Sepax S-100 Research Articles

Autologous bone marrow-derived nucleated cell (aBMNC) transplantation improves endometrial function in patients with refractory Asherman's syndrome or with thin and dysfunctional endometrium.

The purpose was to evaluate the effect of intrauterine injection of aBMNC on the endometrial function in patients with refractory Asherman's syndrome (AS) and/or thin and dysfunctional endometrium (TE). This is a prospective, experimental, non-controlled study MATERIAL AND METHODS: The study was carried out between December 2018 and December 2020 on 20 patients, who were of age < 45 years and had oligo/amenorrhea and primary infertility due to refractory AS and/or TE. One hundred ml BM was extracted. aBMNC cells were separated according to generic volume reduction protocol by using the Cell Separation System SEPAX S-100 table top centrifuge system. We have evaluated CD34+, mononuclear cell (MNC), and total nucleated cell (TNC) counts. The transplantation aBMNC was performed by two intrauterine injections at an interval of one week, transvaginally into the endometrial-myometrial junction by an ovum aspiration needle. Midcyclic endometrial thickness (ET) and gestations after transplantation were evaluated. The mean TNC, MNC, and CD34+ cells were 11.55 ± 4.7 × 108, 3.85 ± 2.01 × 108, and 7.00 ± 2.88 × 106 at first injection, respectively, and 6.85 ± 2.67 × 108, 2.04 ± 1.11 × 108, and 3.44 ± 1.31 × 106 at second injection, respectively. The maximum posttransplantation ET was significantly higher than the maximum pretransplantation ET: 2.97 ± 0.48 vs. 5.76 ± 1.19 (mean ± standard deviation, p < 0.01). Twelve patients had frozen-thaw embryo transfers after the study. In 42% (n = 5 of 12) of the patients, pregnancy was achieved. One of the five patients delivered a healthy baby at term. Autologous BMNC transplantation may contribute to endometrial function in patients with AS and/or TE.

P1338: INFUSION OF DIMETHYL SULFOXIDE (DMSO) - DEPLETED AUTOLOGOUS PERIPHERAL BLOOD PROGENITOR CELLS REDUCES POST-AUTOGRAFTING SEVERE GASTRIC TOXICITIES AND HOSPITALIZATION LENGTH

Background: High-dose chemotherapy (HD CHT) and autologous blood stem cell transplantation (ABSCT) are the cornerstone in the treatment of several hematologic malignancies. Cryopreservation of stem cells needs the use of DMSO as cryoprotectant. Unfortunately, DMSO may induce some severe adverse effects during and after autograft infusion, justifying the attempts to deplete DMSO. Despite several studies have demonstrated that DMSO depletion did not affect engraftment of peripheral blood progenitor cell (PBPC), there is still few and inconclusive data on its impact on post-infusion toxicities. Aims: Since from March 2021 we started to remove DMSO, we are comparing infusion and post-infusion outcome in 55 patients consecutively autografted with DMSO depleted (25 pts; group1) or undepleted (30 pts; group 2) at the hematology unit of the S.M. Goretti Hospital of Latina (Italy) from January 2019 and December 2021, aiming to evaluate whether post-infusion toxicities and clinical outcome may differ between the two groups. Methods: The DMSO was removed by rinsing the units with the Sepax S-100 (Biosafe S.A. Eysins, Switzerland) cellular centrifugation instrument that automatically processes blood or haematic components in a closed and sterile environment followed by the PBSC washing, using the dedicated CS-600 kit. The clinical characteristics, biological parameters of autografted PBPC and post-infusion outcome of the 55 patients entered this study are reported in Table 1. In group 1, 12 patients had a diagnosis of multiple myeloma (MM) and 13 of non Hodgkin’s lymphoma (NHL), whereas in group 2, 17 patients were affected by MM, 6 by Hodgkin’s lymphoma (HL) and 7 by NHL. Results: As reported in Table 1, the amount of harvested and infused CD34+ cells were similar in the two groups. To evaluate viability in group 1 we used a different ISHAGE protocol with lower thresholds. This consideration explains the lower viability rates observed in group 1 respect to those of group 2. No differences between the two groups were observed in the average time to neutrophil (N≥0.5 x 109/L) and platelet (PLTs ≥20x 109/L) recovery (10.4 vs 10 days and 12.5 vs 11.7 days, respectively), as well as in the number of transfused RBCs and PLTs units. No grade 3-4 infusion reactions were observed in any case. By contrast, as concern the post-infusion adverse events, in group 2 we observed a significant higher incidence of grade 3 mucositis (37% in group 2 vs 4% in group 1, p=0.003) and of episodes of nausea associated with vomiting (40% in group 2 vs 8% in group 1, p=0.007). Occurrence of febrile neutropenia, sepsis and other documented infections were similar between the two groups. Overall, the average hospitalization length after the infusion was 17 days (median=16) for group 2 and 14 days (median=13) for group 1 (p=0.059). Image:Summary/Conclusion: Present findings for the first time show a significant lower incidence of severe post infusional gastrointestinal adverse effect in patients receiving depleted DMSO autograft that may result in a reduced hospitalization length. As reported in animal model, this finding may suggest a direct gastrotoxicity of DMSO, that can potentiate that of chemotherapy. Whether these data may be confirmed in future larger studies they further suggest the usefulness of DMSO depletion from PBPC autografts.

