Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Comparison of Different Thawing Protocols in Human Cryopreserved Venous Grafts.

BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

Organization model for allotransplantations of cryopreserved vascular grafts in Czech Republic.

The transplantation of fresh or cryopreserved vascular allografts in patients with a prosthetic graft infection or critical limb ischemia is necessary for their limb salvage and, in many cases, represents a lifesaving procedure. While transplantation of fresh allografts has a long history in the Czech Republic, the standard use of cryopreserved vascular allografts was introduced into the clinical practice in 2011 as a result of the implementation of EU Directive 2004/23/EC into national legislation (Human Cell and Tissue Act No. 296/2008 Coll.). The authors present an organizational model based on cooperation between the majority of Czech Transplant Centers with a tissue establishment licensed by the national competent authority. In various points, we are addressing individual aspects of experimental and clinical studies which affect clinical practice. Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013. The growing number of centers participating in this programme led to a growing number of patients who underwent transplantation of vascular allografts. In 2015 the numbers of transplanted fresh versus cryopreserved allografts in the Czech Republic were almost equal. Cooperation of the participating centers in the Czech Republic with the licensed Tissue Establishment made it possible to achieve a full compliance with the European Union Directives, and harmonized national legal norms and assured a high quality of cryopreserved vascular allografts.

Elution kinetics of vancomycin and gentamicin from carriers and their effects on mesenchymal stem cell proliferation: an in vitro study.

BACKGROUND: Musculoskeletal infections remain a major complication in orthopedic surgery. The local delivery of antibiotics provides the high levels required to treat an infection without systemic toxicity. However, the local toxicity of antibiotic carriers to the mesenchymal stem cells, as a result of both the peak concentrations and the type of carrier, may be significant. METHODS: To address this concern, the elution kinetics of vancomycin and gentamicin from several commercially available antibiotic carriers and several carriers impregnated by a surgeon (10 ml of each sterile carrier were manually mixed with a 500 mg vancomycin and an 80 mg gentamicin solution, and the duration of impregnation was 30 min) were assessed. Moreover, the effects of these antibiotic carriers on stem cell proliferation were investigated. The following two types of stem cells were used: bone marrow and dental pulp stem cells. RESULTS: The high eluted initial concentrations from antibiotic impregnated cancellous allogeneic bone grafts (which may be increased with the addition of fibrin glue) did not adversely affect stem cell proliferation. Moreover, an increased dental pulp stem cell proliferation rate in the presence of antibiotics was identified. In contrast to allogeneic bone grafts, a significant amount of antibiotics remained in the cement. Despite the favorable elution kinetics, the calcium carriers, bovine collagen carrier and freeze-dried bone exhibited decreased stem cell proliferation activity even in lower antibiotic concentrations compared with an allogeneic graft. CONCLUSIONS: This study demonstrated the benefits of antibiotic impregnated cancellous allogeneic bone grafts versus other carriers.

High-Pressure Inactivation of Bacillus cereus in Human Breast Milk.

Although Holder pasteurization is the recommended method for processing breast milk, it does affect some of its nutritional and biological properties and is ineffective at inactivating spores. The aim of this study was to find and validate an alternative methodology for processing breast milk to increase its availability for newborn babies and reduce the financial loss associated with discarding milk that has become microbiologically positive. We prepared two series of breast milk samples inoculated with the Bacillus cereus (B. cereus) strain to verify the effectiveness of two high-pressure treatments: (1) 350 MPa/5 min/38 °C in four cycles and (2) cumulative pressure of 350 MPa/20 min/38 °C. We found that the use of pressure in cycles was statistically more effective than cumulative pressure. It reduced the number of spores by three to four orders of magnitude. We verified that the method was reproducible. The routine use of this method could lead to an increased availability of milk for newborn babies, and at the same time, reduce the amount of wasted milk. In addition, high-pressure treatment preserves the nutritional quality of milk.

Quantitative Risk Assessment of Bacillus cereus Growth during the Warming of Thawed Pasteurized Human Banked Milk Using a Predictive Mathematical Model.

