Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton's jelly: single-use stirred tank bioreactors versus planar culture systems.

BACKGROUND AIMS: The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. METHODS: MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. RESULTS: After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. CONCLUSIONS: In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.

A hub-and-spoke model to deliver effective access to chimeric antigen receptor T-cell therapy in a public health network: the Catalan Blood and Tissue Bank experience.

BACKGROUND AIMS: To describe and analyze whether a hub-and-spoke organizational model could efficiently provide access to chimeric antigen receptor (CAR) T-cell therapy within a network of academic hospitals and address the growing demands of this complex and specialized activity. METHODS: The authors performed a retrospective evaluation of activity within the Catalan Blood and Tissue Bank network, which was established for hematopoietic stem cell transplantation to serve six CAR T-cell programs in academic hospitals of the Catalan Health Service. Procedures at six hospitals were followed from 2016 to 2021. Collection shipments of starting materials, CAR T-cell returns for storage and infusions for either clinical trials or commercial use were evaluated. RESULTS: A total of 348 leukocytapheresis procedures were performed, 39% of which were delivered fresh and 61% of which were cryopreserved. The network was linked to seven advanced therapy medicinal product manufacturers. After production, 313 CAR T-cell products were shipped back to the central cryogenic medicine warehouse located in the hub. Of the units received, 90% were eventually administered to patients. A total of 281 patients were treated during this period, 45% in clinical trials and the rest with commercially available CAR T-cell therapies. CONCLUSIONS: A hub-and-spoke organizational model based on an existing hematopoietic stem cell transplantation program is efficient in incorporating CAR T-cell therapy into a public health hospital network. Rapid access and support of growing activity enabled 281 patients to receive CAR T cells during the study period.

Clinical effects of intrathecal administration of expanded Wharton jelly mesenchymal stromal cells in patients with chronic complete spinal cord injury: a randomized controlled study.

BACKGROUND AIMS: Spinal cord injury (SCI) represents a devastating condition leading to severe disability related to motor, sensory and autonomic dysfunction. Stem cell transplantation is considered a potential emerging therapy to stimulate neuroplastic and neuroregenerative processes after SCI. In this clinical trial, the authors investigated the safety and clinical recovery effects of intrathecal infusion of expanded Wharton jelly mesenchymal stromal cells (WJ-MSCs) in chronic complete SCI patients. METHODS: The authors designed a randomized, double-blind, crossover, placebo-controlled, phase 1/2a clinical trial (NCT03003364). Participants were 10 patients (7 males, 3 females, age range, 25-47 years) with chronic complete SCI (American Spinal Injury Association A) at dorsal level (T3-11). Patients were randomly assigned to receive a single dose of intrathecal ex vivo-expanded WJ-MSCs (10 × 106 cells) from human umbilical cord or placebo and were then switched to the other arm at 6 months. Clinical evaluation (American Spinal Injury Association impairment scale motor and sensory score, spasticity, neuropathic pain, electrical perception and pain thresholds), lower limb motor evoked potentials (MEPs) and sensory evoked potentials (SEPs), Spinal Cord Independence Measure and World Health Organization Quality of Life Brief Version were assessed at baseline, 1 month, 3 months and 6 months after each intervention. Urodynamic studies and urinary-specific quality of life (Qualiveen questionnaire) as well as anorectal manometry, functional assessment of bowel dysfunction (Rome III diagnostic questionnaire) and severity of fecal incontinence (Wexner score) were conducted at baseline and at 6 months after each intervention. RESULTS: Intrathecal transplantation of WJ-MSCs was considered safe, with no significant side effects. Following MSC infusion, the authors found significant improvement in pinprick sensation in the dermatomes below the level of injury compared with placebo. Other clinically relevant effects, such as an increase in bladder maximum capacity and compliance and a decrease in bladder neurogenic hyperactivity and external sphincter dyssynergy, were observed only at the individual level. No changes in motor function, spasticity, MEPs, SEPs, bowel function, quality of life or independence measures were observed. CONCLUSIONS: Intrathecal transplantation of human umbilical cord-derived WJ-MSCs is a safe intervention. A single intrathecal infusion of WJ-MSCs in patients with chronic complete SCI induced sensory improvement in the segments adjacent to the injury site.

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.

Assessing the Performance of EUHFORIA Modeling the Background Solar Wind.

