Recovery Of Mononuclear Cells Research Articles

Establishing a method for the cryopreservation of viable peripheral blood mononuclear cells in the International Space Station

The analysis of cells frozen within the International Space Station (ISS) will provide crucial insights into the impact of the space environment on cellular functions and properties. The objective of this study was to develop a method for cryopreserving blood cells under the specific constraints of the ISS. In a ground experiment, mouse blood was directly mixed with a cryoprotectant and gradually frozen at −80 °C. Thawing the frozen blood sample resulted in the successful recovery of viable mononuclear cells when using a mixed solution of dimethylsulfoxide and hydroxyethyl starch as a cryoprotectant. In addition, we developed new freezing cases to minimize storage space utilization within the ISS freezer. Finally, we confirmed the recovery of major mononuclear immune cell subsets from the cryopreserved blood cells through a high dimensional analysis of flow cytometric data using 13 cell surface markers. Consequently, this ground study lays the foundation for the cryopreservation of viable blood cells on the ISS, enabling their analysis upon return to Earth. The application of this method in ISS studies will contribute to understanding the impact of space environments on human cells. Moreover, this method may find application in the cryopreservation of blood cells in situations where research facilities are inadequate.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Optimizing a fully automated and closed system process for red blood cell reduction of human bone marrow products

Background aimsHematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process. MethodsThe authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors’ center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This “bone marrow-like” product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs. ResultsRBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85–96.39%) in the 10×, 98.80% (range of 98.1–99.5%) in the 12× and 98.89% (range of 98.80–98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0–93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9–62.35%) in the 10×, 14.56% (range of 14.34–14.78%) in the 12× and 27.97% (range of 24.7–31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17–76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0–84.36%) and WBC recovery of 43.37% (range of 37.48–49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process. ConclusionsThe fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from “bone marrow-like” products and a supplemented bone marrow product using a 10× reduction rate.

Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42–0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32–35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6–56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study

IntroductionThe isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. ObjectiveThe aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). MethodFor cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). ResultsThe yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. ConclusionAccording to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

ABO-mismatched marrow processing for transplantation: Comparative results of 80 procedures performed with Cobe Spectra and Spectra Optia

Backgroundremoval of incompatible red blood cells (RBCs) or plasma is usually required to avoid hemolysis during infusion of ABO incompatible bone marrow (BM) allogeneic transplants. This process often involves separation of buffy coat (BC) by centrifugation in automated devices. We have evaluated the Spectra Optia™ (Optia) apheresis system to determine its effectiveness in BC concentration, volume reduction and RBCs depletion of ABO-incompatible BM compared with our previous method using Cobe Spectra™ (Cobe). Materials and methods28 processes were performed with Optia and 52 with Cobe. We compared volume reduction, RBCs depletion, and recovery of total nucleated cells (TNCs), mononuclear cells (MNCs), CD34+ and CD3+ cells in the final product. Hematopoietic engraftment was ascertained. We used Saphiro-Wilks and Kolmorgorov- Smirnov tests to test normality and Mann-Whitney’s U test to compare means between both groups. ResultsWe found statistically significant differences favoring Optia versus Cobe in TNCs recovery (62% vs. 37%), CD34+ cell recovery (98 vs 84%), volume reduction (91 vs 84%), and RBCs depletion (99 vs. 97%), but not in processing time or time to engraftment. ConclusionOptia achieves high RBCs and volume depletion of BM, while providing excellent CD34+ recovery in clinical routine. Some parameters compare favorably with Cobe Spectra.

Separation and Lysing Agents Effect Peripheral Blood Mononuclear Cell Recovery Pre-and Post-Cryopreservation

Separation and Lysing Agents Effect Peripheral Blood Mononuclear Cell Recovery Pre-and Post-Cryopreservation

Collection, processing and freezing of equine bone marrow cells

There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3–24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentration on the prevention of cell aggregation, and cell viability after freezing in liquid nitrogen. Bone marrow aspirations were done with syringes pre-filled with Iscove's modified Dulbecco's medium and different concentrations of sodium heparin. BMMCs were counted, cell viability was determined, and samples were frozen. Bone marrow collection from the sternum is safe, even at large volumes and from horses of advanced age, and the number of cells recovered decreases with successive aspirations (p < 0.0001). Heparin concentration influenced cell aggregation, and recovered cells continued to be commercially viable after 150 days in frozen storage.

