Clinical experience with the delivery of thawed and washed autologous blood cells, with an automated closed fluid management device: CytoMate

BACKGROUND: Washing out of thawed autologous grafts, before reinfusion in poor‐prognosis cancer patients who undergo high‐dose chemotherapy, is desirable. The procedure allows for the reduction of infused dimethyl sulfoxide (DMSO) quantities and the performance of biologic controls on the infused cell product. STUDY DESIGN AND METHODS: Three‐hundred four patients were treated with intensified chemotherapy and autologous transplantation at a single institution. Fifty‐four of them received washed cell products, because three or more bags were to be reinfused. The recently available, closed, automated, and current good manufacturing practice–compliant device (CytoMate, Baxter Oncology) was used for this purpose. RESULTS: The performances of the device were similar to previously reported results, with greater than 75 percent CD34+ cell recovery. Neutrophil and platelet (PLT) recoveries were similar in the group of patients receiving washed cells and in the group of patients for whom cell products were extemporaneously thawed at the bedside. Adverse events that are typically reported after DMSO infusion were significantly less frequent and less severe in patients who received washed cells. Finally, the nurse staff on the transplant ward reported a decreased workload and more satisfactory procedure when infusing washed cell products. CONCLUSION: The CytoMate device allows for a significant reduction in DMSO infusion, with a diminished frequency and severity of immediate side effects and does not compromise neutrophil or PLT engraftment.     

Comparison of cord blood thawing methods on cell recovery, potency, and infusion

BACKGROUND: Umbilical cord blood (UCB) products have traditionally been thawed using a washing method intended to stabilize the cells, reduce dimethyl sulfoxide (DMSO) toxicity, and remove potentially ABO‐incompatible red blood cell (RBC) stroma and plasma. Concerns with this approach include loss of total nucleated cells (TNCs), bag breakage during centrifugation, and poor reproducibility by transplant centers unfamiliar with this technique. We rationalized that a simple 1:1 dilution without centrifugation would stabilize the product and reduce the DMSO concentration by 50%, allowing for a controlled thaw in the laboratory without the risks of cell loss. STUDY DESIGN AND METHODS: We compared the traditional wash method with albumin reconstitution (dilution) and thaw only (no dilution or wash), assessing measurements of viability, TNC, CD34, and colony‐forming cell (CFC) recovery post‐thaw. Ten cryopreserved UCB products were thawed, split equally into three parts, and treated using each method. Product stability was measured at multiple time intervals up to 48 hours post‐thaw. RESULTS: Throughout the entire evaluation, traditional wash and dilution methods performed equally well with no significant differences observed in 7‐aminoactinomycin viability, TNC, CD34, or CFC recovery. For 163 patients in which diluted products were administered, there were no serious adverse effects at infusion and similar time to engraftment was observed when compared to historical experiences with traditional wash and direct infusion. CONCLUSION: We conclude that removing DMSO, RBC stroma, and plasma post‐thaw using a wash method is not necessary when UCB products are RBC and plasma reduced before cryopreservation.     

Evaluation of an automated cell processing device to reduce the dimethyl sulfoxide from hematopoietic grafts after thawing

