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American Journal of Physiology.... Feb 2022Although short-term machine perfusion (≤24 h) allows for resuscitation and viability assessment of high-risk donor livers, the donor organ shortage might be further...
Although short-term machine perfusion (≤24 h) allows for resuscitation and viability assessment of high-risk donor livers, the donor organ shortage might be further remedied by long-term perfusion machines. Extended preservation of injured donor livers may allow reconditioning, repairing, and regeneration. This review summarizes the necessary requirements and challenges for long-term liver machine preservation, which requires integrating multiple core physiological functions to mimic the physiological environment inside the body. A pump simulates the heart in the perfusion system, including automatically controlled adjustment of flow and pressure settings. Oxygenation and ventilation are required to account for the absence of the lungs combined with continuous blood gas analysis. To avoid pressure necrosis and achieve heterogenic tissue perfusion during preservation, diaphragm movement should be simulated. An artificial kidney is required to remove waste products and control the perfusion solution's composition. The perfusate requires an oxygen carrier, but will also be challenged by coagulation and activation of the immune system. The role of the pancreas can be mimicked through closed-loop control of glucose concentrations by automatic injection of insulin or glucagon. Nutrients and bile salts, generally transported from the intestine to the liver, have to be supplemented when preserving livers long term. Especially for long-term perfusion, the container should allow maintenance of sterility. In summary, the main challenge to develop a long-term perfusion machine is to maintain the liver's homeostasis in a sterile, carefully controlled environment. Long-term machine preservation of human livers may allow organ regeneration and repair, thereby ultimately solving the shortage of donor livers.
Topics: Homeostasis; Humans; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; Time Factors
PubMed: 34756122
DOI: 10.1152/ajpgi.00257.2021 -
Journal of Reconstructive Microsurgery Jun 2023For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this...
BACKGROUND
For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model.
METHODS
Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days.
RESULTS
Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure ( = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group ( = 0.01613).
CONCLUSION
To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.
Topics: Animals; Rats; Rats, Inbred Lew; Organ Preservation; Perfusion; Liver Transplantation; Cold Ischemia
PubMed: 35764315
DOI: 10.1055/a-1886-5697 -
Parasites & Vectors Aug 2020Different methods have been used to preserve phlebotomine sand flies for research purposes, including for taxonomic studies and detection of Leishmania spp. Here, we...
BACKGROUND
Different methods have been used to preserve phlebotomine sand flies for research purposes, including for taxonomic studies and detection of Leishmania spp. Here, we evaluated the effect of various preservation methods at different storage times on phlebotomine sand fly DNA concentration and purity.
METHODS
Field-collected phlebotomine sand flies were individually stored in 70% ethanol (G1) and 95% ethanol (G2) at room temperature, 70% ethanol (G3) and 95% ethanol (G4) at 8 °C or frozen dry (i.e. no preservation solution) at - 20 °C (G5). DNA concentration and purity were assessed at various storage times (T1, ≤ 12 h; T2, 3 months; T3, 6 months; T4, 9 months; and T5, 12 months). Fragments of the cytochrome c oxidase subunit 1 (cox1) and cacophony (CAC) genes of phlebotomine sand flies were also amplified.
RESULTS
Mean DNA concentration (P = 0.178) and 260/280 purity ratios (P = 0.584) did not vary significantly among various preservation methods and storage times. Within each group, DNA concentration varied in G1 (Kruskal-Wallis H-test, P = 0.009) for T3 vs T4 (Dunn's post-hoc, P < 0.05), and in G2 (Kruskal-Wallis H-test, P = 0.004) for T1 vs T2 and T1 vs T4 (Dunn's post-hoc, P < 0.05). For 260/280 purity ratios, the only statistically significant difference was found for G5 (Kruskal-Wallis H-test, P = 0.020) between T1 vs T4 (Dunn's post-hoc test, P < 0.05). The cox1 and CAC genes were successfully amplified, regardless of the preservation method and storage time; except in one sample from G2 at T1, for which the CAC gene failed to amplify.