In order to address the growing need for more accurate space-weather predictions, a new model named EUHFORIA (EUropean Heliospheric FORecasting Information Asset) was recently developed. We present the first results of the performance assessment for the solar-wind modeling with EUHFORIA and identify possible limitations of its present setup. Using the basic EUHFORIA 1.0.4 model setup with the default input parameters, we modeled background solar wind (no coronal mass ejections) and compared the obtained results with Advanced Composition Explorer (ACE) in-situ measurements. For the purposes of statistical study we developed a technique of combining daily EUHFORIA runs into continuous time series. The combined time series were derived for the years 2008 (low solar activity) and 2012 (high solar activity), from which in-situ speed and density profiles were extracted. We find for the low-activity phase a better match between model results and observations compared to the high-activity time interval considered. The quality of the modeled solar-wind parameters is found to be rather variable. Therefore, to better understand the results obtained we also qualitatively inspected characteristics of coronal holes, i.e. the sources of the studied fast streams. We discuss how different characteristics of the coronal holes and input parameters to EUHFORIA influence the modeled fast solar wind, and suggest possibilities for the improvement of the model.

On Flare-CME Characteristics from Sun to Earth Combining Remote-Sensing Image Data with In Situ Measurements Supported by Modeling.

We analyze the well-observed flare and coronal mass ejection (CME) from 1 October 2011 (SOL2011-10-01T09:18) covering the complete chain of effects - from Sun to Earth - to better understand the dynamic evolution of the CME and its embedded magnetic field. We study in detail the solar surface and atmosphere associated with the flare and CME using the Solar Dynamics Observatory (SDO) and ground-based instruments. We also track the CME signature off-limb with combined extreme ultraviolet (EUV) and white-light data from the Solar Terrestrial Relations Observatory (STEREO). By applying the graduated cylindrical shell (GCS) reconstruction method and total mass to stereoscopic STEREO-SOHO (Solar and Heliospheric Observatory) coronagraph data, we track the temporal and spatial evolution of the CME in the interplanetary space and derive its geometry and 3D mass. We combine the GCS and Lundquist model results to derive the axial flux and helicity of the magnetic cloud (MC) from in situ measurements from Wind. This is compared to nonlinear force-free (NLFF) model results, as well as to the reconnected magnetic flux derived from the flare ribbons (flare reconnection flux) and the magnetic flux encompassed by the associated dimming (dimming flux). We find that magnetic reconnection processes were already ongoing before the start of the impulsive flare phase, adding magnetic flux to the flux rope before its final eruption. The dimming flux increases by more than 25% after the end of the flare, indicating that magnetic flux is still added to the flux rope after eruption. Hence, the derived flare reconnection flux is most probably a lower limit for estimating the magnetic flux within the flux rope. We find that the magnetic helicity and axial magnetic flux are lower in the interplanetary space by ∼ 50% and 75%, respectively, possibly indicating an erosion process. A CME mass increase of 10% is observed over a range of [Formula: see text]. The temporal evolution of the CME-associated core-dimming regions supports the scenario that fast outflows might supply additional mass to the rear part of the CME.

Autologous platelet-rich plasma for treating chronic wounds.

BACKGROUND: Autologous platelet-rich plasma (PRP) is a treatment that contains fibrin and high concentrations of growth factors with the potential to improve the healing of chronic wounds. This is the first update of a review first published in 2012. OBJECTIVES: To determine whether autologous PRP promotes the healing of chronic wounds. SEARCH METHODS: In June 2015, for this first update, we searched the Cochrane Wounds Specialised Register; the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library): Ovid MEDLINE; Ovid MEDLINE (In-Process & Other Non-Indexed Citations); Ovid EMBASE; and EBSCO CINAHL. We also searched for ongoing and unpublished clinical trials in the WHO International Clinical Trials Registry Platform (ICTRP) (searched January 2015). We did not impose any restrictions with respect to language, date of publication, or study setting. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared autologous PRP with placebo or alternative treatments for any type of chronic wound in adults. We did not apply any date or language restrictions. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methodology, including two reviewers independently selecting studies for inclusion, extracting data, and assessing risk of bias. MAIN RESULTS: The search identified one new RCT, making a total of 10 included RCTs (442 participants, 42% women). The median number of participants per RCT was 29 (range 10 to 117). Four RCTs recruited people with a range of chronic wounds; three RCTs recruited people with venous leg ulcers, and three RCTs considered foot ulcers in people with diabetes. The median length of treatment was 12 weeks (range 8 to 40 weeks).It is unclear whether autologous PRP improves the healing of chronic wounds generally compared with standard treatment (with or without placebo) (risk ratio (RR) 1.19, 95% confidence interval (CI) 0.95 to 1.50; I(2) = 27%, low quality evidence, 8 RCTs, 391 participants). Autologous PRP may increase the healing of foot ulcers in people with diabetes compared with standard care (with or without placebo) (RR 1.22, 95% CI 1.01 to 1.49; I(2) = 0%, low quality evidence, 2 RCTs, 189 participants). It is unclear if autologous PRP affects the healing of venous leg ulcers (RR 1.02, 95% CI 0.81 to 1.27; I(2) = 0% ). It is unclear if there is a difference in the risk of adverse events in people treated with PRP or standard care (RR 1.05, 95% CI 0.29 to 3.88; I(2) = 0%, low quality evidence from 3 trials, 102 participants). AUTHORS' CONCLUSIONS: PRP may improve the healing of foot ulcers associated with diabetes, but this conclusion is based on low quality evidence from two small RCTs. It is unclear whether PRP influences the healing of other chronic wounds. The overall quality of evidence of autologous PRP for treating chronic wounds is low. There are very few RCTs evaluating PRP, they are underpowered to detect treatment effects, if they exist, and are generally at high or unclear risk of bias. Well designed and adequately powered clinical trials are needed.