Bacillus cereus is relatively resistant to pasteurization. We assessed the risk of B. cereus growth during warming and subsequent storage of pasteurized banked milk (PBM) in the warmed state using a predictive mathematical model. Holder pasteurization followed by storage below -18 °C was used. Temperature maps, water activity values, and B. cereus growth in artificially inoculated PBM were obtained during a simulation of manipulation of PBM after its release from a Human Milk Bank. As a real risk level, we chose a B. cereus concentration of 100 CFU/mL; the risk was assessed for three cases: 1. For an immediate post-pasteurization B. cereus concentration below 1 CFU/mL (level of detection); 2. For a B. cereus concentration of 10 CFU/mL, which is allowed in some countries; 3. For a B. cereus concentration of 50 CFU/mL, which is approved for milk formulas. In the first and second cases, no risk was detected after 1 h of storage in the warmed state, while after 2 h of storage, B. cereus concentrations of 102 CFU/mL were occasionally encountered. In the third case, exceeding the B. cereus concentration of 102 CFU/mL could be regularly expected after 2 h of storage. Based on these results, we recommend that post-pasteurization bacteriological analysis be performed as recommended by the European Milk Bank Association (EMBA) and using warmed PBM within 1 h after warming (no exceptions).

Bacillus cereus as a Major Cause of Discarded Pasteurized Human Banked Milk: A Single Human Milk Bank Experience.

A systematic study, performed from 2017-2020 looked at the rate of positive post-pasteurization B. cereus findings, the quantity of B. cereus in pasteurized banked human milk (PBM), and the rate of B. cereus toxicogenic isolates from PBM. During the study period, 6815.71 L (30,943 tested bottles) of PBM were tested, with an average amount per year of 1703.93 L (7736 tested bottles). The PBM discard rate per year due to bacterial contamination varied between 8.7-10.0% and contamination with B. cereus was the most frequent reason. The total number of B. cereus positive tests was 2739 and the proportion of its positivity from all positive tests was between 56.7-66.6%. The prevalence of B. cereus positive tests rose significantly in the summer months. The production of enterotoxin was found in 3 of the 20 tested samples (15.0%). The B. cereus CFU-quantities in the PBM were below 10 CFU/mL in 80% of cases (16 of 20 samples tested). The quantitative data can be used in the risk assessment of cold storage of PBM at temperatures above zero and manipulation of PBM prior to its administration.

The Effect of Different Thawing Rates on Cryopreserved Human Iliac Arteries Allograft's Structural Damage and Mechanical Properties.

INTRODUCTION: The rate of thawing of cryopreserved human iliac arteries allografts (CHIAA) directly affects the severeness of structural changes that occur during this process. METHOD: The experiment was performed on ten CHIAA. The 10% dimethylsulphoxide in 6% hydroxyethyl starch solution was used as the cryoprotectant; all CHIAA were cooled at a controlled rate and stored in the vapor phase of liquid nitrogen (-194°C). Two thawing protocols were tested: (1) placing the CHIAA in a water bath at 37°C, and (2) the CHIAA were thawed in a controlled environment at 5°C. All samples underwent analysis under a scanning electron microscope. Testing of the mechanical properties of the CHIAA was evaluated on a custom-built single axis strain testing machine. Longitudinal and circumferential samples were prepared from each tested CHIAA. RESULTS: Ultrastructural analysis revealed that all five CHIAA thawed during the thawing protocol 1 which showed significantly more damage to the subendothelial structures when compared to the samples thawed in protocol 2. Mechanical properties: Thawing protocol 1-longitudinal UTS 2, 53 ± 0, 47 MPa at relative strain 1, 27 ± 0, 12 and circumferential UTS 1, 94 ± 0, 27 MPa at relative strain 1, 33 ± 0, 09. Thawing protocol 2-longitudinal ultimate tensile strain (UTS) 2, 42 ± 0, 34 MPa at relative strain 1, 32 ± 0, 09 and circumferential UTS 1, 98 ± 0, 26 MPa at relative strain 1, 29 ± 0, 07. Comparing UTS showed no statistical difference between thawing methods. CONCLUSION: Despite the significant differences in structural changes of presented thawing protocols, the ultimate tensile strain showed no statistical difference between thawing methods.

Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

OBJECTIVES AND DESIGN: At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS: Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS: Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION: Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity?

Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings.

Current vascular allograft procurement, cryopreservation and transplantation techniques in the Czech Republic.