CONCLUSIONS
The preservation methods and storage times herein evaluated did not affect the concentration and purity of DNA samples obtained from field-collected phlebotomine sand flies, for up to 12 months. Furthermore, these preservation methods did not interfere with PCR amplification of CAC and cox1 genes, being suitable for molecular analyses under the conditions studied herein.
Topics: Animals; DNA Barcoding, Taxonomic; Humans; Leishmania; Phlebotomus; Preservation, Biological
PubMed: 32762709
DOI: 10.1186/s13071-020-04270-4 -
Nature Communications Feb 2020Most mono- and co-culture bioprocess applications rely on large-scale suspension fermentation technologies that are not easily portable, reusable, or suitable for...
Most mono- and co-culture bioprocess applications rely on large-scale suspension fermentation technologies that are not easily portable, reusable, or suitable for on-demand production. Here, we describe a hydrogel system for harnessing the bioactivity of embedded microbes for on-demand small molecule and peptide production in microbial mono-culture and consortia. This platform bypasses the challenges of engineering a multi-organism consortia by utilizing a temperature-responsive, shear-thinning hydrogel to compartmentalize organisms into polymeric hydrogels that control the final consortium composition and dynamics without the need for synthetic control of mutualism. We demonstrate that these hydrogels provide protection from preservation techniques (including lyophilization) and can sustain metabolic function for over 1 year of repeated use. This approach was utilized for the production of four chemical compounds, a peptide antibiotic, and carbohydrate catabolism by using either mono-cultures or co-cultures. The printed microbe-laden hydrogel constructs' efficiency in repeated production phases, both pre- and post-preservation, outperforms liquid culture.
Topics: Coculture Techniques; Escherichia coli; Hydrogels; Preservation, Biological; Saccharomyces cerevisiae
PubMed: 32019917
DOI: 10.1038/s41467-020-14371-4 -
Microbiology Spectrum Dec 2021A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for...
A critical step in studies of the intestinal microbiome using meta-omics approaches is the preservation of samples before analysis. Preservation is essential for approaches that measure gene expression, such as metaproteomics, which is used to identify and quantify proteins in microbiomes. Intestinal microbiome samples are typically stored by flash-freezing and storage at -80°C, but some experimental setups do not allow for immediate freezing of samples. In this study, we evaluated methods to preserve fecal microbiome samples for metaproteomics analyses when flash-freezing is not possible. We collected fecal samples from C57BL/6 mice and stored them for 1 and 4 weeks using the following methods: flash-freezing in liquid nitrogen, immersion in RNA, immersion in 95% ethanol, immersion in a RNA-like buffer, and combinations of these methods. After storage, we extracted protein and prepared peptides for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis to identify and quantify peptides and proteins. All samples produced highly similar metaproteomes, except for ethanol-preserved samples that were distinct from all other samples in terms of protein identifications and protein abundance profiles. Flash-freezing and RNA (or RNA-like treatments) produced metaproteomes that differed only slightly, with less than 0.7% of identified proteins differing in abundance. In contrast, ethanol preservation resulted in an average of 9.5% of the identified proteins differing in abundance between ethanol and the other treatments. Our results suggest that preservation at room temperature in RNA or an RNA-like solution performs as well as freezing for the preservation of intestinal microbiome samples before metaproteomics analyses. Metaproteomics is a powerful tool to study the intestinal microbiome. By identifying and quantifying a large number of microbial, dietary, and host proteins in microbiome samples, metaproteomics provides direct evidence of the activities and functions of microbial community members. A critical step for metaproteomics workflows is preserving samples before analysis because protein profiles are susceptible to fast changes in response to changes in environmental conditions (air exposure, temperature changes, etc.). This study evaluated the effects of different preservation treatments on the metaproteomes of intestinal microbiome samples. In contrast to prior work on preservation of fecal samples for metaproteomics analyses, we ensured that all steps of sample preservation were identical so that all differences could be attributed to the preservation method.
