combination with cytarabine in mantle cell lymphoma cell lines

Bendamustine is clinically useful in mantle-cell lymphoma (MCL). Its favorable toxicity profile in-vivo favors its combination with other cytotoxic drugs. Cytarabine is a key drug in the treatment of younger patients with MCL. The current study investigated the in-vitro cytotoxic effect of bendamustine and cytarabine, alone or combined, on two MCL cell lines representing the classic and blastoid variant of the lymphoma subtype (JEKO-1 and GRANTA-519). Cell lines were exposed to each drug alone, or simultaneously and consecutively to both drugs, for different time schedules. Apoptosis was measured by flow cytometry. Mitochondrial damage, cell proliferation/metabolic activity, and cell cycle analysis were also assessed. The synergistic, additive, or antagonistic effects of the drugs were calculated with the combination index (CI) method. Bendamustine and cytarabine alone exhibited relevant cytotoxic activity on both cell lines. Both drugs induced cell cycle arrest in S phase. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone. Among the combination schedules, the consecutive incubation of bendamustine followed by cytarabine was associated with the lower CI, being 10-100-fold lower than with simultaneous incubations. The cytotoxic effect of the consecutive combination was prominent on both cell lines, indicating a very strong and highly significant synergy in inducing apoptosis. Similar results were obtained measuring mitochondrial damage or the decline of the metabolic activity in all cell lines. The strong synergistic effect of bendamustine and cytarabine on MCL cells provides a rationale for developing schedules combining these agents in the treatment of MCL

Effect of Platelet Lysate on the Functional and Molecular Characteristics of Mesenchymal Stem Cells Isolated from Adipose Tissue

Abstract: Background: Mesenchymal stem cells (MSCs) are non-hematopoietic, adult, fibroblast-like, multipotent cells that are plastic adherent in standard culture conditions. They can be isolated from several tissues, but it is always necessary to expand them for clinical practice. Aim: We investigated the effect of human platelet lysate (hPL) on the expansion of human MSCs isolated from adipose tissue (AT), comparing it with fetal bovine serum (FBS) and human platelet-poor plasma (hPPP). Materials and Methods: Human AT-MSCs, hPL and hPPP were obtained from 7 healthy subjects. AT-MSCs were seeded at 1500 cells/cm 2 and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 10% hPPP or 10% hPL. Cells were harvested, counted and analyzed by flow cytometry every 7 days for 5 passages (P). The differentiation assays, RNA isolation and co-culture with allogeneic lymphocytes were performed at the end of P2. Results: AT-MSCs achieved a better proliferation rate when cultured with hPL than with hPPP or FBS (20 ±2 versus 8 ±3 and 6 ±3, respectively, at the end of P5 [p<0.01]). hPL preserved the differentiation capacity and typical expression of surface antigens, avoiding the risks associated with the use of animal derivatives. AT-MSCs demonstrated a stronger inhibitory effect on lymphocyte proliferation with hPL than with other culture conditions, even at a AT-MSCs:T cells ratio of 1:10. The transcriptional level of matrix metalloproteinase 2, used to evaluate stemness, was very high in all conditions tested. Conclusions: hPL represents an effective and safe supplement for MSC expansion to be used in the clinical setting

Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future

The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing-thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users