Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors.

Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47 × 109 cells in a real-scale 2.4-L batch.

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.

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds.

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105+ /CD45,- 10.3% HLA-DR,+ 100.0% CD90,+ and 99.2% CD73+ /CD31- expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 ± 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation.

Optimisation of processing methods to improve success in the derivation of human multipotent mesenchymal stromal cells from cryopreserved umbilical cord tissue fragments.

Wharton's Jelly (WJ)-derived Mesenchymal Stromal Cells (MSC) are currently in the spotlight for the development of innovative MSC-based therapies due to their ease of sourcing, high proliferation capacity and improved immunopotency over MSC from other tissue sources. However, the short time window for derivation from donated fresh umbilical cord (UC) tissue fragments does not allow to consider biological features of the donor beyond serological safety testing. This limits the scope of MSC banking to rapid, prospective derivation of MSC, WJ lines without considering biological and genetic characteristics of the donor that may influence their suitability for clinical use (e.g. HLA type, inherited gene variants). In the present study, we describe a simple, efficient and reproducible approach for the cryopreservation of UC tissue fragments, compatible with established workflows in existing public frameworks for cord blood and tissue collection while guaranteeing pharmaceutical grade of starting materials for further processing under GMP standards. Herein we demonstrated the feasibility of time and cost-saving methods for cryopreservation of unprocessed UC tissue fragments directly at reception of the donated tissues using 10% Me2SO-based cryosolution and a commercial clinical-grade defined cryopreservation medium (Cryostor®), showing the preservation of all Critical Quality Attributes in terms of identity, potency and kinetic parameters. In summary, our study provides evidence that cryopreservation of large unprocessed UC tissue fragments (5-13.5 cm) supports subsequent progenitor cell isolation and derivation of MSC,WJ, preserving their viability, identity, proliferation rates and potency.

Epidemiology and Associated Factors in Transfusion Management in Intensive Care Unit.

Severe traumatic injury is one of the main global health issues which annually causes more than 5.8 million worldwide deaths. Uncontrolled haemorrhage is the main avoidable cause of death among severely injured individuals. Management of trauma patients is the greatest challenge in trauma emergency care, and its proper diagnosis and early management of bleeding trauma patients, including blood transfusion, are critical for patient outcomes. AIM: We aimed to describe the epidemiology of transfusion practices in severe trauma patients admitted into Spanish Intensive Care Units. MATERIAL AND METHODS: We performed a multicenter cross-sectional study in 111 Intensive Care Units across Spain. Adult patients with moderate or severe trauma were eligible. Distribution of frequencies was used for qualitative variables and the mean, with its 95% CI, for quantitative variables. Transfusion programmes, the number of transfusions performed, and the blood component transfused were recorded. Demographic variables, mortality rate, hospital stay, SOFA-score and haemoglobin levels were also gathered. RESULTS: We obtained results from 109 patients. The most transfused blood component was packet red blood cells with 93.8% of total transfusions versus 43.8% of platelets and 37.5% of fresh plasma. The main criteria for transfusion were analytical criteria (43.75%), and acute anaemia with shock (18.75%) and without haemodynamic impact (18.75%). CONCLUSION: Clinical practice shows a ratio of red blood cells, platelets, and Fresh Frozen Plasma (FFP) of 2:1:1. It is necessary to implement Massive Transfusion Protocols as they appear to improve outcomes. Our study suggests that transfusion of RBC, platelets and FFP in a 2:1:1 ratio could be beneficial for trauma patients.

The role of routine FIBERoptic bronchoscopy monitoring during percutaneous dilatational TRACHeostomy (FIBERTRACH): a study protocol for a randomized, controlled clinical trial.