BACKGROUND: The direct transfusion of thawed hematopoietic progenitor cells (HPCs) is associated to transfusion-related side effects that are thought to be dose-dependent on the infused dimethyl sulfoxide (DMSO). Both the effectiveness of a fully automated cell processing device to washing out DMSO and the effects of DMSO elimination over the recovered cells were evaluated. STUDY DESIGN AND METHODS: Twenty cryopre-served peripheral blood HPC bags (HPC apheresis [HPC-A]) were thawed and processed for washing with an automated cell-processing device. Viability, colony-forming units (CFUs), and absolute count of recovered cells were evaluated by flow cytometry immediately after washing as well as at different times after washing and compared with a sample taken just after thawing (control) but maintained at 4 degrees C. DMSO content was measured by high-performance liquid chromatography and the osmolarity with an osmometer. RESULTS: The median recovery of viable total nucleated cells, viable CD34+ cells, and CFU colonies was 89 (range, 74-115), 103 (range, 62-126), and 91 percent (range, 46%-196%), respectively, in the washing group. Recovery of viable CD3+ cells was 97 percent (range, 42%-131%) and CD14+ cells was 82 percent (54%-119%). The percentages of DMSO elimination and osmolarity reduction were 98 (range, 96-99) and 90 percent (range 86%-95%), respectively. Moreover, elimination of the cryoprotectant improved CFU count, viability, and cell recoveries along the time when compared with the control group. CONCLUSION: Washing out DMSO in thawed HPC-A by use of this approach is safe and efficient in terms of recovery and viability of nucleated and progenitor cells. Additionally, the removal degree of DMSO is very high and therefore might ameliorate the transfusion-related side effects.     

Differentiation of the human liver progenitor cell line (HepaRG) on a microfluidic‐based biochip

HepaRG is a bipotent stem cell line that can be differentiated towards hepatocyte‐like and biliary‐like cells. The entire cultivation process requires 1 month and relies on the addition of 2% dimethyl sulfoxide (DMSO) to the culture. Our motivation in this research is to differentiate HepaRG cells (progenitor cells and undifferentiated cells) towards hepatocyte‐like cells by minimizing the cultivation time and without using DMSO treatment by instead using a microfluidic device combined with the following strategies: (a) comparison of extracellular matrices (matrigel and collagen I), (b) types of flow (one or both sides), and (c) effects of DMSO. Our results demonstrate that matrigel promotes the differentiation of progenitor cells towards hepatocytes and biliary‐like cells. Moreover, the frequent formation of HepaRG cell clusters was observed by a supply of both sides of flow, and the cell viability and liver specific functions were influenced by DMSO. Finally, differentiated HepaRG progenitor cells cultured in a microfluidic device for 14 days without DMSO treatment yielded 70% of hepatocyte‐like cells with a highly polarized organization that reacted to stimulation with IL‐6 to produce C‐reactive protein (CRP). This culture model has high potential for investigating cell differentiation and liver pathophysiology research.     

Infusion of P-Capt prion-filtered red blood cell products demonstrate acceptable in vivo viability and no evidence of neoantigen formation

BACKGROUND: Transmission of variant Creutzfeldt‐Jacob disease (vCJD) is a major concern in blood transfusion. The P‐Capt filter has been shown to remove around 4 log ID₅₀ prion infectivity from prion‐spiked human red blood cells (RBCs). STUDY DESIGN AND METHODS: Two independent, single‐center, randomized, open‐label studies were designed to analyze the safety of P‐Capt–filtered RBCs. RBCs prepared from leukoreduced whole blood from 43 eligible subjects were randomly assigned to P‐Capt filtration and/or storage in plasma or SAGM and stored for 28 or 42 days. Stored RBCs were analyzed for in vivo 24‐hour recovery, hemolysis, metabolic variables, blood group antigen expression, neoantigen formation, and safety after autologous infusion. RESULTS: Mean P‐Capt filtration times for leukoreduced RBCs were 41 (SAGM) to 51 (plasma) minutes. Thirteen of 14 subjects receiving P‐Capt–filtered RBCs had 24‐hour RBC recoveries of 75% or more after 42‐day storage, with a mean hemolysis of less than 0.6%. No loss of RBC antigen expression or formation of neoantigens was observed. In both studies, RBCs had white blood cell counts of less than 1 × 10⁶/unit after leukofiltration. P‐Capt prion filtration provided an additional greater than 0.8 log leukoreduction. No serious or unexpected adverse events were observed after infusion of P‐Capt–filtered full‐volume RBC units. CONCLUSIONS: P‐Capt–filtered, stored RBCs demonstrated acceptable viability and no detectable neoantigen expression, immunogenic responses. or safety issues after infusion of a complete unit. The additional filtration time and modest reduction in RBC content are within acceptable levels for implementation in countries with transfusion transmission of vCJD.     

