Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing

Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4+ T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from na?ve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4+ T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. The cytokine treatment resulted in a significant inhibition (p<0.01) of apoptosis-induced cell death and rescued CD4+ T-cell survival (p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.     

Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis after 24-h culture

Studies performed with malaria patients living in endemic areas are frequently conducted in laboratories located hundreds of kilometer away from research centers, due to the difficulties in performing the assays in field conditions. Thus, we considered the potential indication of cryopreservation of peripheral blood mononuclear cells (PBMC), in most fieldwork, and decided to evaluate the effect of cryopreservation of PBMC on spontaneous apoptosis. The membrane integrity of PBMC was tested using three previously described protocols of cryopreservation. Cell samples were obtained from 19 healthy volunteers. Percentage of apoptotic nuclei in short-term PBMC cultures was determined by a sensitive method using 7-aminoactinomycin D followed by flow cytometry. Our results indicate that although cryopreservation can to some extent affect lymphocyte membrane integrity rates, flow cytometry analysis showed that frequencies of spontaneous apoptosis in cryopreserved cells were not significantly modified after 24-h culture. It is concluded that cryopreserved PBMC could be used for measuring spontaneous apoptosis and therefore, could be employed for the study of populations living in areas distant from research centers, allowing the comparative evaluation of samples obtained at different time.     

The cryopreservation of high concentrated PBMC for dendritic cell (DC)-based cancer immunotherapy

Peripheral blood mononuclear cells (PBMC) have been accepted as a unique material for cancer immunotherapy using dendritic cells (DC) or activated lymphocytes that are being developed as an alternative or adjuvant to conventional therapies such as surgery, chemotherapy and radiation treatment. Although successful cryopreservation of large numbers of PBMC is critical for the immunotherapy, subsequent functional study of the effects of PBMC cryopreservation on differentiation into immune cells has not been well defined. In this study, over 1.0 × 10⁸ cells/ml PBMC were cryopreserved as long as 52 weeks using a controlled-rate freezer (CRF) and stored in a vapor phase of liquid nitrogen tank. The effect of PBMC cryopreservation on differentiation into DC was studied by comparing the phenotypic and functional properties of immature DC (iDC) and mature DC (mDC) derived from cryopreserved PBMC to those from fresh PBMC. The results show that cryopreservation of PBMC at a fairly high cell concentration does not significantly affect cell recovery, viability, or phenotypes of PBMC. After differentiation into DC, iDC and mDC derived from cryopreserved PBMC had their typical phenotypes and function equivalent to those derived from fresh PBMC. Therefore, the improved cryopreservation process of PBMC described in this study is available for DC-based cancer immunotherapy.     

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (αCD3/αCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or αCD3/αCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or αCD3/αCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time.In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or αCD3/αCD28 as positive control.     

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD₆₅ or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD₆₅ induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.     

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD₆₅ or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD₆₅ induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.     

Effects of long-term cryopreservation on peripheral blood progenitor cells

Background aimsThe long-term stability of cryopreserved peripheral blood progenitor cells is an important issue for patients experiencing disease relapse. However, there is no consensus on how to evaluate the long-term effects of cryopreservation. We describe the effect of cryopreservation on viability and progenitor colony activity from 87 individual samples processed at the Scripps Green Hospital Stem Cell Processing Center (La Jolla, CA, USA).MethodsWe randomly selected 87 peripheral blood hematopoietic stem cell (PBHSC) samples from 60 patients and evaluated the effect of cryopreservation on sample viability and red and white cell colony activity after < 24 h and 7, 10 and 15 years of cryopreservation. Viability was assayed via trypan blue dye exclusion and activity was measured following 14 days of culture.ResultsAn age at collection older than 50 years may result in suboptimal activity and viability following long-term cryopreservation, while gender and disease status had no effect. Cryopreservation did not significantly affect white or red cell activity following 10 years of cryopreservation. However, for samples stored longer than 10 years, viability and activity significantly decreased. We noted a positive association between higher pre-cryopreservation %CD34 count and colony activity.ConclusionsCryopreservation of peripheral blood progenitor cells for up to 10 years results in no loss of clonogenic capacity, as determined by culture activity, although longer durations of storage may affect activity. Until validated methods are developed, cryopreserved grafts should be evaluated based on pre-freeze CD34⁺ cell counts as assayed by flow cytometry, and post-thaw sample evaluation should be reserved for patients identified as poor mobilizers.     