BACKGROUND: Tracheostomy is one of the most frequent techniques in intensive care units (ICU). Fiberoptic bronchoscopy (FB) is a safety measure when performing a percutaneous dilatational tracheostomy (PDT), but the controversy surrounding the routine use of FB as part of the procedure remains open. National surveys in some European countries showed that the use of FB is non-standardized. Retrospective studies have not shown a significant difference in complications between procedures performed with or without a bronchoscope. International guidelines have not been able to establish recommendations regarding the use of FB in PDT due to lack of evidence. DESIGN: This is a multicenter (three centers at the time of  publishing this paper) randomized controlled clinical trial to examine the safety of percutaneous tracheostomy using FB. We will include all consecutive adult patients admitted to the ICU in whom percutaneous tracheostomy for prolonged mechanical ventilation is indicated and with no exclusion criteria for using FB. Eligible patients will be randomly assigned to receive blind PDT or PDT under endoscopic guidance. All procedures will be performed by experienced intensivists in PDT and FB. A Data Safety and Monitoring Board (DSMB) will monitor the trial. The primary outcome is the incidence of perioperative complications. DISCUSSION: FB is a safe technique when performing PDT although its use is not universally accepted in all ICUs as a routine practice. Should PDT be monitored routinely with endoscopic guidance? This study will assess the role of FB monitoring during PDT. TRIAL REGISTRATION: ClinicalTrials.gov NCT04265625. Registered on February 11, 2020.

Derivation of Mesenchymal Stromal Cells from Ovine Umbilical Cord Wharton's Jelly.

The methods described herein allow for the isolation and expansion of fibroblastic-like ovine Wharton's jelly-derived mesenchymal stromal cells (oWJ-MSC) that, similarly to their human counterparts, adhere to standard plastic surfaces in culture; show a mesenchymal profile for specific surface antigens (i.e., positive for CD44 and CD166); and lack expression of endothelial (CD31) and hematopoietic (CD45) markers as well as major histocompatibility complex (MHC) class-II. Homogeneous cell cultures result from a two-phase bioprocess design that starts with the isolation of mesenchymal stromal cells (MSC) from the Wharton's jelly of ovine umbilical cords up to a first step of cryopreservation. The second phase allows for further expansion of ovine WJ-MSC up to sufficient numbers for further studies. Overall, this methodology encompasses a 2-week bioprocess design that encompasses two cell culture passages ensuring sufficient cells for the generation of a Master Cell Bank. Further thawing and scale expansion results in large quantities of oWJ-MSC that can be readily used in proof of efficacy and safety studies in the preclinical development stage of the development of cell-based medicines. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation and expansion of ovine mesenchymal stromal cells from Wharton's jelly of the umbilical cord Basic Protocol 2: Characterization of ovine mesenchymal stromal cells Basic Protocol 3: Growth profile determination of ovine mesenchymal stromal cells from Wharton's jelly.

Advances in translational orthopaedic research with species-specific multipotent mesenchymal stromal cells derived from the umbilical cord.

Compliance with current regulations for the development of innovative medicines require the testing of candidate therapies in relevant translational animal models prior to human use. This poses a great challenge when the drug is composed of cells, not only because of the living nature of the active ingredient but also due to its human origin, which can subsequently lead to a xenogeneic response in the animals. Although immunosuppression is a plausible solution, this is not suitable for large animals and may also influence the results of the study by altering mechanisms of action that are, in fact, poorly understood. For this reason, a number of procedures have been developed to isolate homologous species-specific cell types to address preclinical pharmacodynamics, pharmacokinetics and toxicology. In this work, we present and discuss advances in the methodologies for derivation of multipotent Mesenchymal Stromal Cells derived from the umbilical cord, in general, and Wharton's jelly, in particular, from medium to large animals of interest in orthopaedics research, as well as current and potential applications in studies addressing proof of concept and preclinical regulatory aspects.

Effect of Allogeneic Cell-Based Tissue-Engineered Treatments in a Sheep Osteonecrosis Model.

