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.

Immunomodulation Driven by Theranova Filter during a Single HD Session.

Background: Patients with end-stage kidney disease (ESKD) have altered immunity. Patients on hemodialysis (HD) present a coexistence of immunodeficiency and activation of the immune system. We evaluated the immunophenotypic profile induced by the medium cut-off of Theranova filter during a single HD session in the same individual. Methods: This pilot observational study explored 11 patients (75 ± 8 years and 73% male). Blood samples were collected prior to (predialytic, PRE) and after 4 h (postdialytic, POST) standard HD session with a medium cut-off, polyarylethersulfone and polyvinylpyrrolidone blend, BPA-free membrane. We performed an immunophenotyping characterization by using flow cytometry. We evaluated eryptosis RBCs and HLA-DR expression on monocytes and Treg cells. Results: The percentages of eryptosis in lymphocytes (CD3+), lymphocyte T helper (CD3+ and CD4+) cells, and monocytes (CD45+ and CD14+) were similar pre- and post-HD. On the contrary, HLA-DR expression and Treg cell numbers significantly decreased after HD. Conclusions: Many studies have focused on the comparison between healthy volunteers and HD patients, but very few have focused on the changes that occur after an HD session in the same individual. With this pilot observational study, we have revealed an immunomodulation driven by HD treatment with Theranova filter. Our preliminary results can be considered to be a hypothesis, generating and stimulating further studies with better designs and larger populations.

Enalaprilat and losartan decrease erythroid precursors frequency in cells from patients with polycythemia vera.

OBJECTIVE: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by the overproduction of red blood cells. First-line therapies are directed at lowering hematocrit levels. After the discovery of a mutation in the Janus kinase 2 (JAK2V617F), JAK2 inhibitors have been tested as second-line therapies. Despite these approaches, there is still the need for a major comprehension of the mechanisms involved in PV erythrocytosis and of more effective therapies. Angiotensin-converting enzyme (ACE) stimulates hematopoietic precursors proliferation and erythroid differentiation. We thus hypothesized that ACE inhibition could help in controlling erythrocytosis in PV. METHODS: We assessed the clonogenic potential by colony-forming unit (CFU) assay of mononuclear cells isolated from PV JAK2 positive or JAK2 negative patients with erythrocytosis treated with enalaprilat or losartan. RESULTS: Treatment with drugs led to a decrease of erythroid precursor frequency both in the presence and absence of JAK2 mutation, with a high extent in JAK2 positive cells and without affecting other types of precursors. No dose-dependent effect was observed. CONCLUSIONS: Our results demonstrate that ACE inhibition reduces erythroid precursor frequency, confirming the involvement of ACE in erythrocytosis despite the presence of JAK2 mutation and encouraging the hypothesis that ACE inhibitors and AT1R antagonists could help in directly managing erythrocytosis in PV.

Selective cell cycle arrest in glioblastoma cell lines by quantum molecular resonance alone or in combination with temozolomide.

BACKGROUND: Glioblastoma is the most aggressive form of brain cancer, characterised by high proliferation rates and cell invasiveness. Despite advances in surgery and radio-chemotherapy, patients continue to have poor prognoses, with a survival rate of 14-15 months. Thus, new therapeutic strategies are needed. Non-ionising electromagnetic fields represent an emerging option given the potential advantages of safety, low toxicity and the possibility to be combined with other therapies. METHODS: Here, the anticancer activity of quantum molecular resonance (QMR) was investigated. For this purpose, three glioblastoma cell lines were tested, and the QMR effect was evaluated on cancer cell proliferation rate and aggressiveness. To clarify the QMR mechanism of action, the proteomic asset after stimulation was delineated. Mesenchymal stromal cells and astrocytes were used as healthy controls. RESULTS: QMR affected cancer cell proliferation, inducing a significant arrest of cell cycle progression and reducing cancer tumorigenicity. These parameters were not altered in healthy control cells. Proteomic analysis suggested that QMR acts not only on DNA replication but also on the machinery involved in the mitotic spindle assembly and chromosome segregation. Moreover, in a combined therapy assessment, QMR significantly enhanced temozolomide efficacy. CONCLUSIONS: QMR technology appears to be a promising tool for glioblastoma treatment.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells.