Treatment of non-hypertrophic pseudoarthrosis of long bones with a Tissue Engineered Product loaded with autologous bone marrow-derived Mesenchymal Stromal Cells: Results from a phase IIa, prospective, randomized, parallel, pilot clinical trial comparing to iliac crest autograft.

BACKGROUND: Atrophic pseudoarthrosis is a serious complication with an incidence of 5-10 % of bone fractures located in the diaphysis of long bones. Standard treatments involve aggressive surgical procedures and re-interventions requiring the use of autografts from the iliac crest as a source of bone-forming biological activity (Standard of Care, SoC). In this context, regenerative ex vivo expanded osteogenic cell-based medicines could be of interest. Particularly, Mesenchymal Stromal Cells (MSC) offer new prospects to promote bone tissue repair in pseudoarthrosis by providing biological activity in an osteoconductive and osteoinductive environment. METHODS: We conducted a phase IIa, prospective, randomised, parallel, two-arms, open-label with blinded assessor pilot clinical trial to compare SoC vs. a tissue-engineered product (TEP), composed of autologous bone marrow (BM)-derived MSCs loaded onto allogeneic decellularised, lyophilised spongy bone cubes, in a cohort of 20 patients with non-hypertrophic pseudoarthrosis of long bones. Patients were followed up for 12 months. Radiological bone healing was evaluated by standard X-ray and computed tomography (CT) scanning. Quality of life was measured using the EUROQOL-5D questionnaire. RESULTS: Ten patients were randomized to TEP and 10 to SoC with iliac crest autograft. Manufacturing of TEP was feasible and reproducibly achieved. TEP implantation in the bone defect was successful in all cases and none of the 36 adverse events (AE) reported were related to the treatment. Efficacy analyses were performed in the Full Analysis Set (FAS) population, which included 17 patients after 3 patients withdrew from the study. The degree of consolidation, estimated by measuring Hounsfield units (HU) on CT, showed no significant differences between the two treatment groups at 12 months post treatment (main efficacy variable) (p = 0.4835) or at 6 months. CONCLUSIONS: Although only a small number of patients were included in our study, it is notable that no significant differences were observed between the experimental treatment and SoC, thus suggesting TEP as an alternative where autograft is not available or contraindicated.

Optimized reagents for immunopotency assays on mesenchymal stromal cells for clinical use.