BACKGROUND: Vascular allotransplantations are performed worldwide in selected patients suffering from vascular prosthesis infection or critical limb ischemia. Either fresh or cryopreserved vascular allograft may be used. OBJECTIVES: In various points, we address several aspects (allograft procurement, cryopreservation and transplantation technique) of the program of vascular allotransplantations in the Czech Republic. MATERIAL AND METHODS: Vascular grafts retrieval has been done within multiorgan harvests using no-touch technique. Very short time of cold ischemia is achieved due to close cooperation with Tissue Establishment where the following processing of cryopreservation is performed. Meeting all necessary quality criteria is a prerequisity for releasing grafts for clinical application. Standardized thawing protocol and surgical handling aims to minimize microfractures before implantation. RESULTS: Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013 in the Czech Republic. The developement of storage of vascular tissue in banks was stimulated in 2000-2010 by the issue of EU directives and national harmonized norms, aimed at assurance of high quality and safety of cells and tissues used for transplantations in humans. CONCLUSIONS: There are several crucial moments affecting final quality, including graft retrieval within a multiorgan harvest, short ischemic time, cryopreservation and thawing technique used. The recommended surgical handling during implantation may also affect results and graft-related complications.

Comparative Study on the Application of Mesenchymal Stromal Cells Combined with Tricalcium Phosphate Scaffold into Femoral Bone Defects.

This prospective study sought to evaluate the healing quality of implanted ultraporous ß-tricalcium phosphate sown with expanded autologous mesenchymal stromal cells (MSCs) into femoral defects during revision hip arthroplasty. A total of 37 osseous defects in 37 patients were treated and evaluated concerning bone regeneration. Nineteen subjects received ß-tricalcium phosphate graft material serving as a carrier of expanded autologous MSCs (the trial group A), nine subjects received ß-tricalcium phosphate graft material only (the study group B) and nine subjects received cancellous allografts only (the control group C). Clinical and radiographic evaluations were scheduled at 6 weeks, 3, 6, and 12 months post-operatively, and performed at the most recent visit as well. All observed complications were recorded during follow-up to assess the use of an ultraporous ß-tricalcium phosphate synthetic graft material combined with expanded MSCs in bone defect repair. The resulting data from participants with accomplished follow-up were processed and statistically evaluated with a Freeman-Halton modification of the Fischer's exact test, a P < 0.05 value was considered to be significant. Whereas no significant difference was observed between the trial group A with ß-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs and control group C with cancellous impaction allografting in terms of the bone defect healing, significant differences were documented between the study group B with ß-tricalcium phosphate graft material only and control group C. Regarding adverse effects, six serious events were recorded during the clinical trial with no causal relationship to the cell product. ß-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs appears safe and promotes the healing of bone defects in a jeopardized and/or impaired microenvironment. This clinical trial was registered at the EU Clinical Trials Register before patient recruitment (Registration number: EudraCT number 2012-005599-33; Date of registration: 2013-02-04).

Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats.

OBJECTIVES AND DESIGN: The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. MATERIALS AND METHODS: Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. RESULTS: The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, p < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, p < 0.001, anti-MHC class II 80.9±2.7%). CONCLUSION: Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol.

Structural changes arising from different thawing protocols on cryopreserved human allograft's aortic valve leaflets.

BACKGROUND: The aim of our experimental work was to assess the impact and morphological changes that arise during different thawing protocols on human aortic valve (AV) leaflets resected from cryopreserved aortic root allografts (CARAs). OBJECTIVES: Two thawing protocols were tested: 1. CARAs were thawed at a room temperature (23°C); 2. CARAs were placed directly into a water bath at a temperature of 37°C. After all the samples were thawed, non-coronary AV leaflets were sampled from each specimen and fixed in a 4% formaldehyde solution before they were sent for morphological analysis. MATERIAL AND METHODS: All the samples were washed in distilled water for 5 min and dehydrated in a graded ethanol series (70%, 85%, 95%, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane (HMDS) for 10 min, and then air-dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs and coated with gold. Histological analysis was performed with the use of an electron microscope on a scanning mode operating at 25 kV - BS 301. RESULTS: Thawing protocol 1 (room temperature at 23°C): 6 (100%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. Thawing protocol 2 (water bath at 37°C): 5 (83%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. One (17%) sample showed loss of the endothelial covering the basal membrane with significant damage to the basal membrane. CONCLUSIONS: Based on our experimental work, we can clearly conclude that cryopreserved AV leaflet allografts show identical structural changes at different rates of thawing.

Cryopreserved human aortic root allografts arterial wall: Structural changes occurring during thawing.

BACKGROUND: The aim of our experimental work was to assess morphological changes of arterial wall that arise during different thawing protocols of a cryopreserved human aortic root allograft (CHARA) arterial wall. METHODS: The experiment was performed on CHARAs. Two thawing protocols were tested: 1, CHARAs were thawed at a room temperature at +23°C; 2, CHARAs were placed directly into a water bath at +37°C. MICROSCOPIC SAMPLES PREPARATION: After fixation, all samples were washed in distilled water for 5 min, and dehydrated in a graded ethanol series (70, 85, 95, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane for 10 minutes and air dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs, coated with gold. RESULTS: Thawing protocol 1: All 6 (100%) samples showed loss of the endothelium and damage to the subendothelial layers with randomly dispersed circular defects and micro-fractures without smooth muscle cells contractions in the tunica media. Thawing protocol 2: All 6 (100%) samples showed loss of endothelium from the luminal surface, longitudinal corrugations in the direction of blood flow caused by smooth muscle cells contractions in the tunica media with frequent fractures in the subendothelial layer. CONCLUSION: All the samples thawed at the room temperature showed smaller structural damage to the CHARA arterial wall with no smooth muscle cell contraction in tunica media when compared to the samples thawed in a water bath.

Transplantation of bone marrow derived progenitor cells in acute myocardial infarction. The first results.

The intracoronary administration of autologous bone marrow cells (BMCs) has been shown to improve the left ventricle function in the course of acute myocardial infarction. Therefore we have started a clinical trial using transplantation of BMCs in the acute phase of myocardial infarction. The aim of our study is to assess the feasibility and safety of this procedure, and effect on the left ventricle function of these patients. We describe the first experience in two patients with acute myocardial infarction reperfused using direct stenting. The aspiration of bone marrow from the sternum provided sufficient amount of the cells for transplantation. No serious ischemia and no changes in coronary artery patency were detected after intracoronary infusion. The left ventricle ejection fraction was increasing throughout the time of three-month follow-up. No other complications (ventricular arrhythmias, reinfarction, thrombus formation) were detected.

In vitro studies on transdermal permeation of butorphanol.

The influence of the donor vehicles pH and the addition of laurocapram or transkarbam 12 as permeation enhancers on the transdermal permeation of butorphanol through human skin were examined with the aim of finding out about its possible use in the transdermal delivery system. As the pH of the donor vehicles rises, the mean value of butorphanol skin fluxes declines; an exponential relationship of the means of butorphanol flux values against the pH of the buffered aqueous donor vehicles has been demonstrated. The presence of 1% of transkarbam 12 (T12) or 5% of laurocapram (LC), respectively, in an isopropylmyristate (IPM) donor vehicle increased transdermal fluxes of butorphanol almost 2.5 times (58.1+/-5.7 microg cm-2 hr-1) or 1.5 times (36.4+/-7.0 microg cm-2 hr-1), respectively, when compared to blank donors. Considering clinical and pharmacokinetic data on butorphanol, it is possible to expect that a transdermal preparation sized 20 cm2 and possessing flux values ranging between 5.1 and 15.3 microg cm-2 hr-1 should be sufficient to achieve effective butorphanol transdermal fluxes, namely using IPM donors containing T12. In conclusion, butorphanol is a suitable candidate for transdermal administration and T12 is a very a suitable enhancer for it.

The risk of infection transmission from blood progenitor cell concentrates.

The possibility of infection transmission by infusion of cryopreserved peripheral blood stem cells concentrates or bone marrow is well known. For this reason the European Blood and Marrow Transplantation Group (EBMT) and International Society for Haemotherapy and Graft Engineering (ISHAGE) standards include a panel of serological tests to be performed in donors and patients with the aim to lower the likelihood of infection transmission. The study was performed on a group of 71 patients and 22 donors. No laboratory signs of active infection were found in 15 donors (13 related, 2 unrelated), i.e., in 68.2% and in 55 patients (77.5%). The active infection from herpes viruses was the most common (in patients 13, in donors 7). Hepatitis B was found in only one case. The cytomegalovirus (CMV) immunoglobulin G (IgG) test was the most common marker of previous infection, and it was found in 14 donors and 55 patients. We can conclude that the rate of clinically unsuspected infections in donors and patients, including cases requiring immediate treatment among the patient groups, is relatively high and fully justifies the practice of prophylactic serological testing using the whole palette of tests according to EBMT and ISHGE in both autologous and allogenous transplantations of hematopoietic stem cells.