Tumour-derived extracellular vesicle based vaccines for melanoma treatment

The interest of extracellular vesicles (EVs) in cancer immunotherapy is increasing every day. EVs are lipid bilayer vesicles released by most cells, which contain the molecular signature of their parent cell. Melanoma-derived EVs present antigens specific to this aggressive type of cancer, but they also exert immunomodulatory and pro- metastatic activity. Until now, most reviews focus on the immunoevasive characteristics of tumour-derived EVs, but do not help to overcome the issues related to them. In this review, we describe isolation methods of EVs from melanoma patients and most interesting markers to oversee their effect if they are used as antigen carriers. We also discuss the methods developed so far to overcome the lack of immunogenicity of melanoma-derived EVs, which includes EV modification or adjuvant co-administration. In summary, we conclude that EVs can be an interesting antigen source for immunotherapy development once EV obtaining is optimised and the understanding of the mechanisms behind their multiple effects is further understood.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Basque Government (ELKARTEK project ONKOtools (KK20/00069), ONKOVAC (2021111042), and Consolidated Groups, IT1448-22). Lorena Gonzalez-Melero thanks the University of the Basque Country (UPV/EHU) for the PhD grant (PIF18-295)

3D Bioprinting of Functional Skin Substitutes: From Current Achievements to Future Goals

The aim of this review is to present 3D bioprinting of skin substitutes as an efficient approach of managing skin injuries. From a clinical point of view, classic treatments only provide physical protection from the environment, and existing engineered scaffolds, albeit acting as a physical support for cells, fail to overcome needs, such as neovascularisation. In the present work, the basic principles of bioprinting, together with the most popular approaches and choices of biomaterials for 3D-printed skin construct production, are explained, as well as the main advantages over other production methods. Moreover, the development of this technology is described in a chronological manner through examples of relevant experimental work in the last two decades: from the pioneers Lee et al. to the latest advances and different innovative strategies carried out lately to overcome the well-known challenges in tissue engineering of skin. In general, this technology has a huge potential to offer, although a multidisciplinary effort is required to optimise designs, biomaterials and production processes.This research was funded by the Spanish Ministry of Economy and Competitiveness through the “RETOS” Program (NANOGROW project, RTC-2017-6696-1) and by the Basque Government (Grupos Consolidados, IT 907-16) and through the PhD grant conceded to Paula Gabriela Maniţă (PRE_2020_2_0261)

High resolution and fidelity 3D printing of Laponite and alginate ink hydrogels for tunable biomedical applications

The formulation of hydrogels that meet the necessary flow characteristics for their extrusion-based 3D printing while providing good printability, resolution, accuracy and stability, requires long development processes. This work presents the technological development of a hydrogel-based ink of Laponite and alginate and evaluates its printing capacity. As a novelty, this article reports a standardizable protocol to quantitatively define the best printing parameters for the development of novel inks, providing new printability evaluation parameters such as the Printing Accuracy Escalation Index. As a result, this research develops a printable Laponite-Alginate hydrogel that presents printability characteristics. This ink is employed for the reproducible manufacture of 3D printed scaffolds with versatile and complex straight or curved printing patterns for a better adaptation to different final applications. Obtained constructs prove to be stable over time thanks to the optimization of a curing process. In addition, the study of the swelling and degradation behavior of the Laponite and alginate 3D printed scaffolds in different culture media allows the prediction of their behavior in future in vitro or in vivo developments. Finally, this study demonstrates the absence of cytotoxicity of the printed formulations, hence, setting the stage for their use in the field of biomedicine.This project has been partially supported by Spanish government Ministerio de Ciencia e Innovación Grant PID2021-122577OB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. Grant IT1448-22 funded by Basque Government and Fundación Vital Fundazioa (vital21/28). Elena Munoz-Perez thanks the Basque Government for the predoctoral grant (PRE_2022_2_0115). Arantza Perez-Valle thanks the Spanish Government for the postdoctoral grant (MARSA55/21)

Mesenchymal Stromal Cell Secretome for the Treatment of Immune-Mediated Inflammatory Diseases: Latest Trends in Isolation, Content Optimization and Delivery Avenues

Considering the high prevalence and the complex pharmacological management of immune-mediated inflammatory diseases (IMIDs), the search for new therapeutic approaches for their treatment is vital. Although the immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSCs) have been extensively studied as a potential therapy in this field, direct MSC implantation presents some limitations that could slow down the clinical translation. Since the beneficial effects of MSCs have been mainly attributed to their ability to secrete a plethora of bioactive factors, their secretome has been proposed as a new and promising pathway for the treatment of IMIDs. Formed from soluble factors and extracellular vesicles (EVs), the MSC-derived secretome has been proven to elicit immunomodulatory effects that control the inflammatory processes that occur in IMIDs. This article aims to review the available knowledge on the MSC secretome, evaluating the advances in this field in terms of its composition, production and application, as well as analyzing the pending challenges in the field. Moreover, the latest research involving secretome administration in IMIDs is discussed to provide an updated state-of-the-art for this field. Finally, novel secretome delivery alternatives are reviewed, paying special attention to hydrogel encapsulation as one of the most convenient and promising strategies.This research was funded by the projects SAF2017-82292-R (MINECO), AEI/FEDER, UE, ICTS “NANBIOSIS” Drug Formulation Unit (U10) and the Basque Country Government (Grupos Consolidados, No ref: IT907-16). Elena Munoz-Perez thanks the Basque Government for the predoctoral grant (PRE_2020_1_0279). A. Gonzalez-Pujana thanks the University of the Basque Country (UPV/EHU) for the postdoctoral grant (ESPDOC20/119)