Freezing human platelets with 6 percent dimethyl sulfoxide with removal of the supernatant solution before freezing and storage at -80 degrees C without postthaw processing

BACKGROUND: Platelets (PLTs) can be frozen with 6 percent dimethyl sulfoxide (DMSO) at -80 degrees C for up to 2 years. This method has been modified by concentrating the PLTs and removing the supernatant before freezing. STUDY DESIGN AND METHODS: High-yield leukoreduced PLTs stored at 22 degrees C for up to 5 days were divided into three equal volumes: one was frozen with 6 percent DMSO at -80 degrees C, thawed, washed, and resuspended in plasma (old method with DMSO); the second was treated with 6 percent DMSO, concentrated to remove the supernatant DMSO, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method with DMSO); and the third was treated with 0.9 percent NaCl without DMSO, concentrated to remove the supernatant solution, frozen at -80 degrees C, thawed, and diluted with 0.9 percent NaCl (new method without DMSO). RESULTS: Freeze-thaw-wash recovery of PLTs frozen by the old method with DMSO was 74 +/- 2 percent with 5 percent PLT microparticles. Freeze-thaw recovery was 94 +/- 2 percent with 7 percent PLT microparticles (new method with DMSO) and 69 +/- 9 percent with 15 percent PLT microparticles (new method without DMSO). Total DMSO in washed PLTs was 400 and 600 mg in PLTs concentrated before freezing. In vivo recovery of PLTs frozen by the new method with DMSO and transfused into normal volunteers was 30 percent and the life span was 7 days. CONCLUSION: Concentrating PLTs before freezing simplified the procedure by eliminating postthaw washing. PLTs frozen by this method had more PLTs with reduced GPIb and increased annexin V binding than those frozen by the old method.     

A novel automated cell‐seeding device for tissue engineering of tubular scaffolds: design and functional validation

Obtaining an efficient, uniform and reproducible cell seeding of porous tubular scaffolds constitutes a major challenge for the successful development of tissue‐engineered vascular grafts. In this study, a novel automated cell‐seeding device utilizing direct cell deposition, patterning techniques and scaffold rotation was designed to improve the cell viability, uniformity and seeding efficiency of tubular constructs. Quantification methods and imaging techniques were used to evaluate these parameters on the luminal and abluminal sides of fibrous polymer scaffolds. With the automated seeding method, a high cell‐seeding efficiency (~89%), viability (~85%) and uniformity (~85–92%) were achieved for both aortic smooth muscle cells (AoSMCs) and aortic endothelial cells (AoECs). The duration of the seeding process was < 8 min. Initial cell density, cell suspension in matrix‐containing media, duration of seeding process and scaffold rotation were found to affect the seeding efficiency. After few days of culture, a uniform longitudinal and circumferential cell distribution was achieved without affecting cell viability. Both cell types were viable and spread along the fibres after 28 h and 6 days of static incubation. This new automated cell‐seeding method for tubular scaffolds is efficient, reliable and meets all the requirements for clinical applicability. Copyright © 2011 National Research Council Canada and John Wiley & Sons, Ltd.     

二甲亚砜与海藻糖联用对冷冻保存血小板的保护作用

本研究观察二甲亚砜（DMSO）与海藻糖联用对冷冻保存血小板的保护效果。实验分设空白组、海藻糖组、DMSO组、5％DMs0加海藻糖联用组、2．5％DMSO加海藻糖联用组。各组血小板置-80℃冰箱保存,37℃水浴融化。采用血细胞计数仪测定血小板回收率和MPV值、电子显微镜观察血小板超微结构变化和流式细胞仪测定血小板的CD41、CD42b、CD61及CD62p的表达水平。结果表明：海藻糖单独应用对提高回收率的保护作用不强,但海藻糖处理后冷冻保存的血小板的形态接近正常。DMSO在保证冷冻保存血小板的回收率和血小板整体完好性方面作用较为突出,但其血小板形态偏向于肿胀,仍有部分血小板呈异形性改变。DMSO和海藻糖合用对维持血小扳外部形态和内部结构接近正常稳态方面具有保护作用,同时保证冷冻保存血小板具有理想的回收率和较高的CD41、CD42b、CD61、CD62p表达水平。结论：DMSO和海藻糖联合应用对冷冻保存血小板具有协同保护作用,但DMSO和海藻糖单用或合用都较难抑制冷冻保存血小板的活化,两者合用作为血小板冷冻保护剂有望促使冷冻保存血小板临床输注效果的进一步提高。     