Osteonecrosis of the femoral head (ONFH) is defined as a tissue disorder and successive subchondral bone collapse resulting from an ischemic process, which may progress to hip osteoarthritis. Cell therapy with multipotent bone marrow mesenchymal stromal cells (BM-MSC) of autologous origin appears to be safe and has shown regenerative potential in previous preclinical and clinical studies. The use of allogeneic cells is far more challenging, but may be a promising alternative to use of autologous cells. Moreover, an optimized dosage of cells from an allogeneic source is needed to obtain off-the-shelf tissue engineering products (TEPs). The purpose of this study was to evaluate the efficacy of a TEP composed of undifferentiated ex vivo expanded BM-MSC of allogeneic origin, combined with bone matrix particles in variable doses. A comparative analysis of TEP's bone regenerative properties against its autologous counterpart was performed in an early-stage ONFH preclinical model in mature sheep. Allogeneic BM-MSC groups demonstrated bone regeneration capacity in osteonecrotic lesions equivalent to autologous BM-MSC groups 6 weeks after treatment. Likewise, stimulation of bone regeneration by a low cell dose of 0.5 × 106 BM-MSC/cm3 was equivalent to that of a high cell dose, 5 × 106 BM-MSC/cm3. Neither local nor systemic immunological reactions nor tumorigenesis were reported, strengthening the safety profile of allogeneic BM-MSC therapy in this model. Our results suggest that low-dose allogeneic BM-MSC is sufficient to promote bone regeneration in femoral head osteonecrotic lesions, and should be considered in translation of new allogeneic cell-based TEPs to human clinics. Impact statement Cell therapy and tissue engineering hold promise as novel regenerative therapies for musculoskeletal diseases, and particularly in bone regeneration strategies. In this article, we report the evaluation of the efficacy of an allogeneic cell-based tissue engineering product (TEP) in an early-stage osteonecrosis of the femoral head preclinical model in skeletally mature sheep. Moreover, we demonstrate its bone regeneration capacity and safety in vivo and its equivalence to autologous counterparts. These findings have important implications for the translation of new allogeneic cell-based TEPs to human clinics.

Sterilization Procedure for Temperature-Sensitive Hydrogels Loaded with Silver Nanoparticles for Clinical Applications.

Hydrogels (HG) have recognized benefits as drug delivery platforms for biomedical applications. Their high sensitivity to sterilization processes is however one of the greatest challenges regarding their clinical translation. Concerning infection diseases, prevention of post-operatory related infections is crucial to ensure appropriate patient recovery and good clinical outcomes. Silver nanoparticles (AgNPs) have shown good antimicrobial properties but sustained release at the right place is required. Thus, we produced and characterized thermo-sensitive HG based on Pluronic® F127 loaded with AgNPs (HG-AgNPs) and their integrity and functionality after sterilization by dry-heat and autoclave methods were carefully assessed. The quality attributes of HG-AgNPs were seriously affected by dry-heat methods but not by autoclaving methods, which allowed to ensure the required sterility. Also, direct sterilization of the final HG-AgNPs product proved more effective than of the raw material, allowing simpler production procedures in non-sterile conditions. The mechanical properties were assessed in post mortem rat models and the HG-AgNPs were tested for its antimicrobial properties in vitro using extremely drug-resistant (XDR) clinical strains. The produced HG-AgNPs prove to be versatile, easy produced and cost-effective products, with activity against XDR strains and an adequate gelation time and spreadability features and optimal for in situ biomedical applications.

Vascularized Periosteal Flaps Accelerate Osteointegration and Revascularization of Allografts in Rats.