Industria farmazeutikoak ingurumenean duen inpaktua murrizteko estrategiak

Industria farmazeutikoa, bere ekoizpen-bolumena kontuan izanda, gehien kutsatzen duten industriako sektoreen artean kokatzen da. Hori dela eta, lan honetan, egun martxan dagoen Nazio Batuen Erakundearen (NBE) 2030 Agendarekin lerrokatuz, eta bertan aipatzen diren helburuak betetzeko asmoz, industria farmazeutikoak ingurumenean duen inpaktua murrizteko hainbat estrategia aztertuko dira, arreta medikamentuen ekoizpenari jarriz batez ere. Lehenik, ekoizpen iraunkorra bultzatu nahian, disolbatzaile edo eszipiente organikoak fluido superkritiko eta likido ionikoen bidez ordezkatzea proposatuko da. ondoren, medikamentu edo materia organikoek ingurune urtarrean duten bilakaera aztertuko da. Horren konponbide gisa eta baliabide hidrikoen eraginkortasuna bultzatuz, oxidazio-teknika bidezko erreakzioen erabilera proposatuko da, ur-hondakinen tratamenduan konposatu kimikoak ezabatu ahal izateko. Horrez gain, eta egungo aldaketa klimatikoak ingurumenean duen inpaktua aintzat hartuz, berotegi-efektua duten gasen isurketa murrizteko energia berriztagarrien erabilera bultzatu eta erabiltzen den energiaren efizientziaren hobekuntzarako hainbat estrategia aipatuko dira. Horretaz gain, beste zenbait etapa ere dira, medikamentua sortzen denetik medikamentua birziklatua izan arte. Hortaz, ekoizpen eta kontsumo arduratsuago baten bila, etapa horietako inpaktua ere murriztea beharrezkoa litzateke, medikamentuen kutxen diseinua aldatuz, kutxa ekologikoagoak lortzeko eta garraiorako furgoneta ekologikoen erabilera bultzatuz, besteak beste. Amaitzeko, industria farmazeutikoa I+G+b-n gehien inbertitzen duen sektorea dela aintzat hartuz, hausnarketa txiki bat egiten da, baliabide eta teknologia horiek etorkizunean ekar ditzaketen onurak azpimarratuz.; The pharmaceutical industry, in terms of the volume of production it handles, is among the most polluting industrial sectors. Therefore, in line with the current United Nations (UN) Agenda 2030, and in order to meet the objectives set out therein, in this work we will examine various strategies to reduce the environmental impact of the pharmaceutical industry, focusing especially on drug production. First, in an attempt to promote sustainable production, replacement of organic solvents or excipients with supercritical fluids and ionic liquids is proposed. Next, the evolution of drugs or organic matter in the aquatic environment will be examined. As a solution to this, and by promoting the efficiency of water resources, the use of oxidation technique-based reactions will be proposed to eliminate chemical compounds in the treatment of waste water. In addition, and taking into account the impact of current climate change on the environment, several strategies based on promoting the use of renewable energy will be analysed, in order to reduce the emission of greenhouse gases and improve the efficiency of the energy used. Additionally, other stages of the production are also examined, from the time the drug is created until the drug is discarded and recycled. Therefore, in pursuit of a more responsible production and consumption model, it would also be necessary to reduce the impact of these stages by changing the design of medicine boxes, achieving greener packaging and encouraging the use of environmentally friendly transport, among others. Finally, given that the pharmaceutical industry is the sector that invests the most in R and D and I, a small reflection is made, highlighting the benefits that these resources and technologies can bring in the future

Cell Microencapsulation Technologies for Sustained Drug Delivery: Latest Advances in Efficacy and Biosafety

The development of cell microencapsulation systems began several decades ago. However, today few systems have been tested in clinical trials. For this reason, in the last years, researchers have directed efforts towards trying to solve some of the key aspects that still limit efficacy and biosafety, the two major criteria that must be satisfied to reach the clinical practice. Regarding the efficacy, which is closely related to biocompatibility, substantial improvements have been made, such as the purification or chemical modification of the alginates that normally form the microspheres. Each of the components that make up the microcapsules has been carefully selected to avoid toxicities that can damage the encapsulated cells or generate an immune response leading to pericapsular fibrosis. As for the biosafety, researchers have developed biological circuits capable of actively responding to the needs of the patients to precisely and accurately release the demanded drug dose. Furthermore, the structure of the devices has been subject of study to adequately protect the encapsulated cells and prevent their spread in the body. The objective of this review is to describe the latest advances made by scientist to improve the efficacy and biosafety of cell microencapsulation systems for sustained drug delivery, also highlighting those points that still need to be optimized