Multipotent mesenchymal stromal cells (MSC) offer new therapeutic opportunities based on their ability to modulate an imbalanced immune system. Immunomodulatory potency is typically demonstrated in vitro by measuring the presence of surrogate markers (i.e., indoleamine-2,3-dioxygenase, IDO; tumor necrosis factor receptor type 1, TNFR1) and/or functional assays in co-cultures (i.e., inhibition of lymphoproliferation, polarization of macrophages). However, the biological variability of reagents used in the latter type of assays leads to unreliable and difficult to reproduce data therefore making cross-comparison between batches difficult, both at the intra- and inter-laboratory levels. Herein, we describe a set of experiments aiming at the definition and validation of reliable biological reagents as a first step towards standardization of a potency assay. This approach is based on the co-culture of Wharton's jelly (WJ)-derived MSC and cryopreserved pooled peripheral blood mononuclear cells. Altogether, we successfully defined a robust and reproducible immunopotency assay based on previously described methods incorporating substantial improvements such as cryopreservation of multiple vials of pooled peripheral blood mononuclear cells (PBMC) from 5 individual donors that enable a number of tests with same reagents, also reducing waste of PBMC from individual donors and therefore contributing to a more efficient and ethical method to use substances of human origin (SoHO). The new methodology was successfully validated using 11 batches of clinical grade MSC,WJ. Methods described here contribute to minimize PBMC donor variability while reducing costs, streamlining assay setup and convenience and laying the foundations for harmonization of biological reagents usage in standardized immunopotency assays for MSC. HIGHLIGHTS: • The use of pools of peripheral blood mononuclear cells (PBMCs) in potency assays contributes to robust and reproducible results, which is key in the assessment of mesenchymal stroma cells (MSC) potency for batch release. • Cryopreservation of PBMCs does not impact negatively on their activation and proliferation abilities. • Cryopreserved pools of PBMC constitutes convenient off-the-shelf reagents for potency assays. • Cryopreservation of pooled PBMCs from multiple donors is a way to reduce waste of donated PBMC and its associated costs, as well as reducing the impact of individual donor variability of substances of human origin (SoHO).

EPIDEMIOLOGY OF TRAUMATIC FRACTURES OF THE SPINE IN MARIO COVAS HOSPITAL BETWEEN 2015 AND 2020.

Objectives: Analyze the epidemiological profile of patients with traumatic spinal fractures treated at Mário Covas State Hospital between 2015 and 2020. Methodology: This is an epidemiological, descriptive, retrospective, quantitative, comparative, medical records review-type study. Data collection was carried out between May and June 2022 at the Mário Covas State Hospital, the following characteristics being evaluated: age, sex, lesion topography, trauma mechanism, origin and treatment. Results: Data from 252 patients with traumatic spinal fractures were analyzed. The mean age of patients was 48.7 years, 74.7% were male. The mechanism of trauma from falls from a height and the topography of the lumbar vertebrae have a highly significant trend. The most affected vertebrae are lumbar L1, thoracic T12 and cervical C6. The crossing of the age group with the male sex is higher than expected in those over 60 years of age. The crossing of the age group with the trauma mechanism is higher than expected, between 20 and 39 years. Conclusion: There are few published works on the epidemiology of traumatic fractures of the spine, which points to the need for further studies on the subject. Level of Evidence III; Retrospective comparative study.

Objetivos: Analisar o perfil epidemiológico dos pacientes com fratura traumática de coluna vertebral atendidos no Hospital Estadual Mário Covas entre os anos de 2015 e 2020. Metodologia: Trata-se de um estudo epidemiológico, descritivo, retrospectivo, quantitativo, comparativo, do tipo revisão de prontuário médico. A coleta de dados foi realizada entre maio e junho de 2022 no Hospital Estadual Mário Covas, sendo avaliadas as características: idade, sexo, topografia da lesão, mecanismo de trauma, procedência e tratamento. Resultados: Foram analisados dados de 252 pacientes com fratura traumática da coluna vertebral. A média da idade dos pacientes foi de 48,7 anos, 74,7% eram do sexo masculino. O mecanismo de trauma tipo queda de altura e a topografia das vértebras lombares têm tendência altamente significativa. As vértebras mais afetadas são lombar L1, torácica T12 e cervical C6. O cruzamento da faixa etária com sexo masculino está acima do esperado nos maiores de 60 anos. O cruzamento da faixa etária com mecanismo do trauma está acima do esperado, entre 20 a 39 anos. Conclusão: São poucos os trabalhos publicados a respeito da epidemiologia das fraturas traumáticas de coluna vertebral, o que aponta para a necessidade de novos estudos acerca da temática. Nível de evidência III; Estudo retrospectivo comparativo.

Autologous platelet-rich plasma for treating chronic wounds.