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

Background: 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. Objectives: 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. Methods/Materials: We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery. Results: 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. Conclusion: 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.     

A new automated cell washer device for thawed cord blood units

BACKGROUND: The current available techniques to wash out DMSO from thawed umbilical cord blood (UCB) units are based essentially on standard centrifugation in an open system with various degrees of cell loss. STUDY DESIGN AND METHODS: We evaluated the capacity of a new automated closed device (Cytomate, Baxter, IL) to wash out the DMSO from thawed UCB units, saving at the same time the progenitor and accessory cells in terms of CD34+ cells and MNCs. We modified the standard software of the device and calculated the cell recovery on 25 UCB units. Moreover, we set up a new gas chromatographic method to exactly detect the DMSO removal rate. RESULTS: To evaluate the efficiency of the Cytomate device, we considered the postthawing (prewashing) versus postwashing cell recovery. The average recovery (%) in terms of total nucleated cells was 63.30 (range, 40.12-89.00), CD34+ cells was 70.20 (range, 11.51-89.01), CD3+ cells was 61.01 (range, 28.80-87.08), CD4+ cells was 62.53 (range, 30.62-96.73), CD8+ cells was 57.4 (range, 26.87-94.72), CD19+ cells was 63.33 (range, 39.10-90.33), CD16+/56+ cells was 70.67 (range, 8.91-98.40), CFU-GM was 74.33 (range, 20.23-98.60), total CFUs was 82.34 (range, 14.83-247.12), and viability was 89.67(range, 70.74-98.30). The total working time required was, on average, 15 minutes (range, 7-20). CONCLUSIONS: The Cytomate device demonstrated a satisfying efficiency in cell recovery and in maintaining the clonogenic power of the UCB graft. The removal rate of DMSO was practically complete with evident advantages for the recipient. Finally, the entire manipulation performed in a closed system revealed to be safe, maintaining the sterility of the graft.     

Concentration of bone marrow mononuclear cells using a programmable blood cell separator.

Bone marrow was collected from adult patients with various solid tumors who consented to participate in a study of myelo-ablative chemotherapy followed by autologous bone marrow rescue. Twenty marrow suspensions were processed by using standard Procedure 3 (PRO-3) for lymphocytapheresis without modification. A modified Procedure 1 (M-PRO-1) for plateletpheresis was employed for processing 34 marrow suspensions. For PRO-3, mononuclear cell (MNC) recovery was 68 +/- 22% of the starting marrow suspension (baseline), in a concentrate volume of 234 +/- 53 ml. MNC represented 59 +/- 27% of the total WBC count of the concentrate. The residual volume of RBC was 49 +/- 47 ml. For M-PRO-1, MNC recovery was 63 +/- 22% of the baseline in a concentrate volume of 200 +/- 8 ml. MNC comprised 94 +/- 7% of the total WBC count of the concentrate. RBC contamination was 7 +/- 3 ml. Hematopoietic recovery, defined as the post-transplant days when a sustained granulocyte count of 500/microL and a platelet count of 50,000/microL were achieved, was similar in the two groups and comparable to other reports utilizing other methods and equipment for bone marrow concentration. Personnel time was significantly reduced compared to other procedures for bone marrow concentration due to increased automation. Although there was no significant difference in MNC recovery between the two groups (P greater than 0.5), M-PRO-1 was clearly superior to PRO-3 because of the consistently high degree of purity of the MNC in the concentrate and minimal RBC contamination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotary orbital suspension culture of embryonic stem cell‐derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield,morphology and cell phenotype