The role of osmolarity adjusting agents in the regulation of encapsulated cell behavior to provide a safer and more predictable delivery of therapeutics

[Abstract] Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.Gobierno Vasco; IT-907-16Universidad del País Vasco; UFI11/3

Bioactive and degradable hydrogel based on human platelet-rich plasma fibrin matrix combined with oxidized alginate in a diabetic mice wound healing model

[EN] In the present study we developed an injectable, bioactive and degradable hydrogel composed of alginate at 2.5% oxidation degree and calcium-activated platelet rich plasma (PRP) for wound healing applications (PRP-HG-2.5%). The alginate gives mechanical support to the hydrogel while the activated PRP provides growth factors that enhance wound healing and fibrin which creates an adequate microenvironment for cell migration and proliferation. The rheological and mechanical properties of the hydrogel were characterized. Further characterization revealed that PRPHG-2.5% showed a faster hydrolitic degradation rate than unmodified alginate and a similar platelet derived growth factor (PDGF-BB) release profile. In vitro efficacy studies, carried out in human fibroblasts and keratinocytes, showed that PRP-HG-2.5% was not cytotoxic and that it was able to promote cell adhesion and proliferation. Thereafter, in an in vivo full thickness wound healing study conducted in diabetic mice, no differences were found among PRP-HG-2.5% and its counterpart without PRP, likely due to the xenogeneic origin of the PRP. This hypothesis was validated in vitro, since a cytotoxic effect was observed after human PRP application to mouse fibroblasts. Therefore, PRP-HG-2.5% might be a promising strategy for chronic woundstreatment, although its effectiveness should be evaluated in a more reliable preclinical model.I. Garcia-Orue thanks University of the Basque Country (UPV/EHU) for the Dokberri grant (DOCREC19/10). The authors are thankful for the technical and human support provided by SGIker of UPV/EHU. Authors also thank ICTS "NANBIOSIS", specifically the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBERBBN) at the UPV/EHU in Vitoria-Gasteiz. Finally, the authors thank the Open Access funding provided by University of the Basque Country

Extracellular vesicles from hair follicle-derived mesenchymal stromal cells: isolation, characterization and therapeutic potential for chronic wound healing

[EN] Background Mesenchymal stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have demonstrated to elicit immunomodulatory and pro-regenerative properties that are beneficial for the treatment of chronic wounds. Thanks to different mediators, MSC-EVs have shown to play an important role in the proliferation, migration and cell survival of different skin cell populations. However, there is still a big bid to achieve the most effective, suitable and available source of MSC-EVs. Methods We isolated, characterized and compared medium-large EVs (m-lEVs) and small EVs (sEVs) obtained from hair follicle-derived MSCs (HF-MSCs) against the gold standard in regenerative medicine, EVs isolated from adipose tissue-derived MSCs (AT-MSCs). Results We demonstrated that HF-EVs, as well as AT-EVs, expressed typical MSC-EVs markers (CD9, CD44, CD63, CD81 and CD105) among other different functional markers. We showed that both cell types were able to increase human dermal fibroblasts (HDFs) proliferation and migration. Moreover, both MSC-EVs were able to increase angiogenesis in human umbilical vein endothelial cells (HUVECs) and protect HDFs exposed to a hyperglycemic environment from oxidative stress and cytotoxicity. Conclusions Taken together, HF-EVs demonstrated to exhibit comparable potential to that of AT-EVs as promising candidates in the treatment of chronic wounds.This research was funded by the Basque Government (Consolidated Groups, IT907-16 and PRE_2018_1_0412)

Human Hair Follicle-Derived Mesenchymal Stromal Cells from the Lower Dermal Sheath as a Competitive Alternative for Immunomodulation

Mesenchymal stromal cells (MSCs) have unique immunomodulatory capacities. We investigated hair follicle-derived MSCs (HF-MSCs) from the dermal sheath, which are advantageous as an alternative source because of their relatively painless and minimally risky extraction procedure. These cells expressed neural markers upon isolation and maintained stemness for a minimum of 10 passages. Furthermore, HF-MSCs showed responsiveness to pro-inflammatory environments by expressing type-II major histocompatibility complex antigens (MHC)-II to a lesser extent than adipose tissue-derived MSCs (AT-MSCs). HF-MSCs effectively inhibited the proliferation of peripheral blood mononuclear cells equivalently to AT-MSCs. Additionally, HF-MSCs promoted the induction of CD4+CD25+FOXP3+ regulatory T cells to the same extent as AT-MSCs. Finally, HF-MSCs, more so than AT-MSCs, skewed M0 and M1 macrophages towards M2 phenotypes, with upregulation of typical M2 markers CD163 and CD206 and downregulation of M1 markers such as CD64, CD86, and MHC-II. Thus, we conclude that HF-MSCs are a promising source for immunomodulation.This work was supported by the projects SAF2017-82292-R (MINECO), MCIU-AEI/ FEDER, UE, ICTS “NANBIOSIS” Drug Formulation Unit (U10) and the Eusko Jaurlaritza (Grupos Consolidados, No ref: IT907-16)