Topics: Animals; Bacteria; Bacterial Proteins; Chromatography, Liquid; Feces; Female; Gastrointestinal Microbiome; Male; Mice; Mice, Inbred C57BL; Peptides; Preservation, Biological; Proteomics; Tandem Mass Spectrometry
PubMed: 34908431
DOI: 10.1128/Spectrum.01877-21 -
International Journal of Molecular... Dec 2022Renal transplantation is the preferred treatment for patients with end-stage renal disease. The current gold standard of kidney preservation for transplantation is... (Review)
Review
Renal transplantation is the preferred treatment for patients with end-stage renal disease. The current gold standard of kidney preservation for transplantation is static cold storage (SCS) at 4 °C. However, SCS contributes to renal ischemia-reperfusion injury (IRI), a pathological process that negatively impacts graft survival and function. Recent efforts to mitigate cold renal IRI involve preserving renal grafts at higher or subnormothermic temperatures. These temperatures may be beneficial in reducing the risk of cold renal IRI, while also maintaining active biological processes such as increasing the expression of mitochondrial protective metabolites. In this review, we discuss different preservation temperatures for renal transplantation and pharmacological supplementation of kidney preservation solutions with hydrogen sulfide to determine an optimal preservation temperature to mitigate cold renal IRI and enhance renal graft function and recipient survival.
Topics: Humans; Kidney Transplantation; Temperature; Organ Preservation; Kidney; Reperfusion Injury; Cold Temperature
PubMed: 36614006
DOI: 10.3390/ijms24010567 -
Journal of Clinical Laboratory Analysis Jan 2024Platelet concentrate (PC) transfusions are crucial in prevention and treatment of bleeding in infection, surgery, leukemia, and thrombocytopenia patients. Although the... (Review)
Review
BACKGROUND
Platelet concentrate (PC) transfusions are crucial in prevention and treatment of bleeding in infection, surgery, leukemia, and thrombocytopenia patients. Although the technology for platelet preparation and storage has evolved over the decades, there are still challenges in the demand for platelets in blood banks because the platelet shelf life is limited to 5 days due to bacterial contamination and platelet storage lesions (PSLs) at 20-24°C under constant horizontal agitation. In addition, the relations between some adverse effects of platelet transfusions and PSLs have also been considered. Therefore, understanding the mechanisms of PSLs is conducive to obtaining high quality platelets and facilitating safe and effective platelet transfusions.
OBJECTIVE
This review summarizes developments in mechanistic research of PSLs and their relationship with clinical practice, providing insights for future research.
METHODS
Authors conducted a search on PubMed and Web of Science using the professional terms "PSL" and "platelet transfusion." The obtained literature was then roughly categorized based on their research content. Similar studies were grouped into the same sections, and further searches were conducted based on the keywords of each section.
RESULTS
Different studies have explored PSLs from various perspectives, including changes in platelet morphology, surface molecules, biological response modifiers (BMRs), metabolism, and proteins and RNA, in an attempt to monitor PSLs and identify intervention targets that could alleviate PSLs. Moreover, novel platelet storage conditions, including platelet additive solutions (PAS) and reconsidered cold storage methods, are explored. There are two approaches to obtaining high-quality platelets. One approach simulates the in vivo environment to maintain platelet activity, while the other keeps platelets at a low activity level in vitro under low temperatures.
CONCLUSION
Understanding PSLs helps us identify good intervention targets and assess the therapeutic effects of different PSLs stages for different patients.
Topics: Humans; Blood Platelets; Platelet Transfusion; Thrombocytopenia; Hemorrhage; Blood Banks; Blood Preservation
PubMed: 38069592
DOI: 10.1002/jcla.24994 -
Nature Communications Oct 2021Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health,...
Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.