Centrifugal microfluidics for sorting immune cells from whole blood

Sorting and enumeration of immune cells from blood are critical operations involved in many clinical applications. Conventional methods for sorting and counting immune cells from blood, such as flow cytometry and hemocytometers, are tedious, inaccurate, and difficult for implementation for point-of-care (POC) testing. Herein we developed a microscale centrifugal technology termed Centrifugal Microfluidic Chip (CMC) capable of sorting immune cells from blood and in situ cellular analysis in a laboratory setting. Operation of the CMC entailed a blood specimen layered on a density gradient medium and centrifuged in microfluidic channels where immune cell subpopulations could rapidly be sorted into distinct layers according to their density differentials. We systematically studied effects of different blocking molecules for surface passivation of the CMC. We further demonstrated the applicability of CMCs for rapid separation of minimally processed human whole blood without affecting immune cell viability. Multi-color imaging and analysis of immune cell distributions and enrichment such as recovery and purity rates of peripheral blood mononuclear cells (PBMCs) were demonstrated using CMCs. Given its design and operation simplicity, portability, blood cell sorting efficiency, and in situ cellular analysis capability, the CMC holds promise for blood-based diagnosis and disease monitoring in POC applications.

Cord blood collection and processing with hydroxyethyl starch or non–hydroxyethyl starch

BackgroundCollection and processing characteristics influencing quality of cord blood (CB) units play an essential role to cord blood banks (CBBs). At many CBBs, volume reduction is performed using hydroxyethyl starch (HES) and the Sepax (Biosafe) automated cell processing system. Due to the withdrawal of HES from the European market, a validation of the nonHES protocol was performed. MethodsThis partially retrospective study identified CB characteristics such as gestational age and CB volume/cell count correlated with higher quality. For the nonHES validation, CB was analyzed for total nucleated cell (TNC), mononuclear cell (MNC) recovery, hematocrit (HCT) and colony-forming units (CFUs). Viabilities of CD34+ and CD45+ cells were determined by 7-aminoactinomycin D (7-AAD) and AnnexinV (AnnV) staining and compared for 21 mL and 42 mL buffy coat (BC) samples applying the HES/nonHES protocol. ResultsFactors affecting the potency of CB transplants were the gestational age and the volume reduction to a defined BC volume. High initial cell counts and CB volumes correlated negatively with post-processing TNC recovery for lower BC volumes. Post-processing HES and nonHES results were comparable, but nonHES revealed a significantly lower post-thaw recovery of viable CD34+ cells measured by 7-AAD/AnnV (21 mL: 45.4 ± 16.4%; 42 mL: 67.3 ± 14.5%) as compared with HES (21 mL: 72.7 ± 14.4%, P = 0.0164; 42 mL: 83.4 ± 14.7%, P = 0.0203). DiscussionDue to the lower post-thaw CD34+ cell viability (AnnV−/7-AAD−) for nonHES samples, the use of HES is recommended, ideally combined with a high BC volume. The post-processing HCT has no statistically significant impact on the post-thaw CD34+ cell viability (AnnV−/7-AAD−).

Equine bone marrow volume reduction, red blood cell depletion, and mononuclear cell recovery using the PrepaCyte-CB processing system.

Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture. The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens. One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduction, and total nucleated cell (TNC) and MNC recoveries were determined. Bone marrow volume was reduced from 120 mL to 21 mL with a median RBC depletion of 90.1% (range, 62.0-96.7%). The median preprocessing total TNC count was 2.2 × 10(9) (range, 0.46-7.9 × 10(9)) and the median postprocessing TNC count was 1.7 × 10(9) (range, 0.3-4.4 × 10(9); P < .0001), with a median recovery of 73.5% (range, 22.4-216.7%). The median preprocessing total MNC count was 0.9 × 10(9) (range, 0.1-4.7 × 10(9)) and median postprocessing total MNC count was 0.8 × 10(9) (range, 0.1-2.7 × 10(9); P = .06), with a median recovery of 83.7% (range, 15.4-413.9%). The PrepaCyte-CB processing system can be used to deplete both volume and RBC, and recover MNC from equine BM specimens. Further studies assessing the viability of MSC and the efficacy of MSC expansion after using the PrepaCyte-CB processing system are warranted.