BACKGROUND: Autologous platelet-rich plasma (PRP) is a treatment that contains fibrin and high concentrations of growth factors and has the potential to aid wound healing. OBJECTIVES: To determine whether autologous PRP promotes the healing of chronic wounds. SEARCH METHODS: We searched the Cochrane Wounds Group Specialised Register (searched 15 August 2012); The Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, Issue 8); Ovid MEDLINE (1950 to August Week 1 2012); Ovid MEDLINE (In-Process & Other Non-Indexed Citations, August 14, 2012); Ovid EMBASE (1980 to 2012 Week 32); EBSCO CINAHL (1982 to 10 August 2012) and International Clinical Trials Registry Platform (ICTRP)(accessed 22 August 2012). No date or language restrictions were applied. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared autologous PRP with placebo or alternative treatments for any type of chronic wound in adults. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed each study against the inclusion criteria, extracted data and assessed risk of bias for all included trials. We calculated the risk ratio (RR) or the mean difference (MD) and time to wound healing was analysed as survival data using the hazard ratio (HR). We considered heterogeneity as significant when I(2) was >75%. MAIN RESULTS: Nine eligible RCTs were included, with a total of 325 participants of whom 44% were women. The median number of participants per RCT was 26 (range 10 to 86). Four RCTs recruited people with mixed chronic wounds (there were participants with wounds caused by more than one aetiology and participants who had wounds of several aetiologies in the same trial), three RCTs recruited people with venous leg ulcers and two RCTs considered foot ulcers in people with diabetes. The median length of treatment was 12 weeks (range eight to 40 weeks).One study was at low risk of bias, three studies were at high risk of bias with the remainder being at overall unclear risk of bias. The proportion of completely healed chronic wounds was reported in seven RCTs that compared PRP with standard treatment or placebo, with no statistically significant difference between the groups, in diabetic foot ulcers (RR 1.16; 95% CI 0.57 to 2.35), in venous leg ulcers (pooled RR 1.02; 95% CI 0.81 to 1.27; I(2)=0% ) and in mixed chronic wounds (pooled RR 1.85; 95% CI 0.76 to 4.51; I(2)=42%). The total area epithelialised at the end of the intervention was reported in three RCTs of mixed chronic wounds, there was no statistically significant difference between the groups (pooled MD -1.94 cm(2); 95% CI -4.74 to 0.86; I(2)=47%). The percentage of wound area healed was reported in two RCTs of mixed chronic wounds, and results were statistically significant in favour of the PRP group (RR 51.78%; 95% CI 32.70 to 70.86; I(2)= 0%). Wound complications like infection or necrosis were reported by three RCTs, and there was no statistically significant difference between groups (RR 1.08; 95% CI 0.31 to 3.73). Adverse effects were reported by three studies and there was no statistically significant difference between people treated with PRP and those not given PRP (pooled RR 1.07; 95% CI 0.32 to 3.58; I(2)=0%). AUTHORS' CONCLUSIONS: There is currently no evidence to suggest that autologous PRP is of value for treating chronic wounds. However, current evidence is based on a small number of RCTs, most of which are either at high or unclear risk of bias. Well-designed and adequately powered clinical trials are needed.

Use of Multipotent Mesenchymal Stromal Cells, Fibrin, and Scaffolds in the Production of Clinical Grade Bone Tissue Engineering Products.

Tissue engineering products (TEP) are a new type of medicines resulting from the combination of cells, scaffolds, and/or signalling factors, which can be used for the regeneration of damaged tissues thus opening new avenues for the treatment of complex conditions. However, such combination of biologically active elements, particularly living cells, poses an unprecedented challenge for their production under pharmaceutical standards.In the methods presented here, we formulated two types of TEP based on the use of multipotent mesenchymal stromal cells with osteogenic potential combined with osteoinductive and osteoconductive bony particles from tissue bank embedded in a fibrin hydrogel that, altogether, can induce the generation of new tissue while adapting to the diverse architecture of bony defects. In agreement with pharmaceutical quality and regulatory requirements, procedures presented herein can be performed in compliance with current good manufacturing practices and be readily implemented in straightforward facilities at hospitals and academic institutions.

Cord blood and amniotic membrane extract eye drop preparations display immune-suppressive and regenerative properties.

Diseases and injuries that compromise the ocular surface cause considerable patient distress and have long term consequences for their quality of life. Treatment modalities that can address the delicate balance of tissue regeneration, inflammation and maintenance of corneal transparency are therefore needed. We have recently formulated two novel eye drops from placental tissues: cord blood platelet lysate (CBED) and amniotic membrane extract eye drops (AMED), which can be used to treat severe ocular disorders. Here we characterise these two preparations by measuring: (a) growth factors (GF) and cytokines composition, (b) promotion of human corneal epithelial cell (HCEC) growth and (c) effects on immune cells in a lymphocyte culture assay. Finally, their bioavailability was assayed in an ex vivo porcine corneal model. We show that both preparations contain GF and cytokines that were able to promote the in vitro growth of HCEC and support repair in an in vitro scratch test. When assessed in a lymphocyte culture, both favoured immune suppression reducing the cellular expression of NKG2D and CD107a as well as the production of interferon gamma (IFN-γ) in natural killer, NKT and T cells. Regarding bioavailability, CBED active molecules were found mainly in the pre-corneal fraction with some penetration into the corneal fraction, in an ex vivo model. In summary, both placental-derived allogeneic preparations, CBED and AMED, display regenerative and immunomodulatory capabilities. These results will help define mechanisms of action and the best indications and doses of each product for use in a particular patient and support the development of off-the-shelf therapies for ocular surface pathologies in which wound healing defects and inflammatory events are contributing factors.