BACKGROUND: Surgical reconstruction of large bone defects with structural bone allografts can restore bone stock but is associated with complications such as nonunion, fracture, and infection. Vascularized reconstructive techniques may provide an alternative in the repair of critical bone defects; however, no studies specifically addressing the role of vascularized periosteal flaps in stimulating bone allograft revascularization and osseointegration have been reported. QUESTIONS/PURPOSES: (1) Does a vascularized periosteal flap increase the likelihood of union at the allograft-host junction in a critical-size defect femoral model in rats? (2) Does a vascularized periosteal flap promote revascularization of a critical-size defect structural bone allograft in a rat model? (3) What type of ossification occurs in connection with a vascularized periosteal flap? METHODS: Sixty-four rats were assigned to two equal groups. In both the control and experimental groups, a 5-cm critical size femoral defect was created in the left femur and then reconstructed with a cryopreserved structural bone allograft and intramedullary nail. In the experimental group, a vascularized periosteal flap from the medial femoral condyle, with a pedicle based on the descending genicular vessels, was associated with the allograft. The 32 rats of each group were divided into subgroups of 4-week (eight rats), 6-week (eight rats), and 10-week (16 rats) followup. At the end of their assigned followup periods, the animals were euthanized and their femurs were harvested for semiquantitative and quantitative analysis using micro-CT (all followup groups), quantitative biomechanical evaluation (eight rats from each 10-week followup group), qualitative confocal microscopic, backscattered electron microscopic, and histology analysis (4-week and 6-week groups and eight rats from each 10-week followup group). When making their analyses, all the examiners were blinded to the treatment groups from which the samples came. RESULTS: There was an improvement in allograft-host bone union in the 10-week experimental group (odds ratio [OR], 19.29 [3.63-184.50], p < 0.05). In contrast to control specimens, greater bone neoformation in the allograft segment was observed in the experimental group (OR [4-week] 63.3 [39.6-87.0], p < 0.05; OR [6-week] 43.4 [20.5-66.3], p < 0.05; OR [10-week] 62.9 [40.1-85.7], p < 0.05). In our biomechanical testing, control samples were not evaluable as a result of premature breakage during the embedding and assembly processes. Therefore, experimental samples were compared with untreated contralateral femurs. No difference in torsion resistance pattern was observed between both groups. Both backscattered electron microscopy and histology showed newly formed bone tissue and osteoclast lacunae, indicating a regulated process of bone regeneration of the initial allograft in evaluated samples from the experimental group. They also showed intramembranous ossification produced by the vascularized periosteal flap in evaluated samples from the experimental group, whereas samples from the control group showed an attempted endochondral ossification in the allograft-host bone junctions. CONCLUSIONS: A vascularized periosteal flap promotes and accelerates allograft-host bone union and revascularization of cryopreserved structural bone allografts through intramembranous ossification in a preclinical rat model. CLINICAL RELEVANCE: If large-animal models substantiate the findings made here, this approach might be used in allograft reconstructions for critical defects using fibular or tibial periosteal flaps as previously described.

New and safe experimental model of radiation-induced neurovascular histological changes for microsurgical research.

The aim is to create a new and safe experimental model of radiation-induced neurovascular histological changes with reduced morbidity and mortality for use with experimental microsurgical techniques. Seventy-two Sprague-Dawley rats (250-300 g) were divided as follows: Group I: control group, 24 rats clinically evaluated during six weeks; Group II: evaluation of acute side-effects (two-week follow-up period), 24 irradiated (20 Gy) rats; and Group III: evaluation of subacute side-effects (six-week follow-up period), 24 irradiated (20 Gy) rats. Variables included clinical assessments, weight, vascular permeability (arterial and venous), mortality and histological studies. No significant differences were observed between groups with respect to the variables studied. Significant differences were observed between groups I vs II-III regarding survival rates and histological changes to arteries, veins and nerves. Rat body weights showed progressive increases in all groups, and the mortality rate of the present model is 10.4% compared with 30-40% in the previous models. In conclusion, the designed model induces selective changes by radiotherapy in the neurovascular bundle without histological changes affecting the surrounding tissues. This model allows therapeutic experimental studies to be conducted, including the viability of microvascular and microneural sutures post radiotherapy in the cervical neurovascular bundle.

Effect of Previous Irradiation on Vascular Thrombosis of Microsurgical Anastomosis: A Preclinical Study in Rats.

BACKGROUND: The objective of the present investigation was to compare the effect of neoadjuvant irradiation on the microvascular anastomosis in cervical bundle using an experimental model in rats. METHODS: One hundred forty male Sprague-Dawley rats were allocated into 4 groups: group I, control, arterial microanastomosis; group II, control, venous microanastomosis; group III, arterial microanastomosis with previous irradiation (20 Gy); and group IV, venous microanastomosis with previous irradiation (20 Gy). Clinical parameters, technical values of anastomosis, patency, and histopathological parameters were evaluated. RESULTS: Irradiated groups (III and IV) and vein anastomosis groups (II and IV) showed significantly increased technical difficulties. Group IV showed significantly reduced patency rates (7/35) when compared with the control group (0/35). Radiotherapy significantly decreased the patency rates of the vein (7/35) when compared with the artery (1/35). Groups III and IV showed significantly reduced number of endothelial cells and also showed the presence of intimal thickening and adventitial fibrosis as compared with the control group. CONCLUSION: Neoadjuvant radiotherapy reduces the viability of the venous anastomosis in a preclinical rat model with a significant increase in the incidence of vein thrombosis.