BACKGROUND: Patients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with the infusion of CD19 chimeric antigen receptor-T cells, but several limits still restrain the administration to a limited proportion of patients. This unmet clinical need might be fulfilled by an adoptive immunotherapy approach that combines cytokine-induced killer (CIK) cells and monoclonal antibodies (mAb) to the CD20 antigen. Indeed, CIK cells are an effector population endowed with antitumor activity, which can be further improved and antigen-specifically redirected by clinical-grade mAb triggering antibody-dependent cell-mediated cytotoxicity. METHODS: CIK cells were generated from peripheral blood of patients affected by different B-cell malignancies using a blinatumomab-based cell culture protocol. Effector cells were combined with the anti-CD20 mAb obinutuzumab and their therapeutic activity was assessed both in vitro and in vivo. RESULTS: CIK cells were successfully expanded in clinically relevant numbers, starting from small volumes of peripheral blood with extremely low CD3+ counts and high tumor burden. This relied on the addition of blinatumumab in culture, which leads to the simultaneous expansion of effector cells and the complete elimination of the neoplastic component. Moreover, CIK cells were highly cytotoxic in vitro against both B-cell tumor cell lines and autologous neoplastic targets, and had a significant therapeutic efficacy against a B-cell malignancy patient-derived xenograft on in vivo transfer. CONCLUSIONS: The combination of an easily expandable CIK cell effector population with a mAb already in clinical use establishes a tumor antigen-specific redirection strategy that can be rapidly translated into clinical practice, providing an effective therapeutic alternative for B-cell malignancies without any need for genetic modifications. Additionally, the approach can be potentially applied to an extremely vast array of different tumors by simply substituting the targeting mAb.

The immune modulatory effects of umbilical cord-derived mesenchymal stromal cells in severe COVID-19 pneumonia.

Coronavirus disease 2019 (COVID-19) may result in a life-threatening condition due to a hyperactive immune reaction to severe acute respiratory syndrome-coronavirus-2 infection, for which no effective treatment is available. Based on the potent immunomodulatory properties of mesenchymal stromal cells (MSCs), a growing number of trials are ongoing. This prompted us to carry out a thorough immunological study in a patient treated with umbilical cord-derived MSCs and admitted to the Intensive Care Unit for COVID-19-related pneumonia. The exploratory analyses were assessed on both peripheral blood and bronchoalveolar fluid lavage samples at baseline and after cellular infusion by means of single-cell RNA sequencing, flow cytometry, ELISA, and functional assays. Remarkably, a normalization of circulating T lymphocytes count paralleled by a reduction of inflammatory myeloid cells, and a decrease in serum levels of pro-inflammatory cytokines, mostly of interleukin-6 and tumor necrosis factor-α, were observed. In addition, a drop of plasma levels of those chemokines essential for neutrophil recruitment became evident that paralleled the decrease of lung-infiltrating inflammatory neutrophils. Finally, circulating monocytes and low-density gradient neutrophils acquired immunosuppressive function. This scenario was accompanied by an amelioration of respiratory, renal, inflammatory, and pro-thrombotic indexes. Our results provide the first immunological data possibly related to the use of umbilical cord-derived MSCs in severe COVID-19 context.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Logistics of an advanced therapy medicinal product during COVID-19 pandemic in Italy: successful delivery of mesenchymal stromal cells in dry ice.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. METHODS: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. RESULTS: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. CONCLUSIONS: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells.

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4-64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.

High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources.

BACKGROUND: Mesenchymal stromal cells (MSC) are a heterogeneous population of multipotent progenitors used in the clinic because of their immunomodulatory properties and their ability to differentiate into multiple mesodermal lineages. Although bone marrow (BM) remains the most common MSC source, cord blood (CB) can be collected noninvasively and without major ethical concerns. Comparative studies comprehensively characterizing the MSC phenotype across several tissue sources are still lacking. This study provides a 246-antigen immunophenotypic analysis of BM- and CB-derived MSC aimed at identifying common and strongly expressed MSC markers as well as the existence of discriminating markers between the two sources. METHODS: BM-MSC (n = 4) were expanded and analyzed as bulk (n = 6) or single clones isolated from the bulk culture (n = 3). CB-MSC (n = 6) were isolated and expanded as single clones in 5/6 samples. The BM-MSC and CB-MSC phenotype was investigated by flow cytometry using a panel of 246 monoclonal antibodies. To define the markers common to both sources, those showing the smallest variation between samples (coefficient of variation of log2 fold increase ≤ 0.5, n = 59) were selected for unsupervised hierarchical cluster analysis (HCL). Differentially expressed markers were identified by directly comparing the expression of all 246 antigens between BM-MSC and CB-MSC. RESULTS: Based on HCL, 18 markers clustered as strongly expressed in BM-MSC and CB-MSC, including alpha-smooth muscle antigen (SMA), beta-2-microglobulin, CD105, CD13, CD140b, CD147, CD151, CD276, CD29, CD44, CD47, CD59, CD73, CD81, CD90, CD98, HLA-ABC, and vimentin. All except CD140b and alpha-SMA were suitable for the specific identification of ex-vivo expanded MSC. Notably, only angiotensin-converting enzyme (CD143) was exclusively expressed on BM-MSC. CD143 expression was tested on 10 additional BM-MSC and CB-MSC and on 10 umbilical cord- and adipose tissue-derived MSC samples, confirming that its expression is restricted to adult sources. CONCLUSIONS: This is the first study that has comprehensively compared the phenotype of BM-MSC and CB-MSC. We have identified markers that could complement the minimal panel proposed for the in-vitro MSC definition, being shared and strongly expressed by BM- and CB-derived MSC. We have also identified CD143 as a marker exclusively expressed on MSC derived from adult tissue sources. Further studies will elucidate the biological role of CD143 and its potential association with tissue-specific MSC features.

