METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes.

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading to repeated transfer cycles. To reduce time-to-pregnancy and stress for patients, there is a need for a diagnostic tool to better select embryos and oocytes based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging and Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination and AI to deliver the metabolic profile of embryos and oocytes based on intrinsic autofluorescence signals. We used it to classify i) mouse blastocysts cultured under standard conditions or with depletion of selected metabolites (glucose, pyruvate, lactate); and ii) oocytes from young and old mouse females, or in vitro-aged oocytes. The imaging process was safe for blastocysts and oocytes. The METAPHOR classification of control vs. metabolites-depleted embryos reached an area under the ROC curve (AUC) of 93.7%, compared to 51% achieved for human grading using brightfield imaging. The binary classification of young vs. old/in vitro-aged oocytes and their blastulation prediction using METAPHOR reached an AUC of 96.2% and 82.2%, respectively. Finally, organelle recognition and segmentation based on the flavin adenine dinucleotide signal revealed that quantification of mitochondria size and distribution can be used as a biomarker to classify oocytes and embryos. The performance and safety of the method highlight the accuracy of noninvasive metabolic imaging as a complementary approach to evaluate oocytes and embryos based on their physiology.

On the Design of a Network Digital Twin for the Radio Access Network in 5G and Beyond.

A Network Digital Twin (NDT) is a high-fidelity digital mirror of a real network. Given the increasing complexity of 5G and beyond networks, the use of an NDT becomes useful as a platform for testing configurations and algorithms prior to their application in the real network, as well as for predicting the performance of such algorithms under different conditions. While an NDT can be defined for the different subsystems of the network, this paper proposes an NDT architecture focusing on the Radio Access Network (RAN), describing the components to represent and model the operation of the different RAN elements, and to perform emulations. Different application use cases are identified, and among them, the paper puts the focus on the training of Reinforcement Learning (RL) solutions for the RAN. For this use case, the paper introduces a framework aligned with O-RAN specifications and discusses the functionalities needed to integrate the NDT. This use case is illustrated with the description of a RAN NDT implementation used for training an RL-based capacity-sharing solution for network slicing. Presented results demonstrate that the implemented RAN NDT is a suitable platform to successfully train the RL solution, achieving service-level agreement satisfaction values above 85%.

Radiomics-Based Assessment of OCT Angiography Images for Diabetic Retinopathy Diagnosis.

Purpose: To evaluate the diagnostic accuracy of machine learning (ML) techniques applied to radiomic features extracted from OCT and OCT angiography (OCTA) images for diabetes mellitus (DM), diabetic retinopathy (DR), and referable DR (R-DR) diagnosis. Design: Cross-sectional analysis of a retinal image dataset from a previous prospective OCTA study (ClinicalTrials.govNCT03422965). Participants: Patients with type 1 DM and controls included in the progenitor study. Methods: Radiomic features were extracted from fundus retinographies, OCT, and OCTA images in each study eye. Logistic regression, linear discriminant analysis, support vector classifier (SVC)-linear, SVC-radial basis function, and random forest models were created to evaluate their diagnostic accuracy for DM, DR, and R-DR diagnosis in all image types. Main Outcome Measures: Area under the receiver operating characteristic curve (AUC) mean and standard deviation for each ML model and each individual and combined image types. Results: A dataset of 726 eyes (439 individuals) were included. For DM diagnosis, the greatest AUC was observed for OCT (0.82, 0.03). For DR detection, the greatest AUC was observed for OCTA (0.77, 0.03), especially in the 3 × 3 mm superficial capillary plexus OCTA scan (0.76, 0.04). For R-DR diagnosis, the greatest AUC was observed for OCTA (0.87, 0.12) and the deep capillary plexus OCTA scan (0.86, 0.08). The addition of clinical variables (age, sex, etc.) improved most models AUC for DM, DR and R-DR diagnosis. The performance of the models was similar in unilateral and bilateral eyes image datasets. Conclusions: Radiomics extracted from OCT and OCTA images allow identification of patients with DM, DR, and R-DR using standard ML classifiers. OCT was the best test for DM diagnosis, OCTA for DR and R-DR diagnosis and the addition of clinical variables improved most models. This pioneer study demonstrates that radiomics-based ML techniques applied to OCT and OCTA images may be an option for DR screening in patients with type 1 DM. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Description of the first brachypterous Lonchidia species (Hymenoptera: Figitidae: Figitinae), with a key to European species.