Embryonic stem (ES)‐derived neural stem/progenitor cells (ES‐NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single‐cell suspensions of ES‐NSPCs, to obtain homogeneously‐sized ES‐NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES‐NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller‐sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES‐NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N‐cadherin and β‐catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase‐2 (MMP‐2) activity and percentage of neuronal cells. In line with the increased MMP‐2 activity levels found, hydrodynamically‐cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single‐cell suspensions of ES‐NSPCs, besides allowing the formation of homogeneously‐sized neurospheres, promoted ES‐NSPC differentiation and outward migration, possibly by influencing the expression of cell–cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly‐sized ES‐NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd.     

Transplantation of cancerous cell sheets effectively generates tumour‐bearing model mice

Tumour‐bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene‐transfected mammary gland adenocarcinoma cells, 4T1‐luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature‐responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin‐based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase‐gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1‐luc2 engraftment efficiency in living mouse tissues at the initial stage by 13‐fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet‐transplanted mice also accelerated, and the mean tumour volume became 1116 mm³, which was 10 times larger than that in cell suspension‐transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet‐based transplantation is a promising method for efficiently creating cancer‐bearing mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Algorithm‐driven optimization of cryopreservation protocols for transfusion model cell types including Jurkat cells and mesenchymal stem cells

This investigation describes the use of a differential evolution (DE) algorithm to optimize cryopreservation solution compositions and cooling rates for specific cell types. Jurkat cells (a lymphocyte model cell type) and mesenchymal stem cells (MSCs) were combined with non‐DMSO solutions at concentrations dictated by a DE algorithm. The cells were then frozen in 96‐well plates at DE algorithm‐dictated cooling rates in the range 0.5–10°C/min. The DE algorithm was iterated until convergence resulted in identification of an optimum solution composition and cooling rate, which occurred within six to nine generations (seven to 10 experiments) for both cell types. The optimal composition for cryopreserving Jurkat cells included 300 mm trehalose, 10% glycerol and 0.01% ectoine (TGE) at 10°C/min. The optimal composition for cryopreserving MSCs included 300 mm ethylene glycol, 1 mm taurine and 1% ectoine (SEGA) at 1°C/min. High‐throughput concentration studies verified the optimum identified by the DE algorithm. Vial freezing experiments showed that experimental solutions of TGE at 10°C/min resulted in significantly higher viability for Jurkat cells than DMSO at 1°C/min, while experimental solutions of SEGA at 10°C/min resulted in significantly higher recovery for MSCs than DMSO at 1°C/min; these results were solution‐ and cell type‐specific. Implementation of the DE algorithm permits optimization of multicomponent freezing solutions in a rational, accelerated fashion. This technique can be applied to optimize freezing conditions, which vary by cell type, with significantly fewer experiments than traditional methods. Copyright © 2016 John Wiley & Sons, Ltd.     

Cryopreservation of stromal vascular fraction of adipose tissue in a serum‐free freezing medium