Clinical evaluation of allogeneic eye drops from cord blood platelet lysate.

BACKGROUND: Current treatments for several corneal lesions show limited efficacy. Here we report the clinical evaluation of the efficacy of a novel eye drop preparation produced in a public cord blood (CB) bank. MATERIALS AND METHODS: In a multicentre, retrospective, consecutive case study we evaluated 33 patients (46 eyes) unresponsive to conventional treatments who required urgent intervention. The patients were given allogeneic eye drops obtained from cord blood platelet lysate (CBED) to treat severe ocular surface lesions under a compassionate use protocol. The CBED were prepared from CB units donated for haematopoietic stem cell transplantation that did not contain the minimum stem cell dose required for this use. Patients were grouped by acute conditions (neurotrophic ulcers: group I; other corneal ulcers: group II; corneal burns: group III), and chronic conditions (ocular graft-versus-host disease: group IV; severe dry eye syndrome: group V). The patients received one or two drops of the product to the affected eye four to six times per day for 19 days. A further 19-day cycle of treatment could be repeated according to the initial clinical response. RESULTS: Patients received a median of 19 CBED vials (interquartile range 19-57, range 19-442) to complete the therapy. Group I-II-III patients showed full and partial ulcer recovery in 25 (78%) and six (19%) eyes respectively. One eye (3%) did not respond to treatment. For groups IV-V improvement was reported for 12 (85%) eyes and lesions worsened on treatment in both eyes (15%) of one patient. No severe adverse events were directly attributed to CBED. DISCUSSION: Promptly available CBED resulted in a well-tolerated allogeneic treatment that showed evidence of efficacy in this cohort of patients. These positive results support further studies on CBED from platelet lysate as a novel product of CB banks. A prospective clinical trial in neurotrophic keratitis (NCT03084861) is ongoing to confirm these preliminary data.

Understanding the Origins of Problem Geomagnetic Storms Associated with "Stealth" Coronal Mass Ejections.

Geomagnetic storms are an important aspect of space weather and can result in significant impacts on space- and ground-based assets. The majority of strong storms are associated with the passage of interplanetary coronal mass ejections (ICMEs) in the near-Earth environment. In many cases, these ICMEs can be traced back unambiguously to a specific coronal mass ejection (CME) and solar activity on the frontside of the Sun. Hence, predicting the arrival of ICMEs at Earth from routine observations of CMEs and solar activity currently makes a major contribution to the forecasting of geomagnetic storms. However, it is clear that some ICMEs, which may also cause enhanced geomagnetic activity, cannot be traced back to an observed CME, or, if the CME is identified, its origin may be elusive or ambiguous in coronal images. Such CMEs have been termed "stealth CMEs". In this review, we focus on these "problem" geomagnetic storms in the sense that the solar/CME precursors are enigmatic and stealthy. We start by reviewing evidence for stealth CMEs discussed in past studies. We then identify several moderate to strong geomagnetic storms (minimum Dst < - 50  nT) in solar cycle 24 for which the related solar sources and/or CMEs are unclear and apparently stealthy. We discuss the solar and in situ circumstances of these events and identify several scenarios that may account for their elusive solar signatures. These range from observational limitations (e.g., a coronagraph near Earth may not detect an incoming CME if it is diffuse and not wide enough) to the possibility that there is a class of mass ejections from the Sun that have only weak or hard-to-observe coronal signatures. In particular, some of these sources are only clearly revealed by considering the evolution of coronal structures over longer time intervals than is usually considered. We also review a variety of numerical modelling approaches that attempt to advance our understanding of the origins and consequences of stealthy solar eruptions with geoeffective potential. Specifically, we discuss magnetofrictional modelling of the energisation of stealth CME source regions and magnetohydrodynamic modelling of the physical processes that generate stealth CME or CME-like eruptions, typically from higher altitudes in the solar corona than CMEs from active regions or extended filament channels.

Cord Blood Platelet Rich Plasma Derivatives for Clinical Applications in Non-transfusion Medicine.