The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cells in vitro.

BACKGROUND: The use of fetal bovine serum (FBS) as a media supplement for the ex vivo expansion of bone-marrow derived mesenchymal stromal cells (BM-MSC) has been discouraged by regulatory agencies, due to the risk of transmitting zoonoses and to elicit immune reactions in the host once transplanted. Platelet derivatives are valid FBS substitutes due to their content of growth factors that can be released disrupting the platelets by physical methods or physiological stimuli. We compared platelet derivatives produced by freezing/thawing (platelet lysates, PL) or after CaCl2 activation (platelet releasate surnatant rich in growth factors, PR-SRGF) for their content in growth factors and their ability to support the ex vivo expansion of BM-MSC. METHODS: The cytokine content in the two platelet derivatives was evaluated. BM-MSC were expanded in complete medium containing 10, 7.5 and 5% PL or PR-SRGF and the cell phenotype, clonogenic capacity, immunomodulation properties and tri-lineage differentiation potential of the expanded cells in both media were investigated. RESULTS: The concentration of PDGF-AB, PDGF-AA, PDGF-BB in PR-SRGF resulted to be respectively 5.7×, 1.7× and 2.3× higher compared to PL. PR-SRGF promoted a higher BM-MSC proliferation rate compared to PL not altering BM-MSC phenotype. Colony forming efficiency of BM-MSC expanded in PR-SRGF showed a frequency of colonies significantly higher than cells expanded in PL. BM-MSC expanded in PL or PR-SRGF maintained their immunomodulatory properties against activated lymphocytes even if BM-MSC expanded in FBS performed significantly better. CONCLUSIONS: The method used to release platelet factors significantly affects the enrichment in growth factors and overall product performance. The standardization of the production process of platelet derivatives and the definition of their release criteria requires further investigation.

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

BACKGROUND: Increasing evidence suggests the safety and efficacy of mesenchymal stromal cells (MSC) as advanced therapy medicinal products because of their immunomodulatory properties and supportive role in hematopoiesis. Although bone marrow remains the most common source for obtaining off-the-shelf MSC, cord blood (CB) represents an alternative source, which can be collected noninvasively and without major ethical concerns. However, the low estimated frequency and inconsistency of successful isolation represent open challenges for the use of CB-derived MSC in clinical trials. This study explores whether CB may represent a suitable source of MSC for clinical use and analyzes several in vitro parameters useful to better define the quality of CB-derived MSC prior to clinical application. METHODS: CB units (n = 50) selected according to quality criteria (CB volume ≥ 20 ml, time from collection ≤ 24 h) were cultured using a standardized procedure for CB-MSC generation. MSC were analyzed for their growth potential and secondary colony-forming capacity. Immunophenotype and multilineage differentiation potential of culture-expanded CB-MSC were assessed to verify MSC identity. The immunomodulatory activity at resting conditions and after inflammatory priming (IFN-γ-1b and TNF-α for 48 hours) was explored to assess the in vitro potency of CB-MSC prior to clinical application. Molecular karyotyping was used to assess the genetic stability after prolonged MSC expansion. RESULTS: We were able to isolate MSC colonies from 44% of the processed units. Our results do not support a role of CB volume in determining the outcome of the cultures, in terms of both isolation and proliferative capacity of CB-MSC. Particularly, we have confirmed the existence of two different CB-MSC populations named short- and long-living (SL- and LL-) CBMSC, clearly diverging in their growth capacity and secondary colony-forming efficiency. Only LL-CBMSC were able to expand consistently and to survive for longer periods in vitro, while preserving genetic stability. Therefore, they may represent interesting candidates for therapeutic applications. We have also observed that LL-CBMSC were not equally immunosuppressive, particularly after inflammatory priming and despite upregulating priming-inducible markers. CONCLUSIONS: This work supports the use of CB as a potential MSC source for clinical applications, remaining more readily available compared to conventional sources. We have provided evidence that not all LL-CBMSC are equally immunosuppressive in an inflammatory environment, suggesting the need to include the assessment of potency among the release criteria for each CB-MSC batch intended for clinical use, at least for the treatment of immune disorders as GvHD.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

BACKGROUND: Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). MATERIALS AND METHODS: We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. RESULTS: After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. DISCUSSION: We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

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.