Developing effective techniques for the cryopreservation of human adipose‐derived adult stem cells could increase the usefulness of these cells in tissue engineering and regenerative medicine. Unfortunately, the use of serum and a commonly used cryoprotectant chemical, dimethyl sulphoxide (DMSO), during cryopreservation storage restricts the direct translation of adult stem cells to in vivo applications. The objective of this study was to test the hypothesis that the stromal vascular fraction (SVF) of adipose tissue can be effectively cryopreserved and stored in liquid nitrogen, using a freezing medium containing high molecular weight polymers, such as methylcellulose (MC) and/or polyvinylpyrollidone (PVP), as the cryoprotective agent (CPA) instead of DMSO. To this end, we investigated the post‐freeze/thaw viability and apoptotic behaviour of SVF of adipose tissue frozen in 16 different media: (a) the traditional medium containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (b) DMEM with 80% human serum (HS) and 10% DMSO; (c) DMEM with 0%, 2%, 4%, 6%, 8% or 10% DMSO; (d) DMEM with 1% MC and 10% of either HS or FCS or DMSO; (e) DMEM with 10% PVP and varying concentrations of FCS (0%, 10%, 40% or 80%); (f) DMEM with 10% PVP and 10% HS. Approximately 1 ml (10⁶ cells/ml) of SVF cells were frozen overnight in a ?80 °C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 °C water bath (1–2 min agitation), resuspended in culture medium and seeded in separate wells of a six‐well plate for a 24 h incubation period at 37 °C. After 24 h, the thawed samples were analysed by brightfield microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic SVF cells. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing medium with DMSO and serum (HS or FCS), i.e. ～54 ± 14% and ～63 ± 10%, respectively. Adipogenic and osteogenic differentiation behaviour of the frozen thawed cells was also assessed, using histochemical staining. Our results suggest that post‐thaw SVF cell viability and adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum and DMSO but with 10% PVP in DMEM. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of cryopreservation on human mesenchymal stem cells attached to different substrates

There is a need to preserve cell‐seeded scaffolds or cell–matrix constructs for tissue‐engineering and other applications. Cryopreservation is likely to be the most practical method. The aim of this study was to investigate how cryopreservation affects cells attached to different substrates and how they respond differently from those in suspension. Human mesenchymal stem cells (hMSCs) were studied for their close relevance to tissue‐engineering and stem cell therapy applications, in particular how cryopreservation affects cell adherence, cell growth and the viability of hMSCs attached to different substrates, including glass, gelatin, matrigel and a matrigel sandwich. The effects of cryopreservation on F‐actin organization, intracellular pH and mitochondrial localization of the adherent hMSCs were further investigated. It was found that cells attached to a glass surface could hardly survive the common cryopreservation protocol using 10% DMSO and a 1°C/min cooling rate. By contrast, cells attached to gelatin and matrigel could survive to a greater extent. Furthermore, cryopreservation affected the potential of cell attachment and proliferation, resulted in distortion of F‐actin, led to alteration of intracellular pH of the hMSCs for all tested substrates and caused a change in the mitochondrial localization of hMSCs on a matrigel substrate and in a matrigel sandwich. Our results showed that cell attachment and cell viability could be improved by changing the interaction between cell and substrate through modification of the substrate properties, which has implications for scaffold design if cell‐seeded scaffolds or engineered tissues need to be cryopreserved. Copyright © 2012 John Wiley & Sons, Ltd.     

Hydroxyethyl starch is an alternative washing solution for peripheral bloodstem cells products

Cryopreservation of hematopoietic stem cells (HSC) involves slow rate cooling in the presence of a cryoprotectant (DMSO) to avoid the damaging effects of intracellular ice formation. The infusion of DMSO with the thawed product has been related to adverse events. Reduction of DMSO content by washing the HSCs after thawing has been suggested as a method to avoid infusion-related side-effects. Albumin-dextran washing methods have proved useful in thawing HSC products. Dextran40 shortages prompted us to search for suitable alternatives. We report the results of a comparative study of the use of hydroxyethyl starch (HES) as an alternative to dextran40 for washing thawed HSCs products. A total of 10 HSC bags cryopreserved with 10 % DMSO were used. We conducted a paired study; one of the bags was thawed and washed with our standard washing solution (Dextran 40) and the paired bag with HES solution with a final HES and Human Serum Albumin (HSA) concentration of 2.4 % and 4.2 % respectively. Each final product was tested immediately after washing (sample 0’) and after 90 min (sample 90’) for total nucleated cells (TNC) recovery, acridine orange viability, viable CD34+ enumeration, and clonogenicity. No significant difference was found for any of the cell counts, viability tests, cell recovery, or potency. We can state that the washing solution based on 2.4 % HES and 4.2 % HSA is equivalent to that used in our routine practice. Therefore, we could use the solution with HES, paying special attention to the renal function of the recipient.     

