New alternatives to holder pasteurization in processing donor milk in human milk banks.

Infectious and toxicological risks are the main potential hazards that operators of Human Milk Banks (HMBs) encounter and must eliminate. HMBs are trying to implement procedures that allow to manage and sanitize human milk without altering significantly its nutritional and biologically protective components, obtaining a product characterized by a valid balance between safety and biological quality. The history of human milk processing is linked to the origins of HMBs themselves. And although other forms of sterilization were used originally, pasteurization soon became the recognized most effective means for sanitizing milk: all the milk that arrives at the HMB must be pasteurized. Holder pasteurization (HoP) is the most used methodology, and it is performed using low temperature and long time (+62.5°C for 30 min). With HoP some bioactive milk components are lost to varying degrees, but many other precious bioactive compounds are completely or partially preserved. To improve the quality of human milk processed by HMBs, maintaining in the meantime the same microbiological safety offered by HoP, new technologies are under evaluation. At present, High-Temperature Short-Time pasteurization (HTST) and High-Pressure Processing are the most studied methodologies. HTST is already utilized in some HMBs for daily practical activity and for research purposes. They seem to be superior to HoP for a better preservation of some nutritional and biologically protective components. Freeze-drying or lyophilization may have advantages for room temperature storage and transportation. The aim of this study is to evaluate the advancement regarding the processing of DHM with a literature search from 2019 to 2022. The effects of the new technologies on safety and quality of human milk are presented and discussed. The new technologies should assure microbiological safety of the final product at least at the same level as optimized HoP, with an improved preservation of the nutritional and bioactive components of raw human milk.

Intra-articular injection of platelet lysate-derived extracellular vesicles recovers from knee osteoarthritis in an in vivo rat model.

Objective: MSCs and Platelet-Rich Plasma are the main focus in the study of new regenerative treatments aimed to reverse Osteoarthritis (OA). However, extracellular vesicles (EVs) present several advantages to cell-based treatments. Thus, the aim of this study was to compare and evaluate the regenerative potential of MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in an OA cartilage rat model. Design: OA in vivo model was established through injection of 6 mg MIA in the rat knee joints. After 14 and 21 days, OA knee joints were treated with 1 × 1010 particles of pEVs or cEVs. At day 28, the animals were sacrificed, plasma was collected to quantify CTX-II and knee joints were excised to be evaluated by Cone Beam Computed Tomography (CBCT). After decalcification, histology was used to determine the OARSI score and to visualize collagen and glycosaminoglycan content. Results: pEVs and cEVs samples did not show significant differences per se but they did in terms of regenerative effects on OA knee joints. pEVs-treated knee joints showed better subchondral bone integrity in CT-analysed parameters when compared to cEVs or OA group, showing similar values to the healthy control group. Moreover, OARSI score indicated that pEVs showed a greater OA reversion in knee joints, especially in female rats, and so indicated the analysed histological images. Conclusions: pEVs are proposed as a viable regeneration treatment for OA since they are not only capable of exerting their regenerative potential on osteoarthritic cartilage, but also outperform cEVs in terms of efficacy, particularly in females. Significance statement: Osteoarthritis (OA) is one of the most age-related diseases. It is estimated that 500 million people suffer from OA worldwide, representing the principal cause of chronic disability in adults. In the present study we evaluated the therapeutic effect of extracellular vesicles (EVs) from different sources (platelet lysate and human umbilical cord mesenchymal stromal cells) in an in vivo rat model. Our results demonstrate that platelet-derived EVs (pEVs) induce an OA reversion in knee joints, thus evidencing the therapeutic potential of pEVs as cell-free regenerative agents for OA treatment. The translational potential of this article: Platelet-derived extracellular vesicles (pEVs) offer a promising cell-free therapy option for OA treatment. Their production could be easily standardized and reproduced without extensive platelet harvesting and amplification, thus paving the way for their clinical translation.

Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model.

Aims: Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. Methods: pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 109 particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs. Results: Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group. Conclusion: In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine.

Impact of time-temperature combinations on the anti-Cytomegalovirus activity and biological components of human milk.

BACKGROUND: There is extensive evidence that Holder pasteurization (HoP) (30 min at 62.5 °C) has harmful effects on the bioactivities of human milk (HM). We previously demonstrated that lowering HoP temperature is sufficient to inactivate Cytomegalovirus (HCMV). Here, we analyzed the effect of lowering time/temperature on the antiviral activity against HCMV and IgA levels of HM. METHODS: Eighty HM samples from five mothers were pasteurized in a range of temperature (62.5-56 °C) and time (40-10 min) in a conventional setting of Human Milk Bank. Unpasteurized HM from each mother was used as control. The samples were assayed against HCMV-AD169 strain in cell cultures and IgA levels were determined by ELISA. RESULTS: All HM samples exhibited anti-HCMV activity, to a different extent. An improvement of antiviral activity was observed in samples treated at 60, 58 and 56 °C compared to those at 62.5 °C, with ID50 values near those of unpasteurized milk. Similarly, better retention in IgA levels was observed by reducing the temperature of treatment. CONCLUSIONS: We demonstrated that a 2.5 °C reduction of heat treatment significantly preserved the IgA content and fully restored the anti-HCMV activity of HM, supporting this variant of HoP as a valid alternative to preserve HM bioactivities. IMPACT: This work questions the standard HoP and opens the debate on whether the pasteurization temperature commonly used in Human Milk Banks should be lowered to better preserve the biological components of the milk. A reduction of HoP temperature at 60 °C determined a significant preservation of anti-HCMV activity and IgA content of donor HM, compared to standard HoP. This alternative HoP is highly feasible compared to other substitute pasteurization techniques, since it would employ the same pasteurizer equipment found in most Human Milk Banks.

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications.

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

Customizing the extracellular vesicles release and effect by strategizing surface functionalization of titanium.

Metallic material functionalization with Extracellular Vesicles (EVs) is a desirable therapeutic approach to improve regenerative procedures. Among the different functionalization strategies available, here we have compared drop casting on machined Ti surfaces, drop casting on nanostructured TiO2 surfaces and polymeric entrapment with polydopamine. EVs are a heterogeneous population of communication nanovesicles released by cells that are being intensively investigated for their use in therapeutics. We have selected platelet derived EVs for Ti surface coating due to their demonstrated osteoinductive properties. Our results show that each functionalization strategy leads to differences in the size of EV populations attached to and released from the metallic implants, which, in turn, leads to variations in their osteogenic capability measured through alkaline phosphatase activity and calcium deposition. In conclusion, the functionalization strategy used has an important effect on the resulting implant functionality, probably due to the heterogeneous EVs nature. Thus, the methodological approach to metallic material functionalization should be carefully chosen when working with extracellular vesicles in order to obtain the desired therapeutic application.

The legislative framework of donor human milk and human milk banking in Europe.

Data about the regulatory approaches to donor human milk (DHM) in European countries are lacking. The aim of this study is to describe the various regulations of DHM within European countries, to assess its legislative context and its impact in relation to donor milk banking. We performed a cross-sectional survey using a semistructured online questionnaire addressing 29 national European milk-banking representatives from June 2020 to February 2021. Representatives of 26 national DHM services participated in this study. The legal classification and regulatory status of DHM were defined in 9 out of 26 areas of jurisdiction (35%) as either food product (n = 6), product of human origin according to a blood, tissue, cell regulation (n = 2), or medicinal product (n = 1). In the remainder, DHM remains unclassified. Most legislations did not provide a comprehensive framework concerning DHM and costs to cover milk bank operations were rarely reimbursed. In general, the lack of national legislative governance and the actual legislative regulations in place do not support the use of DHM in European countries. National medical guidelines for the use of DHM have been issued in only 11 countries. The current number and distribution of milk banks (n = 239) within participating countries may not provide an equitable access to DHM for eligible infants. These findings could guide stakeholders aiming to establish a regulatory framework for DHM.

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 µm sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Analysis of Thermal Sensitivity of Human Cytomegalovirus Assayed in the Conventional Conditions of a Human Milk Bank.

One of the main concerns in human milk banks (HMB) is the transmission of human cytomegalovirus (HCMV) that could be present in the milk of infected women. There are consistent data showing that this virus is destroyed by Holder pasteurization (62.5°C for 30 min), but there is a lack of information about the response of the virus to the treatment at lower temperatures in strict HMB conditions. In order to analyze the effectiveness of different temperatures of pasteurization to eliminate HCMV in human milk, a preliminary assay was performed incubating HCMV-spiked raw milk samples from donor mothers at tested temperatures in a PCR thermocycler and the viral infectivity was assayed on cell cultures. No signs of viral replication were observed after treatments at temperatures equal or >53°C for 30, 20, and 10 min, 58°C for 5 min, 59°C for 2 min, and 60°C for 1 min. These data were confirmed in a pasteurizer-like model introducing HCMV-spiked milk in disposable baby bottles. No viral infectivity was detected on cell cultures after heating treatment of milk for 30 min at temperatures from 56 to 60°C. Thus, our results show that by using conventional pasteurization conditions, temperatures in the range of 56-60°C are enough to inactivate HCMV. Consequently, we consider that, in order to provide a higher quality product, the current recommendation to pasteurize both mother's own milk and donated milk at 62.5°C must be re-evaluated.

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants.

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as proteins and nucleic acids, which present great potential in regenerative medicine. More recently, EVs derived from Platelet Lysate (PL) have shown an osteogenic capability comparable to PL. Besides, biomaterials are frequently used in orthopedics or dental restoration. Here, we provide a method to functionalize Ti surfaces with PL-derived EVs in order to improve their osteogenic properties. EVs are isolated from PL by size exclusion chromatography, and afterward Ti surfaces are functionalized with PL-EVs by drop casting. Functionalization is proven by EVs release and its biocompatibility by the lactate dehydrogenase (LDH) release assay.

Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.

AIMS: Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). METHODS: EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content. RESULTS: Osteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response. CONCLUSION: PL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations.Cite this article: Bone Joint Res 2020;9(10):667-674.

Intracoronary injection of haematopoietic precursor cells regenerates the borders, but not the core, of old myocardial scars.

AIMS: Cell therapy regenerative potential is hindered by cell access to the infarct zone. We studied function recovery at the scar zone and its impact in global left ventricular function after intracoronary injection of haematopoietic precursor cells. METHODS AND RESULTS: Haematopoietic precursor cells were obtained by blood apheresis in patients with an old myocardial infarction, and the presence of CD34+ and CD133+ cells was quantified. Left ventricular function, volumes, and infarct zone segmental motion were measured by magnetic resonance imaging (MRI) and echo left ventricular segmental strain (LVSS). The aphaeresis product was administered to 20 patients in the coronary artery responsible for the myocardial infarction. High cell yield in blood aphaeresis product allowed us to inject a high number of cells in most patients. Three patients were excluded because of insufficient CD133+ cell number, and one more patient was excluded because of artefacts in MRI images. The remaining 16 patients were compared with 16 controls. After 1 year, infarct zone reduction was related to the number of CD133+ (R = 0.53; P %3C 0.05) and CD34+ (R = 0.63; P %3C 0.01) cells injected. The number of CD133+ cells injected was also related to an improvement in LVSS (R = 0.62; P %3C 0.01). In turn, scar zone reduction was related to an improvement in LVSS (R = 0.64; P %3C 0.01). End-diastolic volume showed a reduction at follow-up in the treated group when compared with control patients. MRI infarct area segments systolic thickness increase improved after treatment in treated patients [expressed as median (interquartile range)] [0.42 (-0.38 to 1.14) vs. 1.06 (-0.10 to 2.12) mm; P %3C 0.01], but not in controls [2.02 (0.75 to 3.4) vs. 1.91 (0.77-3.17) mm; P = not significant (n.s.)]. In cell therapy patients, the borders of the infarct zone, but not the core, showed a significant recovery [proximal rim: 0.48 (-0.18 to 1.33) vs. 1.07 (0.22-2.40) mm; P %3C 0.05, distal rim: 0.75 (0.26-1.40) vs. 1.76 (0.65-2.86) mm; P %3C 0.05, and core: 0.36 (-0.33 to 1.20) vs. 0.60 (-0.18 to 1.62) mm; P = n.s.]. That improvement was not observed in the control group [proximal rim: 1.20 (0.33-2.53) vs. 0.82 (-0.13 to 1.65) mm; P = n.s., distal rim: 1.24 (0.80-1.72) vs. 0.96 (0.19-1.81) mm; P = n.s., and core: 0.30 (-0.42 to 1.64) vs. 0.07 (-0.60 to 1.20) mm; P = n.s.]. Only small size infarcts showed a complete recovery in the cell therapy patients [systolic thickness increase post-treatment increment in infarcts ≤6 segments vs. >6 segments affected: 0.28 (-0.19 to 0.71) vs. -1.21 (-2.60 to -0.53) mm; P %3C 0.01]. CONCLUSIONS: Intracoronary injection of peripheral blood-derived haematopoietic precursor cells produces a complete recovery of the borders and partial regeneration of the infarct core, which is directly related to the number of CD133+ and CD34+ cells injected. Cell therapy infarct zone regeneration prevents ventricular remodelling by preserving segmental contractility and halting left ventricular dilatation.

Generation of a human iPSC line (IMEDEAi008-A) derived from natural homozygous CCR5-Δ32 PBMCs enriched in the pro-erythroblast population.

A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Δ32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Δ32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Δ32/Δ32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Δ32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers.

Effect of Nonthermal Processing on Human Milk Bactericidal Activity Against Escherichia coli.

Nonthermal methods are more efficient at preserving various biological properties of human milk, as compared with holder pasteurization (HoP), which is the most common preservation method. This study was performed to assess the effects of nonthermal processing on bactericidal activity against Escherichia coli in human milk. Milk samples obtained from the Regional Human Milk Bank in Warsaw at Holy Family Hospital were processed by HoP, irradiated with ultraviolet-C (UV-C) for 5, 10, and 15 minutes (6720 J/L each minute), subjected to 2 variations of high-pressure processing (HPP): 450 MPa for 15 minutes and 200 MPa for 10 minutes + 400 MPa for 10 min, with a 10-minutes break. The samples were then evaluated by a bactericidal assay (raw untreated human milk was used as a control). The bactericidal capacity after HoP was preserved in 12.1% of samples, showing a significant reduction in bactericidal properties compared with in raw milk (P < 0.05). The differences between samples preserved by nonthermal methods and raw milk were not significant (P > 0.05). Nonthermal methods of human milk treatment better preserve the bactericidal capacity compared with holder pasteurisation. Those alternative technologies to HoP can be proposed after further investigation for milk processing for Human Milk Banks facilities.

Improved physical and osteoinductive properties of demineralized bone matrix by gelatin methacryloyl formulation.

The demineralized bone matrix (DBM) is the most widely used bone allograft, which is obtained by removing the mineral component of bone, leading to exposure of the proteins responsible for osteoinduction. For clinical use, DBM shall be formulated with a carrier that provides consistency and improves its osteoinduction. In this study, three DBM formulations with glycerol (Gly), hyaluronic acid (HA), and gelatin methacryloyl (GelMA) were evaluated measuring their physicochemical properties (microstructure, compressive strength, and serum cohesivity) and their osteoinductive capacity both in vitro using C2C12 cells and umbilical cord human mesenchymal stem cells and in vivo in an ectopic bone formation model in athymic mice. To assess the effectiveness of DBM in vitro in inducing the differentiation into osteoblasts, alkaline phosphatase (ALP) activity was assessed in combination with a cytotoxicity assay. In vivo, new bone formation was assessed by histological and radiological analysis. In the compression and in the serum cohesive assays, the GelMA DBM formulation showed its superiority over the other formulations. In addition, GelMA showed a more compact structure analysed by scanning electron microscopy. Higher cell toxicity was observed on Gly formulations in vitro, whereas GelMa and HA showed very low toxicity. All formulations significantly improved ALP activity compared with control. In the in vivo studies, GelMA showed the greatest osteoinductive potential with a higher percentage of new bone and bone marrow formation. Our results suggest GelMA is useful as a carrier for DBM designed to promote the formation of the new bone.

Purity Determines the Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) have been recently identified as vital components of cell-based therapies based on the observation that conditioned media from cultured stromal cells reproduce some of the beneficial effects of intact cells. In order to obtain clinically active EVs derived from Mesenchymal Stromal Cells (MSCs) different procedures have been reported in the literature. Usually, non-confluent cells are incubated with culture medium for 48 h either with EV-depleted Fetal Bovine Serum (FBS) or without FBS. Our aim was to compare the effects of EVs isolated by ultracentrifugation from human umbilical cord MSC conditioned media obtained using these two conditions: with EV-depleted FBS (UC) or without FBS (UCw/o) on the mRNA expression levels of extracellular matrix related genes using the mouse chondrogenic cell line ATDC-5. We observed a deleterious effect on chondrogenic cells treated with UCw/o, showing higher mRNA expression levels of different metalloproteinases and decorin (Dcn) and lower collagen (Col1a1 and Col2a1) and aggrecan (Acan) mRNA levels. To elucidate whether this deleterious effect was induced by the EVs or by any proteins co-purified in the EV pellet, we used size exclusion chromatography (SEC) to further purify the EV pellet, obtaining an EV enriched fraction (EV or EVw/o) and a protein enriched fraction (Prot or Protw/o). Our results pointed that the negative effect on the chondrogenic cell line was due to the contaminant proteins coisolated with the EVs by ultracentrifugation and not from the EVs themselves. Thus, these results highlight the importance of working with well purified EV preparations to specifically achieve their therapeutic effect.

New Bicistronic TALENs Greatly Improve Genome Editing.

Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.

Tissue Donors in Regional Hospitals: A Reality.

Progress in transplantation means the process of procuring both human organ and tissues has become a daily challenge. Considering that tissues are usually procured from organ donors who have suffered brain death or after controlled cardiac death, the tissue procurement is done mainly in major hospitals. With the aim of highlighting the potential role of regional hospitals in tissue donation, a prospective descriptive study was carried out of all the patients who died at the Regional Hospital of Inca in Mallorca (Spain) from January 2013 to August 2018. To ensure an early detection of all possible tissue donors, the hospital has implemented a computerized alert system that is activated immediately after a patient dies. This strategy has proven to be very useful as in the analyzed period, the hospital had an average of 280 donors per million population, which is one of the highest rates of cornea donation among the Spanish hospitals. Our data and experience show the important role of regional hospitals in tissue donation and the value of implementing screening programs and early selection of potential tissue donors.

Fortification of Human Milk for Preterm Infants: Update and Recommendations of the European Milk Bank Association (EMBA) Working Group on Human Milk Fortification.

Evidence indicates that human milk (HM) is the best form of nutrition uniquely suited not only to term but also to preterm infants conferring health benefits in both the short and long-term. However, HM does not provide sufficient nutrition for the very low birth weight (VLBW) infant when fed at the usual feeding volumes leading to slow growth with the risk of neurocognitive impairment and other poor health outcomes such as retinopathy and bronchopulmonary dysplasia. HM should be supplemented (fortified) with the nutrients in short supply, particularly with protein, calcium, and phosphate to meet the high requirements of this group of babies. In this paper the European Milk Bank Association (EMBA) Working Group on HM Fortification discusses the existing evidence in this field, gives an overview of different fortification approaches and definitions, outlines the gaps in knowledge and gives recommendations for practice and suggestions for future research. EMBA recognizes that "Standard Fortification," which is currently the most utilized regimen in neonatal intensive care units, still falls short in supplying sufficient protein for some VLBW infants. EMBA encourages the use of "Individualized Fortification" to optimize nutrient intake. "Adjustable Fortification" and "Targeted Fortification" are 2 methods of individualized fortification. The quality and source of human milk fortifiers constitute another important topic. There is work looking at human milk derived fortifiers, but it is still too early to draw precise conclusions about their use. The pros and cons are discussed in this Commentary in addition to the evidence around use of fortifiers post discharge.

Processing of Donor Human Milk: Update and Recommendations From the European Milk Bank Association (EMBA).

Background: A mother's own milk (MOM) is the gold standard for the feeding and nutrition of preterm and full term infants. When MOM is not available or there is not enough, donor human milk (DHM) should be used. Milk delivered to Human Milk Banks (HMBs) should be pasteurized to inactivate viral and bacterial agents. Currently, a pasteurization process at 62.5°C for 30 min (Holder pasteurization, HoP) is recommended in all international HMBs guidelines. State of the art: It is known that HoP affects some of the nutritional and biological components of human milk. Studies have demonstrated that temperature cycle in HoP is not always controlled or calibrated. A better check of these parameters in the pasteurizers on the market today may contribute to an improvement of the quality of HM, still maintaining some of the negative effects of the heat treatment of human milk. So, food industry, and dairy industry in particular, are evaluating innovative methodologies alternative to HoP to better preserve the nutritional and biological properties of fresh human milk, while assuring at least the same microbiological safety of HoP. The most studied processing techniques include High-Temperature-Short-Time (HTST) pasteurization, High Pressure Processing (HPP), and Ultraviolet-C (UV-C) irradiation. HTST is a thermal process in which milk is forced between plates or pipes that are heated on the outside by hot water at a temperature of 72°C for 5-15 s. HPP is a non-thermal processing method that can be applied to solid and liquid foods. This technology inactivates pathogenic microorganisms by applying a high hydrostatic pressure (usually 300-800 MPa) during short-term treatments (<5-10 min). UV irradiation utilizes short-wavelength ultraviolet radiation in the UV-C region (200-280 nm), which is harmful to microorganisms. It is effective in destroying the nucleic acids in these organisms, so that their DNA is disrupted by UV radiation. Aim: The aim of this paper is to present the EMBA recommendations on processing of HM, based on the most recent results obtained with these new technologies. Conclusions: Although research on the most promising technologies that will represent an alternative to HoP (HTST, HPP, UV-C) in the future is progressing, it is now important to recognize that the consistency and quality assurance of the pasteurizers on the market today represent a fundamental component that was previously lacking in the Holder approach.