Optimal conditions for heart cell cryopreservation for transplantation

Cultured myocyte transplantation into an infarcted myocardium has been shown to improve contractile function. Cryopreservation of cultured muscle cells or heart tissue will be important for the technology to be practical. This study, using fetal cardiomyocytes, evaluated the optimal conditions for muscle cell cryopreservation. Study 1: Fetal rat cardiomyocytes were isolated and cultured. The freshly isolated and passage 1, 2, 3 and 4 cells were cryopreserved in a solution containing 70% IMDM, 20% FBS and 10% DMSO and stored in –196°C for 1, 2, 4, 8, 12 and 24 weeks. The cells were thawed and cultured. Cell number and contractility were evaluated at 0, 2, 4, 6, 8 and 10 days of culture. Study 2: Rat myocardium was cryopreserved in sizes of 0.2, 2 and 6 mm³ for 1 week. The tissue was thawed and cells were isolated. Cell growth and contractility were evaluated. (1) Cardiomyocytes grew and contracted after cryopreservation. Storage time did not affect cell survival rate, beating cell numbers and beating rates. Increasing cell passage prior to cryopreservation decreased the percentage of beating cells. (2) Cells isolated from cryopreserved tissue grew in vitro and contracted normally. Cell yield decreased with increased cryopreserved tissue size. Fetal rat cardiomyocytes survived and functioned after in vitro cryopreservation. Viable cells can be isolated from cryopreserved myocardium and cultured. Cryopreservation of small pieces of myocardium is preferred for maximal cell yields.     

Structure and functional alterations in lymphocytes induced by cryopreservation

Surface markers, Con A-induced capping, blastogenic transformation stimulated by PHA and allogeneic mononuclear cells, and natural killer activity of Ficoll — Hypaque-separated lymphocytes were studied before and after varying periods of cryopreservation. An increase was observed in the relative number of E rosetteforming cells and in the incorporation of [³H]thymidine into DNA, by the unstimulated cryopreserved cells after thawing. On the other hand, a substantial drop occurred in the Con A-induced capping and the natural killer activity of cryopreserved cells. The possible causes for the variation in the effects of cryopreservation on lymphocyte functions as reported by different investigators were discussed. It was concluded that until universally accepted, standardized procedures for the assessment of lymphocyte functions in vitro become available, each laboratory should establish the changes induced by cryopreservation in lymphocyte function with the methods employed locally to allow the observations made on cryopreserved lymphocytes to be meaningful.     

Evaluation of a clinical-grade,cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34⁺ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP).MethodscGMP VPA-mediated expansion was initiated with CD34⁺ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts.ResultsThe authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products.ConclusionsThe authors’ results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34⁺ cells for clinical utilization.     

Influence of cryopreservation on human periodontal ligament cells in vitro

Cryopreservation of teeth before autotransplantation may create new possibilities in dentistry. The purpose of this study was to examine the effect of a standardised cryopreservation procedure on human periodontal ligament (PDL) cell cultures. Human PDL fibroblasts obtained from immature third molars of 11 patients were cultured and divided into two groups. The experimental group was cryopreserved and cultured after thawing. The control group was cultured without cryopreservation. A comparison was made between cryopreserved and control cells. To evaluate possible differences in the characteristics of the fibroblasts, the cells in both groups were tested for viability (membrane integrity), growth capacity and alkaline phosphatase (ALP) expression. The Wilcoxon test for paired comparison between cryopreserved and non-cryopreserved cells was performed for each characteristic. The results showed that membrane integrity of cells was not influenced by cryopreservation. There was no statistically significant difference in growth capacity between cryopreserved and control cells. Non-cryopreserved cells were slightly stronger positive for ALP, but the difference was not statistically significant. From these experiments it can be concluded that the observed parameters are not influenced by cryopreservation.     

Viability,proliferation and phenotype maintenance in cryopreserved human iliac apophyseal chondrocytes

Cryopreservation preserves cells at low temperature and creates a reserve for future use while executing the clinical translation. Unlike articular chondrocyte, cryopreservation protocol and its outcome are not described in iliac apophyseal chondrocytes, a potential source of chondrocytes in cartilage engineering. This study for the first time describes the cryopreservation of human iliac apophyseal chondrocytes. Four cartilage samples were procured from iliac crests of children undergoing hip surgery after consent. The total chondrocyte yield was divided into two groups. First group was grown as monolayer while second group was cryopreserved following the slow cooling method in the medium containing 10 % Dimethyl sulfoxide for 3 months. Group two cells were also grown as a monolayer following thawing. Viability, time to confluence, population doubling time and phenotype maintenance were compared for both the groups. Viability was 65.75 % after 3 months of cryopreservation at ?196 °C, as compared to 94.19 % for fresh chondrocytes (p = 0.001). Fresh and cryopreserved cells reached confluence on 10th and 15th day of culture respectively. Population doubling time was significantly more in fresh than cryopreserved chondrocytes on 10th (p = 0.0006) and 15th day (p = 0.0002) in culture. Both fresh and cryopreserved cells maintain their chondrocyte phenotype as assessed by immunocytochemistry. Relative gene expression by real time polymerase chain reaction showed similar upregulation of mRNA of Collagen 2, SOX 9, Aggrecan and Collagen 1 in cryopreserved chondrocyte as compared to fresh chondrocyte. Iliac apophyseal chondrocytes cryopreserved for 3 months maintained the phenotype successfully 2 weeks after thawing in culture. The viability and proliferation rates after thawing were adequate for a clinical translation of these cells.     