Cell encapsulation within PVA‐based hydrogels via freeze‐thawing: a one‐step scaffold formation and cell storage technique

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA‐based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue‐engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell‐laden hydrogels with adjustable mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.     

骨髓细胞液氮保存21-25年后的细胞活力体外研究

为探讨适合临床应用的骨髓细胞保存方法,揭示细胞长期低温保存效果,将20人份骨髓加入DMSO-AuP(10%二甲亚砜、10%自体血浆)或DMSO-HES-HuA(5%DMSO、6%羟乙基淀粉、4%人血白蛋白)保护剂,分装2毫升/管,600-800管/人份,自控程序降温仪或-80℃低温冰箱预冻降温、液氮中保存21-25年。取冷冻骨髓细胞于38℃复温,检测细胞相关指标。结果表明:加保护剂的细胞标本在-80℃低温冰箱中为低速降温,-30℃前,与自控程序降温仪设定1℃/min的低降温速率接近。DMSO-HES-HuA与DMSO-AuP比较,红细胞形态畸形率分别为(3.5±1.5)%和(12.6±4.8)%;溶血率分别为(3.3±1.6)%和(23.1±5.1)%(p0.05);前者渗透性脆性不变,后者减低;红细胞、血小板、粒单系造血祖细胞、长期培养起始细胞回收率,前后者对比分别为(96.1±1.8)%、(70.0±9.5)%、(49.2±10.9)%、(54.2±13.8)%vs(76.3±5.6)%、(52.7±8.1)%、(43.5±12.3)%、(47.2±13.6)%。用上述保护剂配合上述降温法保存后,细胞在间充质干细胞培养上,生长特性良好。同一保护剂用上述两种降温法保存后,细胞各种回收效果差异不显著(p0.05)。结论:细胞在液氮中保存21-25年,其细胞形态和回收活力(率)良好;保存如骨髓这种细胞成分并非单一的标本时,用5%DMSO-6%HES-4%HuA为保护剂的-80℃冰箱预冻降温后液氮保存,方法简便、经济、易操作,适合临床应用。     

Frozen for combat: Quality of deep-frozen thrombocytes,produced and used by The Netherlands Armed Forces 2001–2021

Background

The Netherlands Armed Forces (NLAF) are using −80°C deep-frozen thrombocyte concentrate (DTC) since 2001. The aim of this study is to investigate the effect of storage duration and alterations in production/measurement techniques on DTC quality. It is expected that DTC quality is unaffected by storage duration and in compliance with the European guidelines for fresh and cryopreserved platelets.

Study Design and Methods

Pre-freeze and post-thaw product platelet content and recovery were collected to analyze the effects of dimethyl sulfoxide (DMSO) type, duration of frozen storage (DMSO-1 max 12 years and DMSO-2 frozen DTC max 4 years at −80°C) and type of plasma used to suspend DTC. Coagulation characteristics of thawed DTC, plasma and supernatant of DTC (2× 2500 G) were measured with Kaolin thromboelastography (TEG) and phospholipid (PPL) activity assay.

Results

Platelet content and recovery of DTC is ±10%–15% lower in short-stored products and remained stable when stored beyond 0.5 years. Thawed DTC (n = 1724) were compliant to the European guidelines (98.1% post-thaw product recovery ≥50% from original product, 98.3% ≥200 × 10⁹ platelets/unit). Compared to DMSO-1, products frozen with DMSO-2 showed ±8% reduced thaw–freeze recovery, a higher TEG clot strength (MA 58 [6] vs. 64 [8] mm) and same ±11 s PPL clotting time. The use of cold-stored thawed plasma instead of fresh thawed plasma did not influence product recovery or TEG-MA.

Discussion

Regardless of alterations, product quality was in compliance with European guidelines and unaffected by storage duration up to 12 years of −80°C frozen storage.