Enumeration of residual white blood cells in leukoreduced blood products: Comparing flow cytometry with a portable microscopic cell counter.

Transfusion of blood components is potentially associated to the risk of cell-mediated adverse events and current guidelines require a reduction of residual white blood cells (rWBC) below 1 × 10(6) WBC/unit. The reference method to enumerate rare events is the flow cytometry (FCM). The ADAM-rWBC microscopic cell counter has been proposed as an alternative: it measures leukocytes after their staining with propidium iodide. We have tested the Adam-rWBC for the ability to enumerate rWBC in red blood cells and concentrates. We have validated the flow cytometry (FCM) for linearity, precision accuracy and robustness and then the ADAM-rWBC results have been compared with the FCM. Our data confirm the linearity, accuracy, precision and robustness of the FCM. The ADAM-rWBC has revealed an adequate precision and accuracy. Even if the Bland-Altman analysis of the paired data has indicated that the two systems are comparable, it should be noted that the rWBC values obtained by the ADAM-rWBC were significantly higher compared to FCM. In conclusion, the Adam-rWBC cell counter could represent an alternative where FCM technology expertise is not available, even if the risk that borderline products could be misclassified exists.

Evaluation of lymphocytes inactivation by extracorporeal photopheresis using tetrazolium salt based-assay.

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP.

Multivariate statistical data analysis as a tool to analyze ex vivo expansion dynamics of cytokine-induced killer cells.

BACKGROUND: Cytokine-induced killer (CIK) cells, obtained after mononucleated cell stimulation with interferon-γ, interleukin-2, and anti-CD3 antibody, are constituted by CD3(+) CD56(+) (CIK) cells and a minority of natural killer (NK; CD3(-) CD56(+) ) cells and T-lymphocytes (CD3(+) CD56(-) ) with antitumor effect against hematological malignancies, thus representing a promising immunotherapy strategy. To ensure in vivo antitumor activity it is mandatory to maximize the percentage of CD3(+) 56(+) effector cells, which is highly variable depending on the starting sample and the harvesting day. Based on cytofluorimetric data, we have retrospectively applied multivariate statistical data analysis (MVDA) to 30 expansions building mathematical models able to predict the expansion fate and the optimal CIK harvesting day. METHODS: Cell phenotype was monitored during culture; multivariate batch statistical process control was applied to monitor cell expansion and orthogonal projections to latent structures to predict CIK percentage. RESULTS: Ten expansions had CD3(+) CD56(+) cells ≥ 40% (good batches) and 20 had CD3(+) CD56(+) cells ≤ 40%. In 36.7%, CD3(+) CD56(+) cells reached the highest concentration at day 17 and the others at day 21. We built a highly predictive regression model for estimating CD3(+) CD56(+) cells during culture. Three variables resulted highly informative: NK % at day 0, cytotoxic T-lymphocytes % (CTLs, CD3(+) CD8(+) ) at day 4, and CIK % at day 7. "Good batches" are characterized by a high percentage of CTLs and CD3(+) CD56(+) cells at day 4 and day 7, respectively. CONCLUSION: By applying MVDA it is possible to optimize CIK expansion, deciding the optimal cell harvesting day. A predictive role for CTL and CIK was evidenced.

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

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.

A study on mutual interaction between cytokine induced killer cells and umbilical cord-derived mesenchymal cells: Implication for their in-vivo use.

Recently a number of cellular therapy based-clinical trials have been carried out using mesenchymal stromal cells (MSC) or cytokine-induced-killer (CIK) cells aiming to improve outcome of allogeneic hematopoietic stem cell transplantation. We have isolated MSC from umbilical cord (UC) exploring the interaction between CIK cells and UC-MSC. We found that UC-MSC could suppress CIK cells activity, when co-cultured in a cell-to-cell system. In addition, CIK cells could potentially lyse UC-MSC in a time and ratio dependent manner that could have implications for their in vivo use. Here we provide experimental data on the mutual interaction of CIK cells and UC-MSC, suggesting a negative interference when the two cell types are used in combination. In the light of our observations, when CIK and UC-MSC will be used in clinical trials, timing and sequencing of their infusion should be considered.