Short-Term PTEN Inhibition Improves In Vitro Activation of Primordial Follicles,Preserves Follicular Viability,and Restores AMH Levels in Cryopreserved Ovarian Tissue From Cancer Patients

IntroductionIn vitro activation and growth of primordial dormant follicles to produce fertilizable oocytes would provide a useful instrument for fertility preservation. The employment of Phosphatase and TENsin homolog (PTEN) inhibitors, in combination with Protein kinase B (Akt) stimulating molecules, has been previously employed to increase follicular activation through the stimulation of the PTEN-Akt pathway.MethodsWe aim to establish improved in vitro activation also for cancer patients whose ovarian tissue has already been cryopreserved. Fresh and previously cryopreserved human ovarian cortex were exposed to short-term, low-concentration and ovary-specific treatment with only a PTEN inhibitor.ResultsOur in vitro activation protocol enhances the activation mechanisms of primordial follicles in both fresh and cryopreserved samples, and enlarges growing populations without inducing apoptosis in either follicles or the surrounding stroma. Treatment augments estradiol secretion and restores the expression levels of the previously diminished Anti-Müllerian hormone by means of cryopreservation procedures. Genomic modulation of the relative expression of PTEN pathway genes was found in treated samples.ConclusionThe in vitro activation protocol offers new alternatives for patients with cryopreserved tissue as it increases the pool of viable activated follicles available for in vitro growth procedures. The combination of ovarian tissue cryopreservation and in vitro activation of primordial follicles, the main ovarian reserve component, will be a major advancement in fertility preservation.     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Reduced dimethyl sulfoxide concentrations successfully cryopreserve human hematopoietic stem cells with multi-lineage long-term engraftment ability in mice

Background aimsThe cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured.MethodsCryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33⁺ myeloid, CD19⁺ B-lymphoid, CD235a⁺ erythroid and CD34⁺ progenitors; (ii) engraftment of the same four populations plus CD41⁺CD42b⁺ platelets; (iii) engraftment of CD34⁺⁺CD133⁺ cells; and (iv) engraftment of CD34⁺⁺CD38⁻ cells.ResultsHematopoietic colony-forming, CD34^++/+, CD34⁺⁺CD133⁺ and CD34⁺⁺CD38⁻ cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs.ConclusionCryopreservation of HSCs in DMSO concentrations as low as 5% is effective.     

Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)–T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.     

Cytotoxicity of human peripheral blood monocytes

Native tumoricidal activity of human peripheral blood mononuclear cells was examined before and after their separation by counterflow centrifugation elutriation (CCE). Tumoricidal activity was found in the subpopulation of small mononuclear cells but not within the relatively pure subpopulation of large monocytes. Addition of lymphokine and/or lipopolysaccharide demonstrated that large monocytes were resistant to activation for tumor killing, in contrast to small mononuclear cells. However, cryopreservation or simply exposure to dimethyl sulfoxide (DMSO) rendered the large monocytes sensitive to activating agents without altering their unstimulated tumoricidal activity. Cryopreservation was not detrimental to small or large monocytes either in number or tumoricidal function but did decrease the number of large granular lymphocytes (LGL). The small mononuclear cell fraction was enriched for small monocytes to 80% by combining CCE with Percoll gradient separation. HNK-1 mouse monoclonal antibody against human LGL was used with complement to remove virtually all LGL from cryopreserved cells as judged by morphology and tumoricidal activity against K-562 human lymphoblastoid cells. Such treatment actually augmented rather than suppressed tumoricidal activity against P-815 mastocytoma cells. Therefore, we conclude that small monocytes but not large monocytes possess native tumoricidal activity distinct from that attributed to LGL or natural killer lymphocytes. Further, small monocytes are readily activated for tumor killing and can be cryopreserved without loss of tumoricidal activity.     

Purification of circulating plasmacytoid dendritic cells using counterflow centrifugal elutriation and immunomagnetic beads

Background aimsPlasmacytoid dendritic cells (pDC) are a dendritic cell (DC) subset specialized in the production of high amounts of interferon (IFN) type I (IFN-α, -β) in response to viruses. They can be purified from peripheral blood mononuclear cells (PBMC), usually using magnetic bead sorting.MethodsIn this study, we set up a counterflow centrifugal elutriation (CCE) procedure to enrich pDC from PBMC. We first analyzed each CCE fraction for the presence of pDC using CD123 and BDCA-2 as markers. We then purified pDC using CCE and magnetic beads and verified that their functions were not affected by this procedure.ResultspDC were sorted by CCE into intermediate fractions between those containing lymphocytes and monocytes. The pDC frequency in these intermediate fractions was 3-fold that in PBMC. Using negative-magnetic bead sorting, starting with the same number of cells and beads, we obtained more than twice as many pDC from intermediate fractions as from PBMC. The phenotypes and IFN-α production capacities of sorted pDC from PBMC and from intermediate fractions were similar, both immediately after sorting and after stimulation with CpG-A oligodeoxynucleotides. In addition, we showed that intermediate fractions could be cryopreserved and that magnetic bead sorting could be performed with the same efficiency after thawing.ConclusionsAltogether, our results show that CCE can be used to enrich lymphocytes, monocytes and pDC from the same donor, without magnetic beads on their surface. Our method should be useful for the purification of these cells for experimental research and may also be adaptable for clinical use in immunotherapy.