C/EBPδ deficiency sensitizes mice to ionizing radiation-induced hematopoietic and intestinal injury.

Knowledge of the mechanisms involved in the radiation response is critical for developing interventions to mitigate radiation-induced injury to normal tissues. Exposure to radiation leads to increased oxidative stress, DNA-damage, genomic instability and inflammation. The transcription factor CCAAT/enhancer binding protein delta (Cebpd; C/EBPδ is implicated in regulation of these same processes, but its role in radiation response is not known. We investigated the role of C/EBPδ in radiation-induced hematopoietic and intestinal injury using a Cebpd knockout mouse model. Cebpd−/− mice showed increased lethality at 7.4 and 8.5 Gy total-body irradiation (TBI), compared to Cebpd+/+ mice. Two weeks after a 6 Gy dose of TBI, Cebpd−/− mice showed decreased recovery of white blood cells, neutrophils, platelets, myeloid cells and bone marrow mononuclear cells, decreased colony-forming ability of bone marrow progenitor cells, and increased apoptosis of hematopoietic progenitor and stem cells compared to Cebpd+/+ controls. Cebpd−/− mice exhibited a significant dose-dependent decrease in intestinal crypt survival and in plasma citrulline levels compared to Cebpd+/+ mice after exposure to radiation. This was accompanied by significantly decreased expression of γ-H2AX in Cebpd−/− intestinal crypts and villi at 1 h post-TBI, increased mitotic index at 24 h post-TBI, and increase in apoptosis in intestinal crypts and stromal cells of Cebpd−/− compared to Cebpd+/+ mice at 4 h post-irradiation. This study uncovers a novel biological function for C/EBPδ in promoting the response to radiation-induced DNA-damage and in protecting hematopoietic and intestinal tissues from radiation-induced injury.

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device

Background aimsThe CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ+) and CD19 positive (CD19+) cells from apheresis products. MethodsInvestigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2–10.4) × 1010 mononuclear cells were used with a median 358 (92–1432) × 106 CD34+ cells. There were 24.8% (6.1–45.7%) median TCRαβ+ cells and 4.4% (1.2–11.7%) median B cells in the apheresis product. ResultsAfter depletion, a median 0.00097% (0.00025–0.0048%) of TCRαβ+ cells could be detected, and B cells, as determined as CD20+ cells, were reduced to 0.0072% (0.0008–0.072%). TCRαβ+ cells were depleted by log 4.7 (3.8–5.5), and B cells were depleted by log 4.1 (3.0–4.7). Recovery of mononuclear cells was 55% (33–77%), and recovery of CD34+ cells was 73% (43–98%). Recovery of CD56+/3− natural killer cells was 80% (35–142%), recovery of TCR gamma/delta positive (TCRγδ+) T cells was 83% (39–173%) and recovery of CD14+ cells was 79% (22–141%). Viability of cells was 98% (93–99%) after separation (all values median). ConclusionsProfound depletion of TCRαβ+ T cells can be achieved with the CliniMACS system. Recovery of CD34+ stem cells is in the same range than after CD34+ enrichment and CD3/CD19 depletion. Transplantation with >4 × 106 CD34+ cells/kg can be performed for every patient with 1–5 × 104 TCRαβ+ cells/kg and about 5–10 × 106 TCRγδ+ cells/kg with two rounds of apheresis.

Methods of freezing cord blood hematopoietic stem cells

Cord blood (CB) is a valuable source of hematopoietic stem cells (HSCs). Extended storage of CB is possible provided that validated cryopreservation procedures are used. The study objective was to determine optimal methods of CB cryopreservation. In the study we 1) compared the effect of two-step cryopreservation and controlled-rate freezing method on the postthaw quality of CB (Study A) and 2) evaluated the postthaw quality of HSC fractions isolated from CB with various methods and frozen with controlled-rate freezing method (Study B). The same cryoprotectant mixture was used for 20 CB units (Study A) and 122 CB units (Study B). In Study A, 13.79 × 10(8) and 13.29 × 10(8) initial white blood cell (WBC) counts decreased to 6.38 × 10(8) and 6.02 × 10(8) after thaw for the two methods, respectively. The mononuclear cell (MNC) counts decreased from 5.90 × 10(8) to 3.71 × 10(8) and from 5.64 × 10(8) to 3.47 × 10(8) dependent on the method. MNC viability decreased from 99.0% to 97.4% for the former and from 98.5% to 97.2% for the latter method. The differences were insignificant. In Study B, postthaw WBC recovery in HSC fractions was 74.4% to 103.5%, MNC recovery 106.4% to 118.5%, CD34+ cell recovery 102.5% to 150.2%, and MNC viability 94.1% to 97.4%. Neither the cryopreservation procedure nor the freezing of isolated HSCs affected product quality, which may indicate that various freezing methods can be used for cell banking provided the they follow recommendations of good manufacturing practice and Directive 2004/33/EC.

Qualitative and quantitative cell recovery in umbilical cord blood processed by two automated devices in routine cord blood banking: a comparative study.

Volume reduction is a widely used procedure in umbilical cord blood banking. It concentrates progenitor cells by reducing plasma and red blood cells, thereby optimising the use of storage space. Sepax and AXP are automated systems specifically developed for umbilical cord blood processing. These systems basically consist of a bag processing set into which cord blood is transferred and a device that automatically separates the different components during centrifugation. The aim of this study was to analyse and compare cell recovery of umbilical cord blood units processed with Sepax and AXP at Valencia Cord Blood Bank. Cell counts were performed before and after volume reduction with AXP and Sepax. When analysing all the data (n =1,000 for AXP and n= 670 for Sepax), the percentages of total nucleated cell recovery and red blood cell depletion were 76.76 ± 7.51% and 88.28 ± 5.62%, respectively, for AXP and 78.81 ± 7.25% and 88.32 ± 7.94%, respectively, for Sepax (P <0.005 for both variables). CD34(+) cell recovery and viability in umbilical cord blood units were similar with both devices. Mononuclear cell recovery was significantly higher when the Sepax system was used. Both the Sepax and AXP automated systems achieve acceptable total nucleated cell recovery and good CD34(+) cell recovery after volume reduction of umbilical cord blood units and maintain cell viability. It should be noted that total nucleated cell recovery is significantly better with the Sepax system. Both systems deplete red blood cells efficiently, especially AXP which works without hydroxyethyl starch.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery

The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.

Standardized Serum-Free Cryomedia Maintain Peripheral Blood Mononuclear Cell Viability, Recovery, and Antigen-Specific T-Cell Response Compared to Fetal Calf Serum-Based Medium

The ability to analyze cryopreserved peripheral blood mononuclear cells (PBMCs) from biobanks for antigen-specific T-cell immunity is necessary to evaluate responses to immune-based therapies. Comprehensive studies have demonstrated that the quality of frozen PBMCs is critical and the maintenance of cell viability and functionality by using appropriate cryopreservation techniques is a key to the successful outcome of assays using PBMCs. Different cryomedia additives affect cell viability. The most common additive is fetal calf serum (FCS), although it is widely known that each FCS lot has to be tested before usage to prevent nonspecific stimulation of T-cells. Also, shipping of samples containing FCS is critical because of many import restrictions. Often, dimethyl sulfoxide (DMSO) is added as a cryoprotectant. However, DMSO concentration has to be reduced significantly because of its toxic effect on cells at room temperature. Therefore, we have developed freezing approaches to minimize cytotoxicity of cryoprotectants and maintain T-cell functionality. We compared different additives to the widely used FCS and found bovine serum albumin fraction V to be an appropriate substitute for the potentially immune-modulating FCS. We also found that DMSO concentration can be reduced by the addition of hydroxyethyl starch. Using our serum-free cryomedia, the PBMC recovery was more than 83% and the PBMC viability was more than 98%. Also, the T-cell functionality measured by enzyme-linked immunospot (ELISpot) was optimal after cryopreservation with our new cryomedia. On the basis of our experimental results, we could finally design 2 different, fully working cryomedia that are standardized, serum free, and manufactured under GMP conditions.

Optimization of the Isolation, Culture, and Characterization of Equine Umbilical Cord Blood Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.