The male and female of a new species of Lonchidia Thomson, 1862, L. atypica Pujade-Villar & de Paz sp. nov., are described from the provinces of Salamanca and Cceres (Western Spain). Specimens were collected with a G-Vac suction sampler and pitfall traps from traditional almond and cherry orchards. The diagnostic characters of this species are: deep but short notauli, reaching only the anterior part of the mesoscutum, and brachypterous wings. Both sexes are illustrated and an identification key is proposed herein to differentiate the European species. The possibility that the male of L. clavicornis Thomson, 1862 corresponds to a different species is also discussed.

A catalogue, revision, and regional perspective of Eastern Palaearctic and Oriental oak gall wasps and their inquilines (Hymenoptera: Cynipidae: Cynipini, Synergini, Ceroptresini).

Recent years have seen rapid advances in the study of Fagaceae-associated gall wasps (Hymenoptera: Cynipidae) of the Eastern Palaearctic and the Oriental (EPO) regions, for both the gall inducing Cynipini (commonly termed oak gall wasps though many species gall non-oak Fagaceae) and the predominantly inquiline tribes Synergini and Ceroptresini. This process has propagated some taxonomic errors and involves many taxa whose taxonomic status is uncertain. To provide a stable foundation for further advance, here we review the taxonomic status of the 212 species (133 oak gall wasps and 79 oak cynipid inquilines) that have been described or recorded in these regions. Of this total, we treat 171 as valid species names (103 oak gall wasps and 68 oak cynipid inquilines) in 20 genera; 22 as synonym names, 13 as incertae sedis, three as nomen dubium, and three as species inquirenda. Callirhytis kunugicola Shinji, 1944 is proposed as syn. nov. of C. kunugicola Shinji, 1943, as Shinji described the same species twice. For all valid species names, we provide taxonomic references, synonyms, and geographical distributions. We summarize what is known of host plant associations and gall locations for gall inducers, and host associations for inquilines. We discuss geographic patterns in the known richness of currently valid species for both groups of organisms, and place this in the context of the biogeography of available Fagaceae host plants. We provide a brief historical review of the study of oak gall wasps and their inquilines in the EPO regions.

A Deep Q-Network-Based Algorithm for Multi-Connectivity Optimization in Heterogeneous Cellular-Networks.

The use of multi-connectivity has become a useful tool to manage the traffic in heterogeneous cellular network deployments, since it allows a device to be simultaneously connected to multiple cells. The proper exploitation of this technique requires to adequately configure the traffic sent through each cell depending on the experienced conditions. This motivates this work, which tackles the problem of how to optimally split the traffic among the cells when the multi-connectivity feature is used. To this end, the paper proposes the use of a deep reinforcement learning solution based on a Deep Q-Network (DQN) in order to determine the amount of traffic of a device that needs to be delivered through each cell, making the decision as a function of the current traffic and radio conditions. The obtained results show a near-optimal performance of the DQN-based solution with an average difference of only 3.9% in terms of reward with respect to the optimum strategy. Moreover, the solution clearly outperforms a reference scheme based on Signal to Interference Noise Ratio (SINR) with differences of up to 50% in terms of reward and up to 166% in terms of throughput for certain situations. Overall, the presented results show the promising performance of the DQN-based approach that establishes a basis for further research in the topic of multi-connectivity and for the application of this type of techniques in other problems of the radio access network.

A new species of the inquilinous gall wasp genus Saphonecrus Dalla-Torre & Kieffer (Hymenoptera: Cynipidae: Synergini) from South Korea, Saphonecrus jejuensis Kang & Lobato-Vila, sp. nov.

A new species of the inquilinous gall wasp genus Saphonecrus (Hymenoptera: Cynipidae: Synergini) reared from undetermined cryptic stem galls on Quercus glauca Thunb., Saphonecrus jejuensis Kang & Lobato-Vila, sp. nov., is described from South Korea. A detailed description, diagnosis, data on biology and pictures of both the new species and the host galls are provided. An identification key to the Saphonecrus species found in South Korea is also given.

A new cynipid inquiline of the genus Lithosaphonecrus (Hymenoptera: Cynipidae: Synergini) from India, with an updated key to all known species.

A new species of cynipid inquiline, Lithosaphonecrus nagalandi Melika, Lobato-Vila Pujade-Villar, sp. nov. (Hymenoptera: Cynipidae: Synergini), is described from the state of Nagaland (India) reared from undescribed bud galls on an undetermined species of Lithocarpus (Fagaceae), thus being the first Lithosaphonecrus known from India. Description, diagnosis, data on phenology and host associations, and illustrations of the new species are given. An identification key to all known Lithosaphonecrus is also provided. The biology of the genus Lithosaphonecrus and the distribution range of Saphonecrus and Lithosaphonecrus in Asia and Oceania are discussed.

A Taxonomic Review of the Gall Wasp Genus Saphonecrus Dalla-Torre and Kieffer and other Oak Cynipid Inquilines (Hymenoptera: Cynipidae) from Mainland China, with Updated Keys to Eastern Palaearctic and Oriental Species.

After the examination of the oak cynipid inquilines deposited in the Parasitic Hymenoptera Collection of the Agriculture and Forestry University of Zhejiang (ZAFU, China), we provide a revision of the species of Saphonecrus, Lithosaphonecrus, Ufo (Hymenoptera: Cynipidae: Synergini) and Ceroptres (Hymenoptera: Cynipidae: Ceroptresini) found in mainland China. Two new species of Saphonecrus are described: S. albidus Lobato-Vila and Pujade-Villar, sp. nov. and S. segmentatus Lobato-Vila and Pujade-Villar, sp. nov. Four Saphonecrus species (S. gilvus Melika and Schwéger, 2015, S. globosus Schwéger and Tang, 2015, S. leleyi Melika and Schwéger, 2015, and S. nantoui Tang, Schwéger and Melika, 2015) are new records for this region. We also provide new data on the biology and distribution, redescriptions, illustrations and corrections to some of the species, as well as updated keys to Eastern Palaearctic and Oriental species of Saphonecrus and Ufo. Three species of Saphonecrus are considered to have an uncertain status: S. gemmariae (Ashmead, 1885) incertae sedis from the USA, S. excisus (Kieffer, 1904) nomen dubium from India, and S. sinicus Belizin, 1968 incertae sedis from China. The validity of Saphonecrus in the Nearctic region as well as the current status of S. serratus Weld, 1926 from the Philippines and S. favanus Weld, 1944 from the USA are discussed.

The genus Synergus Hartig (Hymenoptera: Cynipidae: Synergini) in the New World: a complete taxonomic revision with a key to species.

A complete revision of the genus Synergus Hartig (Hymenoptera: Cynipidae: Synergini) in the New World (Nearctic and Neotropical regions) is conducted for the first time in order to stabilize its taxonomy. A total of 11 new species are described from Mexico: S. ashmeadi Lobato-Vila Pujade-Villar, sp. nov.; S. beutenmulleri Lobato-Vila Pujade-Villar, sp. nov.; S. compressus Lobato-Vila Pujade-Villar, sp. nov.; S. diversicolor Lobato-Vila Pujade-Villar, sp. nov.; S. ebenus Lobato-Vila Pujade-Villar, sp. nov.; S. linnei Lobato-Vila Pujade-Villar, sp. nov.; S. macrackenae Lobato-Vila Pujade-Villar, sp. nov.; S. oaxaquensis Lobato-Vila Pujade-Villar, sp. nov.; S. personatus Lobato-Vila Pujade-Villar, sp. nov.; S. ruficephalus Lobato-Vila Pujade-Villar, sp. nov.; and S. weldi Lobato-Vila Pujade-Villar, sp. nov. Synergus splendidus Fullaway, 1911 is proposed as a syn. nov. of S. dorsalis (Provacher, 1889); S. garryana Gillette, 1893 and S. oneratus coloradensis Gillette, 1896 as syn. nov. of S. oneratus (Harris, 1841); and S. magnificus Weld, 1957 as a syn. nov. of S. reniformis McCracken Egbert, 1922. Redescriptions and illustrations are provided for poorly characterized species. A key to species and a summary table including all valid Synergus from the New World, their biology and distribution, are given. Distribution, morphology, and trophic associations are discussed and compared between New World and Palaearctic species.

Taxonomic assessment of the inquiline fauna (Hymenoptera: Cynipidae: Synergini, Ceroptresini) reared from cynipid galls on oaks (Quercus spp.) from South Korea.

A complete taxonomic assessment of the inquiline fauna (Hymenoptera: Cynipidae: Synergini, Ceroptresini) reared from cynipid oak galls collected in South Korea is conducted for the first time. Previously, six species were known from this country (Ceroptres kovalevi Belizin, 1973; Synergus chinensis Melika, Ács Bechtold, 2004; Synergus japonicus Walker, 1874; Ufo koreanus Melika, Pujade-Villar Choi, 2007; and presumably, Saphonecrus chaodongzhui Melika, Ács Bechtold, 2004 and Ufo cerroneuroteri Tang Melika, 2012). The study of new material reared from cynipid galls collected from oaks (Quercus spp.) from various locations throughout South Korea has increased the total number of inquiline species known from this country to 14, of which seven are new country records (Saphonecrus shirakashii (Shinji, 1940); Sa. symbioticus Melika Schwéger, 2015; Synergus abei Melika Schwéger, 2015; S. belizinellus Schwéger Melika 2015; S. formosanus Schwéger Melika, 2015; S. ishikarii Melika Schwéger, 2015; and S. symbioticus Schwéger Melika, 2015), and one is a new species: Synergus minutus Lobato-Vila Pujade-Villar, sp. nov. The new species is formally described and illustrated. New biological and distribution data are provided for both the newly recorded species and the other reared species. The Korean fauna is compared with that from other countries within the Eastern Palearctic and the Oriental regions.

Synergus Hartig species group (Hymenoptera: Cynipidae: Synergini) with partially smooth mesopleurae from the New World.

Inquiline oak gall wasps from the genus Synergus Hartig (Hymenoptera: Cynipidae: Synergini) that occur in the New World should be classified into the following morphological groups: i) species with the radial cell of fore wings open; ii) species with the radial cell of fore wings closed and mesopleurae completely sculptured, always with transversal striae covering its surface; iii) species with the radial cell of fore wings closed and mesopleurae partially smooth, with the speculum always shiny and not sculptured. The latter group, which appears to be unique to the New World's fauna, is the focus of this study. An exhaustive taxonomical revision of all the Synergus species within this morphological group is carried out for the first time after the initial treatment by Lobato-Vila Pujade-Villar (2017). Redescriptions, images and new distribution and biological data are provided. A new species from Mexico, Synergus aurofacies Lobato-Vila Pujade-Villar, sp. nov., is here formally described and illustrated. Synergus variegatus McCracken Egbert, 1922 is a junior synonym of S. flavens McCracken Egbert, 1922 syn. nov.; S. profusus McCracken Egbert, 1922 is a junior synonym of S. pacificus McCracken Egbert, 1922 syn. nov.; and S. flavus Kieffer, 1904 and S. varicolor Fullaway, 1911 are junior synonyms of S. pomiformis (Ashmead, 1885) syn. nov. A key to species of this group is provided. The morphological traits of the species with partially smooth mesopleurae are discussed.

Evaluation of a cell-based osteogenic formulation compliant with good manufacturing practice for use in tissue engineering.

Proper bony tissue regeneration requires mechanical stabilization, an osteogenic biological activity and appropriate scaffolds. The latter two elements can be combined in a hydrogel format for effective delivery, so it can readily adapt to the architecture of the defect. We evaluated a Good Manufacturing Practice-compliant formulation composed of bone marrow-derived mesenchymal stromal cells in combination with bone particles (Ø = 0.25 to 1 µm) and fibrin, which can be readily translated into the clinical setting for the treatment of bone defects, as an alternative to bone tissue autografts. Remarkably, cells survived with unaltered phenotype (CD73+, CD90+, CD105+, CD31-, CD45-) and retained their osteogenic capacity up to 48 h after being combined with hydrogel and bone particles, thus demonstrating the stability of their identity and potency. Moreover, in a subchronic toxicity in vivo study, no toxicity was observed upon subcutaneous administration in athymic mice and signs of osteogenesis and vascularization were detected 2 months after administration. The preclinical data gathered in the present work, in compliance with current quality and regulatory requirements, demonstrated the feasibility of formulating an osteogenic cell-based tissue engineering product with a defined profile including identity, purity and potency (in vitro and in vivo), and the stability of these attributes, which complements the preclinical package required prior to move towards its use of prior to its clinical use.

Characterization of a Cytomegalovirus-Specific T Lymphocyte Product Obtained Through a Rapid and Scalable Production Process for Use in Adoptive Immunotherapy.

Immunosuppressed patients are susceptible to virus reactivation or de novo infection. Adoptive immunotherapy, based on virus-specific T lymphocytes (VST), can prevent or treat viral diseases. However, donor availability, HLA-compatibility restrictions, high costs, and time required for the production of personalized medicines constitute considerable limitations to this treatment. Ex vivo rapid and large-scale expansion of VST, compliant with current good manufacturing practice (cGMP) standards, with an associated cell donor registry would overcome these limitations. This study aimed to characterize a VST product obtained through an expansion protocol transferable to cGMP standards. Antigenic stimulus consisted of cytomegalovirus (CMV) pp65 peptide pool-pulsed autologous dendritic cells (DCs) derived from monocytes. G-Rex technology, cytokines IL-2, IL-7, and IL-15, and anti-CD3 and anti-CD28 antibodies were used for culture. At day 14 of cell culture, the final product was characterized regarding T cell subsets, specificity, and functionality. The final product, comprised mainly CD4+ and CD8+ T lymphocytes (49.2 ± 24.7 and 42.3 ± 25.2, respectively). The culture conditions made it possible to achieve at least a 98.89-fold increase in pp65-specific CD3+ IFN-γ+ cells. These cells were specific, as pp65-specific cytotoxicity was demonstrated. Additionally, in complete HLA mismatch and without the presence of pp65, alloreactivity resulted in <5% cell lysis. In conclusion, a cGMP scalable process for the generation of a large number of doses of CMV-specific cytotoxic T cells was successfully performed.

A new Diplolepis Geoffroy (Hymenoptera, Cynipidae, Diplolepidini) species from China: a rare example of a rose gall-inducer of economic significance.

A new species of the genus Diplolepis Geoffroy, Diplolepis abei Pujade-Villar & Wang sp. nov. is described on host plant Rosa sertata Rolfe × R. rugosa Thunb. from China with an integrative approach based on molecular and morphological data. Diagnosis, distribution and biology of the new species are included and illustrated. This species is the first known rose gall-inducer of economic importance. A review of Eastern Palearctic species of Diplolepis is given and a key to the Chinese fauna is presented.

Revision of world Ceroptresini (Hymenoptera: Cynipidae) with the description of a new genus and five new species.

A taxonomic revision of the tribe Ceroptresini (Hymenoptera: Cynipidae) is conducted for the first time. Prior to this study, the total number of valid species of Ceroptres, the only genus within Ceroptresini to date, was 23. As a result of this revision, 15 Ceroptres species are retained as valid and one species, Amblynotus ensiger Walsh, 1864, is desynonymized from Ceroptres petiolicola (Osten-Sacken, 1861), being considered here as a valid Ceroptres species: C. ensiger (Walsh, 1864) status verified and comb. nov. An additional five new species are described from Mexico: Ceroptres junquerasi Lobato-Vila Pujade-Villar sp. nov.; C. lenis Lobato-Vila Pujade-Villar sp. nov.; C. mexicanus Lobato-Vila Pujade-Villar sp. nov.; C. nigricrus Lobato-Vila Pujade-Villar sp. nov.; C. quadratifacies Lobato-Vila Pujade-Villar sp. nov., increasing the total number of valid Ceroptres species to 21. Ceroptres masudai Abe, 1997 is synonymized with C. kovalevi Belizin, 1973. Ceroptres niger Fullaway, 1911 is transferred to Andricus (Andricus confusus Lobato-Vila Pujade-Villar comb. nov. and nom. nov.). Five species (Amblynotus inermis Walsh, 1864; Cynips quercusarbos Fitch, 1859; Cynips querficus Fitch, 1859; Cynips quercuspisum Fitch, 1859; and Cynips quercustuber Fitch, 1859) are not considered as valid Ceroptres. The status of Ceroptres quereicola (Shinji, 1938), previously classified as an unplaced species, is commented on. In addition, a Nearctic species from the USA, Ceroptres politus Ashmead, 1896, is here proposed as the type species of a new genus within Ceroptresini: Buffingtonella Lobato-Vila Pujade-Villar gen. nov. Redescriptions, biological and distribution data, illustrations and keys to genera and species within Ceroptresini are provided. The diagnostic morphological traits of Ceroptresini, Ceroptres and the new genus are discussed.

What makes implementation intention interventions effective for promoting healthy eating behaviours? A meta-regression.

This study examines the efficacy of implementation intentions (II), a widely used self-regulatory strategy to help people achieve their goals. Although previous research has shown that the effect of II interventions is significantly higher in promoting healthy eating behaviours than in diminishing unhealthy eating behaviours, the factors that can moderate the effectiveness of these interventions remain unclear. In a meta-analysis of 70 interventions (N = 9689), we confirmed that II interventions for healthy eating behaviours yielded a medium significant effect size (d = 0.33) and a low significant effect size for unhealthy eating behaviors (d = 0.18). We show that the moderator variables of II interventions for healthy and unhealthy eating goals are very different. Regarding healthy eating, since moderator variables explain 53% of the variance in the heterogeneity of the effect sizes, the present study helps in gaining an understanding of the previous inconsistent results and offers suggestions for designing more efficient interventions. Effect size was negatively predicted by age, indicating that for younger people the effect size is higher, and II check, showing that if the instructor checks the plan it decreases its efficacy. Moreover, the effect of II interventions on students is significantly smaller than in non-student samples. In contrast, the effect size was positively predicted by initial training, off-line delivered interventions and, specific if-then and action plans versus complex plans. For unhealthy eating behaviours, our results show that there is less room to improve the intervention; there is only one moderator variable (plan formulation), and the heterogeneity found in the studies is lower for unhealthy eating behaviours (I2 = 46.70%) than for healthy eating behaviours (I2 = 73.25%), indicating that the intervention has low efficacy regardless of the design of the intervention.

Levels of IL-17F and IL-33 correlate with HLA-DR activation in clinical-grade human bone marrow-derived multipotent mesenchymal stromal cell expansion cultures.

BACKGROUND AIMS: Multipotent mesenchymal stromal cell (MSC)-based medicines are extensively investigated for use in regenerative medicine and immunotherapy applications. The International Society for Cell and Gene Therapy (ISCT) proposed a panel of cell surface molecules for MSC identification that includes human leukocyte antigen (HLA)-DR as a negative marker. However, its expression is largely unpredictable despite production under tightly controlled conditions and compliance with current Good Manufacturing Practices. Herein, we report the frequency of HLA-DR expression in 81 batches of clinical grade bone marrow (BM)-derived MSCs and investigated its impact on cell attributes and culture environment. METHODS: The levels of 15 cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interferon-γ, soluble CD40 ligand and tumor necrosis factor-α) were determined in sera supplements and supernatants of BM-MSC cultures. Identity, multipotentiality and immunopotency assays were performed on high (>20% of cells) and low (≤20% of cells) HLA-DR+ cultures. RESULTS: A correlation was found between HLA-DR expression and levels of IL-17F and IL-33. Expression of HLA-DR did neither affect MSC identity, in vitro tri-lineage differentiation potential (into osteogenic, chondrogenic and adipogenic lineages), nor their ability to inhibit the proliferation of stimulated lymphocytes. DISCUSSION: Out of 81 batches of BM-MSCs for autologous use analyzed, only three batches would have passed the ISCT criteria (<2%), whereas 60.5% of batches were compliant with low HLA-DR values (≤20%). Although a cause-effect relationship cannot be drawn, we have provided a better understanding of signaling events and cellular responses in expansion culture conditions relating with HLA-DR expression.

Derivation of Multipotent Mesenchymal Stromal Cells from Ovine Bone Marrow.

In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the development of cell-based therapies, the lack of manageable immunocompromised preclinical large animal models prevents the use of human cells, which makes it necessary to produce equivalent homologous cell types for the study of their pharmacodynamics, pharmacokinetics, and toxicology. The methods described herein allow for the isolation, expansion, manipulation, and characterization of fibroblastic-like ovine bone marrow-derived multipotent mesenchymal stromal cells (BM-MSC) that, similar to human BM-MSC, adhere to standard plastic surfaces; express specific surface markers such as CD44, CD90, CD140a, CD105, and CD166; and display trilineage differentiation potential in vitro. Homogeneous cell cultures result from a 3-week bioprocess yielding cell densities in the range of 2-4 × 104 MSC/cm2 at passage 2, which corresponds to ∼8 cumulative population doublings. Large quantities of BM-MSC resulting from following this methodology can be readily used in proof of efficacy and safety studies in the preclinical development stage. © 2018 by John Wiley & Sons, Inc.

Optimisation of a potency assay for the assessment of immunomodulative potential of clinical grade multipotent mesenchymal stromal cells.

Clinical use of multipotent Mesenchymal Stromal Cell (MSC)-based medicinal products requires their production in compliance with Good Manufacturing Practices, thus ensuring that the final drug product meets specifications consistently from batch to batch in terms of cell viability, identity, purity and potency. Potency relates to the efficacy of the medicine in its target clinical indication, so adequate release tests need to be defined and validated as quality controls. Herein we report the design and optimisation of parameters affecting the performance of an in vitro cell-based assay for assessing immunomodulatory potential of clinical grade MSC for human use, based on their capacity to inhibit proliferation of T lymphocytes under strong polyclonal stimuli. The resulting method was demonstrated to be reproducible and relatively simple to execute. Two case studies using clinical grade MSC are presented as examples to illustrate the applicability of the methodology described in this work.