Cord blood platelet rich plasma (CB-PRP) derivatives have been investigated as potential therapeutic agents for the treatment of diverse conditions including ocular surface disease and skin ulcers. We have developed processes for the formulation of several CB-PRP preparations, which have different composition and attributes. Here we describe the molecular characteristics of these preparations and we make recommendations as to their most appropriate clinical application based on functional and immunomodulatory profiles. We show that incubation of adult peripheral blood mononuclear cells (PBMCs) with all three preparations dramatically reduced the production of INFγ and the expression of NKG2D and CD107a in NK, NKT, and T cells thus diminishing their activation, we propose that the likely mechanism is the high levels of soluble NKG2D ligands present in plasma. Of the three preparations we investigated, CB platelet lysate (PL) and platelet releaseate (PR) have higher concentrations of trophic and pro-angiogenic factors, CB platelet poor plasma (PPP) has the lowest concentration of all analytes measured. Based on these finding we propose that CB-PR is the most suitable raw material for skin wound patches, while CB-PL and PPP can be used to prepare eye drops for severe ocular surface pathologies and inflammatory conditions such as corneal ulcers or severe dry eye disease, respectively.

Cord blood-derived platelet concentrates as starting material for new therapeutic blood components prepared in a public cord blood bank: from product development to clinical application.

BACKGROUND: There are many advantages to using cord blood (CB) as a source of therapeutic platelet and plasma derivatives for regenerative medicine. These include availability, universal use, young donor source, and virally safe biological material, rich in tissue regenerative factors. MATERIALS AND METHODS: We aimed to validate a bioprocess design for the production of cord blood-derived platelet concentrates (CBPC) in a public Cord Blood Bank (CBB). CBPC was defined as a product of 10±5 mL, 1,000±200×109/L total platelets, free of erythrocytes and leukocytes. A total of 300 CB units were centrifuged in two steps to enrich for platelets, in compliance with Good Manufacturing Practice. The samples were tested for the degree of platelet activation present, and the levels of growth factor were analysed to evaluate their potential function. CBPC were then activated after thawing with 10% calcium gluconate to generate platelet gels (CBPG) to treat patients with diabetic foot ulcers. RESULTS: After processing, 84% of the products fulfilled the acceptance criteria. Final products contained 1,017±149×106 platelets/mL in 10±3mL of plasma. Platelet recovery was 50±9%. The methods described here ensure depletion of white and red blood cells down to a residual concentration of 0.2±0.1×106/mL and 0.03±0.02×106/mL, respectively. Platelets showed low levels of activation during processing, but were significantly activated after thawing, as indicated by an increase in CD62p expression. The growth factors EGF, VEGF, bFGF, PDGF AB/BB and TGF-ß1 were at concentrations of 1,706±123 pg/mL; 1,602±227 pg/mL; 314±26 pg/mL; 30±1.5 ng/mL; 24±2 ng/mL (mean±standard error of mean), respectively. For clinical evaluation, a total of 21 CBPG were applied in 3 patients, with no reported adverse events and improvement of ulcers in all of them. DISCUSSION: We designed and validated a highly reproducible, closed system method to manufacture high quality CBPC suitable for clinical applications using CB units not suitable for transplantation in a public CBB.

Randomized clinical trial: expanded autologous bone marrow mesenchymal cells combined with allogeneic bone tissue, compared with autologous iliac crest graft in lumbar fusion surgery.