Isolation of nucleated cells fraction from umbilical cord blood – choice of method

The comparison of different techniques for cord blood processing to ensure the highest cell yield based on the “cost-effectiveness” ratio is important.A study of the effectiveness of nucleated cells fraction isolation from umbilical cord blood samples (n = 2898) using double centrifugation, automatic (Sepax S100, Biosafe) or semi-automatic (MacoPress Smart, MacoPharma) system was performed. These include the determining of nucleated cells number (per ml and total), the number of CD34+ cells, and cell viability before samples treatment, after treatment and before freezing.It was shown that semi-automatic and automatic processing methods were more efficient then the double centrifugation by key quality indicators of cell concentrates: the total number of nucleated cells and nucleated cells output. The semi-automatic method of nucleated cells fraction isolation requires the least time for processing a single sample.Considering these data such a system has been proposed as optimal for daily receipt and processing of cord blood samples in a cord blood bank.

Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion

Transplantation of hematopoietic stem cells (HSCs) has been successfully developed as a part of treatment protocols for a large number of clinical indications, and cryopreservation of both autologous and allogeneic sources of HSC grafts is increasingly being used to facilitate logistical challenges in coordinating the collection, processing, preparation, quality control testing and release of the final HSC product with delivery to the patient. Direct infusion of cryopreserved cell products into patients has been associated with the development of adverse reactions, ranging from relatively mild symptoms to much more serious, life-threatening complications, including allergic/gastrointestinal/cardiovascular/neurological complications, renal/hepatic dysfunctions, and so on. In many cases, the cryoprotective agent (CPA) used-which is typically dimethyl sulfoxide (DMSO)-is believed to be the main causal agent of these adverse reactions and thus many studies recommend depletion of DMSO before cell infusion. In this paper, we will briefly review the history of HSC cryopreservation, the side effects reported after transplantation, along with advances in strategies for reducing the adverse reactions, including methods and devices for removal of DMSO. Strategies to minimize adverse effects include medication before and after transplantation, optimizing the infusion procedure, reducing the DMSO concentration or using alternative CPAs for cryopreservation and removing DMSO before infusion. For DMSO removal, besides the traditional and widely applied method of centrifugation, new approaches have been explored in the past decade, such as filtration by spinning membrane, stepwise dilution-centrifugation using rotating syringe, diffusion-based DMSO extraction in microfluidic channels, dialysis and dilution-filtration through hollow-fiber dialyzers and some instruments (CytoMate, Sepax S-100, Cobe 2991, microfluidic channels, dilution-filtration system, etc.) as well. However, challenges still remain: development of the optimal (fast, safe, simple, automated, controllable, effective and low cost) methods and devices for CPA removal with minimum cell loss and damage remains an unfilled need.

A Randomised, Placebo-Controlled, First-In-Human Study of a Novel Clade C Therapeutic Peptide Vaccine Administered Ex Vivo to Autologous White Blood Cells in HIV Infected Individuals

BackgroundPreclinical studies of overlapping 15mer peptides, spanning SIV, SHIV or HIV, pulsed on autologous PBMC ex vivo have demonstrated high level, virus-specific T cell responses and viral suppression in non-human primates (NHP). Opal-HIV-Gag(c) consists of 120 synthetic 15mer peptides spanning Clade C, consensus Gag, manufactured to current good manufacturing practice; having been evaluated in a good laboratory practice toxicology study in Macaca mulatta. We evaluated the safety and preliminary immunogenicity of such peptides administered intravenously after short-duration ex vivo incubation, to HIV-positive adults on suppressive antiretroviral therapy.Methods and FindingsA first-in-human, placebo-controlled, double-blind, dose escalation study was conducted. Twenty-three patients with virus suppressed by antiretroviral therapy were enrolled in four groups 12 mg (n = 6), 24 mg (n = 6), 48 mg (n = 2) or matching placebo (n = 8). Treatment was administered intravenously after bedside enrichment of 120 mL whole blood for white cells using a closed system (Sepax S-100 device), with ex vivo peptide admixture (or diluent alone) and 37°C incubation for one hour prior to reinfusion. Patients received 4 administrations at monthly intervals followed by a 12-week observation post-treatment. Opal-HIV-Gag(c) was reasonably tolerated at doses of 12 and 24 mg. There was an increased incidence of temporally associated pyrexia, chills, and transient/self-limiting lymphopenia in Opal-HIV-Gag(c) recipients compared to placebo. The study was terminated early, after two patients were recruited to the 48 mg cohort; a serious adverse event of hypotension, tachycardia secondary to diarrhoea occurred following a single product administration. An infectious cause for the event could not be identified, leaving the possibility of immunologically mediated product reaction.ConclusionsA serious, potentially life-threatening event of hypotension led to early, precautionary termination of the study. In the absence of a clearly defined mechanism or ability to predict such occurrence, further development of Opal-HIV-Gag(c) will not be undertaken in the current form.RegistrationClinicalTrials.gov NCT01123915; EudraCT: 2008-005142-23

Optimization of the immunomagnetic selection in microcythemic donors enrolled for haploidentical transplantation

BackgroundImmunomagnetic cell selection (ICS) cells is increasingly used in allogeneic hematopoietic transplantation in order to reduce the T cells quantity. The aim of this study was to evaluate an protocol based on Ficoll method before ICS. Study design and methodsThe automated procedure was compared with the standard method. In the group 1 the cell processing involves the extraction of the buffy-coat by Ficoll before incubation with antibodies. This procedure was performed with the Sepax S-100 device. The efficacy of this automated procedure was compared with the group 2. In this group, the cell washing and the incubation were performed through the standard method. The CD34+ cells collected by apheresis (HPC-A) were selected with ICS. ResultsThe results obtained after Ficoll procedure, showed a total nucleated cells (TNCs) and CD34+ cells recovery of 85.73% (75.90–90.63; SD 4.25) and 79.31% (51.77–112.31; SD 18.40), respectively. The TNC and CD34+ cells recovery after the pre-incubation washing performed through the standard method, was 75.54% (38.36–97.76; SD 22.5) and 61.51% (30.87–81.79; SD 19.3), respectively. The CD34+ cells recovery after ICS was 79% (51.77–100; SD 18.40) and 44% (15.57–88.24; SD 25.91) in the group 1 and the group 2, respectively. This difference was statistically significant (p=0.001). ConclusionThe efficacy of the ICS which resulted to be higher in the group 1 compared to the group 2. Overall, our data suggest that the Ficoll procedure before incubation is suitable for the clinical routine in the ICS for haploidentical transplantation in patients affected by thalassemia.

A first-in-man, double blind, placebo controlled study of the candidate therapeutic vaccine Opal-HIV-Gag(c) in HIV infected patients receiving HAART

Methods Synthetic 15mer peptides (n=123, Opal-HIV-Gag(c)) spanning Clade C, consensus Gag were manufactured to current good manufacturing practice and evaluated in a good laboratory practice toxicology study in Macaca mulatta. A first-in-human, single centre, placebo-controlled, doubleblind, dose escalation study was conducted. Twenty three people with well controlled HIV (CD4+ > 350cells/mm3 and a HIV < 400 copies/mL), stratified by clade, were enrolled in four groups: 12mg (n=6), 24mg (n=7), 48mg (n=2) or matching placebo (n=8). Treatment was administered intravenously bedside (closed system) by enrichment of 120mL of whole blood for WBCs using a Sepax S-100 device, ex vivo mixing the peptides (or diluent alone) and incubation at 37°C for one hour prior to reinfusion. Subjects received 4 administrations at 4 weekly intervals followed by a 12 week post-treatment follow up. Immunogencity was assessed by ELIspot.

Cell processing for haplo-identical hematopoietic stem cell transplantation: automated washing and immunomagnetic-positive selection

Immunomagnetic cell selection (ICS) of CD34(+) cells is being used increasingly in allogeneic transplantation in order to reduce T-cell quantity. The aim of this study was to evaluate an automated washing protocol before immunomagnetic selection. The automated method was compared with a conventional washing procedure. In the study group the cell processing using the automated procedure, both before and after antibody incubation, was performed with a Sepax S-100 device. The efficacy of the automated procedure was compared with the control group, where washing were performed using a standard method. The results obtained after pre-incubation washing performed using the automated system showed a total nucleated cell (NC) and CD34(+) cell recovery of 84.87% (71.80-105, SD 8.62; range, standard deviation) and 83.45% (47-109, SD 16.12), respectively. The NC and CD34(+) cell recovery after the pre-incubation washing cycle was performed using the standard method was 75.54% (38.36-97.76, SD 22.5) and 61.51% (30.87-81.79, SD 19.3), respectively. The CD34(+) cell recovery after ICS was 51.27% (13.77-98.82, SD 24.97) and 48.89% (15.57-88.24, SD 25.91) for group 1 and group 2, respectively. The average purity in group 1 was 86.46% (67.4-96.10, SD 13.07) and in group 2 84.97% (58.1-97.8, SD 15.58). The efficacy of the ICS led to an optimal purity without affecting cell recovery, which was higher in group 1. Overall, our data suggest that the automated method is suitable for washing hematopoietic progenitor cell apheresis (HPC-A) concentrates before immunomagnetic cell selection in daily clinical routines.

An automatic wash method for dimethyl sulfoxide removal in autologous hematopoietic stem cell transplantation decreases the adverse effects related to infusion

Products cryopreserved with dimethyl sulfoxide (DMSO) in stem cell transplant (SCT) often cause many adverse effects during their infusion (major cardiovascular events, dyspnea … even death). These are especially frequent in pediatric patients. We tested if a fully automated and closed wash procedure (Sepax S-100, Biosafe) allowed us to maintain the absolute CD34+ cell number, cell viability, and engraftment potential, decreasing the untoward reactions. Forty-six washes of DMSO cryopreserved peripheral blood hematopoietic progenitor (HP) apheresis were studied. Blood aliquots were taken both after thawing and after washing to assess the total nucleated and CD34+ cell counts, as well as cell viability. The washed products were infused in 26 autologous SCTs (ASCTs). Results were compared with the 53 previous SCTs performed without DMSO removal. After washing there were no significant differences between the pre- and postwashing CD34+ cell counts (p=0.08) or viability (p=0.68). No significant differences were observed between washed and nonwashed infusions in relation to the day of the neutrophil (p=0.46) and platelet (p=0.26) engraftment. One adverse event, abdominal pain, occurred during the washed cells infusions. When compared with the 14 untoward reactions that took place during the nonwashed HP infusions, significance was reached (p=0.00043). The automatic method described is effective in terms of CD34+ cell recovery and viability in ASCT. Moreover, Sepax decreased significantly the untoward reactions during the infusion.

Automated washing of human progenitor cells: evaluation of apoptosis and cell necrosis

High-dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. The aim of this study is to evaluate the efficacy of the Sepax S-100 device to process thawed HPC-A products in comparison with manual procedure. We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery. The TNC and CD34+ cells recovery in the automatic procedure was of 91.9% (73-100; SD ± 12.60) and 86.7% (69-100; SD ± 10.21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84.7% (47-100; SD ± 22.9) and 80.29% (23-100; SD ± 25.96). The results, obtained from the assessment of viability of CD34+ both 7-AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system. Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC-A products and can be daily used in clinical routine.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system

Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.

Evaluation of An Automatic Washing Method for DMSO Cryopreserved Peripheral Blood Hematopoietic Progenitors Using the Sepax S-100.

Abstract 3219Poster Board III-156 IntroductionDimethylsulfoxide (DMSO) containing cryoprotective solutions are routinely used for the storage of hematopoietic progenitors (HP). At room temperature, DMSO is toxic for the cells and may produce severe adverse reactions during their infusion, especially in the pediatric patients. These problems can be avoided by washing the cells prior to the infusion. Our objective was to test if an automatic washing method (Sepax S-100, Biosafe) allowed us to preserve the CD34+ cell numbers with an adequate viability and engraftment potential. Material and MethodsForty five peripheral blood HP apheresis that have been cryopreserved using autologous plasma plus 9% DMSO were studied. After rapid thawing in a water bath at 37° C, an automatic wash with the Sepax S-100 (2 washes cycle) was performed. Nucleated cell levels determined by an hematology analyzer, flow cytometry CD34+ cell counts and Trypan Blue cell viability test were performed on aliquots collected prior to and after the washing technique. The paired Student's t-test and the Pearson's correlation coefficient were used for the statistical analysis. ResultsThe mean total nucleated cell (TNC) and CD34+ cell recovery was 75,47% ± 3, and 94,66% ± 4,62 respectively. In spite of the TNC significant loss (p<0,001), there were no significant differences between the pre and post-washing CD34+ cell numbers (p=0,08) or viability (p=0,19). The TNC loss had no correlation with the pre or post-washing number of platelets (p=0,18 and p=0,16 respectively), neither with the loss of platelets during the procedure (p=0,31). In contrast with the 40% of untoward reactions recorded in our historical data of 226 DMSO containing cell infusions, we observed just one adverse effect (2,22%) with the washed cells. One patient reported a transient mild abdominal pain with nausea and one vomit. Median time to neutrophil engraftment (>500 cells /mL) and platelet engraftment (>50.000 cells/mL) were 11 ± 0,2 and 20 ± 1,4 days respectively. ConclusionsThe Sepax S-100 automatic wash protocol of DMSO containing peripheral blood progenitor cells determines a good CD34+ cell recovery and preserves their viability and engraftment potential. This method avoids the DMSO infusion related adverse events and it constitutes a closed and easy to do procedure. DisclosuresNo relevant conflicts of interest to declare.

Separation of adult bone marrow mononuclear cells using the automated closed separation system Sepax

The Düsseldorf-based cardiologist Professor Strauer was the first to present a therapeutic concept for the repair of acute infarcted myocardium in 2001: the autologous intracoronary transplantation of unfractionated human bone marrow (BM) mononuclear cells (MNC). The Division of Cardiology, Pneumology and Angiology, University of Duesseldorf Medical School, Duesseldorf, Germany, was also able to show the regenerative potential of BM stem cell transplantation in patients with chronic heart disease (CHD) and peripheral arterial disease (PAD). In the mean time, several clinical trials have been set up worldwide, predominantly by using MNC isolated manually from BM aspirates via density-gradient centrifugation; 374 patients have been treated here with unselected BM MNC since 2001. Altogether 217 BM aspirates have been processed manually. In order to maintain the high standards required for cellular therapeutics, the Sepax cell-separation system was implemented into routine BM processing in 2006. The closed Sepax system provides a reproducible MNC isolation method, and 157 BM samples have been processed with the Sepax device. The results of manual MNC isolation were compared with the Sepax-mediated MNC isolation. The manual Ficoll separation method was compared with the Sepax density gradient-based separation (DGBS) protocol using Ficoll with the kit CS-900 and the Sepax S-100 main processing unit from Biosafe. Nucleated cell and MNC recovery were significantly higher after Sepax processing (P<0.0001) whereas no significance was found for red blood cell depletion. The Sepax cell-separation system is a time-saving method providing clinical-grade MNC isolated automatically from human BM by Ficoll density centrifugation.