Topics: COVID-19; COVID-19 Nucleic Acid Testing; Coronavirus Nucleocapsid Proteins; Feces; Humans; Phosphoproteins; Preservation, Biological; RNA, Viral; Reagent Kits, Diagnostic; Reference Standards; SARS-CoV-2; Specimen Handling; Viral Load
PubMed: 34599164
DOI: 10.1038/s41467-021-25576-6 -
Frontiers in Immunology 2021Donor organ shortage still remains a serious obstacle for the access of wait-list patients to kidney transplantation, the best treatment for End-Stage Kidney Disease... (Review)
Review
Donor organ shortage still remains a serious obstacle for the access of wait-list patients to kidney transplantation, the best treatment for End-Stage Kidney Disease (ESKD). To expand the number of transplants, the use of lower quality organs from older ECD or DCD donors has become an established routine but at the price of increased incidence of Primary Non-Function, Delay Graft Function and lower-long term graft survival. In the last years, several improvements have been made in the field of renal transplantation from surgical procedure to preservation strategies. To improve renal outcomes, research has focused on development of innovative and dynamic preservation techniques, in order to assess graft function and promote regeneration by pharmacological intervention before transplantation. This review provides an overview of the current knowledge of these new preservation strategies by machine perfusions and pharmacological interventions at different timing possibilities: in the organ donor, during perfusion machine reconditioning or after implementation in the recipient. We will report therapies as anti-oxidant and anti-inflammatory agents, senolytics agents, complement inhibitors, HDL, siRNA and H2S supplementation. Renal delivery of pharmacologic agents during preservation state provides a window of opportunity to treat the organ in an isolated manner and a crucial route of administration. Even if few studies have been reported of transplantation after drugs administration, targeting the biological pathway associated to kidney failure (i.e. oxidative stress, complement system, fibrosis) might be a promising therapeutic strategy to improve the quality of various donor organs and expand organ availability.
Topics: Animals; Humans; Kidney Transplantation; Mice; Organ Preservation; Perfusion; Reperfusion Injury
PubMed: 34295329
DOI: 10.3389/fimmu.2021.673562 -
Indian Journal of Ophthalmology Sep 2021To compare the physical and microbiological characteristics of McCarey-Kaufman (MK), Cornisol, and Optisol-GS media and evaluate the outcomes of keratoplasty performed...
PURPOSE
To compare the physical and microbiological characteristics of McCarey-Kaufman (MK), Cornisol, and Optisol-GS media and evaluate the outcomes of keratoplasty performed using corneas stored in these three media.
METHODS
The study involved 60 donor corneas which were distributed in 3 groups: MK, Cornisol, and Optisol-GS. Corneas in these groups were further analyzed based on the type of keratoplasty performed (full thickness versus endothelial keratoplasty). At baseline, the endothelial cell density and death to preservation time of donor corneas were recorded. Following keratoplasty, patients were evaluated on day 1, at 1 month, 3 months, and 6 months follow-up. Outcomes were assessed in terms of corrected distance visual acuity (CDVA), endothelial cell density, percentage endothelial cell loss, and corneal thickness. The storage media were also assessed for their physical quality and their microbiological characteristics.
RESULTS
Physical characteristics of all three media were found to be within normal limits. Mean CDVA was comparable among the 3 groups at 6-month follow-up. The absolute endothelial cell count values were significantly lower for corneas stored in MK medium (1873.7 ± 261.1 cells/mm) compared to the Cornisol (2085.0 ± 230.3 cells/mm) and Optisol-GS media [(2180.3 ± 217.2 cells/mm) (P = <0.001)]. Corneas stored in Optisol-GS medium were significantly thinner at 1-month follow-up with no significant difference at 6 months (P = 0.66).
CONCLUSION
Optisol-GS and Cornisol media were found to preserve endothelial cell density better and stabilize corneal thickness earlier as compared to the MK medium. However, the functional outcomes were comparable among the three groups.
Topics: Cornea; Culture Media, Serum-Free; Endothelium, Corneal; Humans; Organ Preservation; Tissue Donors
PubMed: 34427243
DOI: 10.4103/ijo.IJO_258_21