BACKGROUND CONTEXT: Although autogenous iliac crest bone graft (AICBG) is considered the gold-standard graft material for spinal fusion, new bone substitutes are being developed to avoid associated complications and disadvantages. By combining autologous bone marrow mesenchymal stromal cells (MSCs) expanded ex vivo and allogenic cancellous bone graft, we obtain a tissue-engineered product that is osteoconductive and potentially more osteogenic and osteoinductive than AICBG, owing to the higher concentration of MSCs. PURPOSE: This study aimed to evaluate the feasibility and safety of implanting a tissue-engineered product consisting of expanded bone marrow MSCs loaded onto allograft bone (MSC+allograft) for spinal fusion in degenerative spine disease, as well as to assess its clinical and radiological efficacy. STUDY DESIGN/SETTING: A prospective, multicenter, open-label, blinded-reader, randomized, parallel, single-dose phase I-II clinical trial. PATIENT SAMPLE: A total of 73 adult patients from 5 hospitals, with Meyerding grade I-II L4-L5 degenerative spondylolisthesis and/or with L4-L5 degenerative disc disease who underwent spinal fusion through transforaminal lumbar interbody fusion (TLIF). OUTCOME MEASURES: Spinal fusion was assessed by plain X-ray at 3, 6, and 12 months and by computed tomography (CT) at 6 and 12 months post-treatment. An independent radiologist performed blinded assessments of all images. Clinical outcomes were measured as change from baseline value: visual analog scale for lumbar and sciatic pain at 12 days, 3, 6, and 12 months posttreatment, and Oswestry Disability Index and Short Form-36 at 3, 6, and 12 months posttreatment. METHODS: Patients who underwent L4-L5 TLIF were randomized for posterior graft type only, and received either MSC+allograft (the tissue-engineered product, group A) or AICBG (standard graft material, group B). Standard graft material was used for anterior fusion in all patients. Feasibility was measured primarily as the percentage of randomized patients who underwent surgery in each treatment group. Safety was assessed by analyzing treatment-emergent adverse events (AEs) for the full experimental phase and appraising their relationship to the experimental treatment. Outcome measures, both radiological and clinical, were compared between the groups. RESULTS: Seventy-three patients were randomized in this study, 36 from the MSC+allograft group and 37 from the AICBG group, and 65 were surgically treated (31 group A, 34 group B). Demographic and comorbidity data showed no difference between groups. Most patients were diagnosed with grade I or II degenerative spondylolisthesis. MSC+allograft was successfully implanted in 86.1% of randomized group A patients. Most patients suffered treatment-emergent AEs during the study (88.2% in group A and 97.1% in group B), none related to the experimental treatment. X-ray-based rates of posterior spinal fusion were significantly higher for the experimental group at 6 months (p=.012) and 12 months (p=.0003). CT-based posterior fusion rates were significantly higher for MSC+allograft at 6 months (92.3% vs 45.7%; p=.0001) and higher, but not significantly, at 12 months (76.5% vs 65.7%; p=.073). CT-based complete response (defined as the presence of both posterior intertransverse fusion and anterior interbody fusion) was significantly higher at 6 months for MSC+allograft than for AICBG (70.6% vs 40%; p=.0038), and remained so at 12 months (70.6% vs 51.4%; p=.023). Clinical results including patient-reported outcomes improved postsurgery, although there were no differences between groups. CONCLUSIONS: Compared with the current gold standard, our experimental treatment achieved a higher rate of posterior spinal fusion and radiographic complete response to treatment at 6 and 12 months after surgery. The treatment clearly improved patient quality of life and decreased pain and disability at rates similar to those for the control arm. The safety profile of the tissue-engineered product was also similar to that for the standard material, and no AEs were linked to the product. Procedural AEs did not increase as a result of BM aspiration. The use of expanded bone marrow MSCs combined with cancellous allograft is a feasible and effective technique for spinal fusion, with no product-related AEs found in our study.

Efficacy and safety of the use of autologous plasma rich in platelets for tissue regeneration: a systematic review.

BACKGROUND: Autologous plasma rich in platelets (PRP) is a derived blood product whose application in clinical practice is growing. A systematic review was conducted to evaluate its efficacy and safety. STUDY DESIGN AND METHODS: A search was performed in electronic databases. Randomized controlled clinical trials (RCTs) in adult patients were included and assessed for methodologic quality. The main outcomes were "tissue regeneration" and "safety." Relative risks (RRs) and standardized mean differences (SMDs) were calculated to show pooled estimates for these outcomes. When the results heterogeneity was more than 50 percent, a sensitivity analysis was performed. RESULTS: Twenty RCTs were included (11 of oral and maxillofacial surgery, 7 of chronic skin ulcers, and 2 of surgery wounds). Four RCTs evaluated the depth reduction in gingival recession in chronic periodontitis; the SMD was 0.54 (95% confidence interval [CI], 0.16 to 0.92) mm, favorable to PRP. Three RCTs evaluated the clinical attachment level in chronic periodontitis; the SMD was 0.33 (95% CI, -0.71 to 1.37) mm. Six RCTs assessed the complete skin epithelialization in wound ulcers; the RR was 1.40 (95% CI, 0.85 to 2.31). Only 6 RCTs reported adverse effects without differences between groups. CONCLUSIONS: PRP improves the gingival recession but not the clinical attachment level in chronic periodontitis. In the complete healing process of chronic skin ulcers, the results are inconclusive. There are little data about PRP safety. There are several methodologic limitations and, consequently, future research should focus on strong and well-designed RCTs that assess the efficacy and safety of PRP.

Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton's Jelly.

BACKGROUND: The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards.