Bioreactors for tendon tissue engineering: challenging mechanical demands towards tendon regeneration

Tendon tissues have very important load-bearing and load-transfer functions, and are also very prone to injuries that can dramatically affect patientâ s quality of life and which are difficult to manage successfully with current available therapies. Regenerative approaches following tendon tissue engineering (TTE) principles have sought to augment the injured tendon with stem cells, scaffolds and mechanical stimulus to improve natural healing response. In fact, combinatorial tenogenic cues may involve adequate topographical, biochemical and mechanical signals for recapitulating native cellular microenvironment and thus promote regeneration. Hence, for the successful implementation of TTE therapies, all aspects of tendon function and requirements should be taken into account in the in vitro maturation of constructs prior implantation. In this sense, bioreactor systems represent attractive tools to provide biomechanical signaling to cells-laden constructs under closely monitored and tightly controlled environments. This chapter discusses specific roles of biomechanical stimulation in tendons and the most frequently used bioreactor systems in tendon tissue engineering field

Monocyte chemoattractant protein-induced protein 1 (MCPIP1) enhances angiogenic and cardiomyogenic potential of murine bone marrow-derived mesenchymal stem cells

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues

Optimization of in vitro culture of murine bone marrow-derived mesenchymal stem cells for the bioactive microvesicle isolation

Mezenchymalne komórki macierzyste (z ang. mesenchymal stem cells; MSC) ze względu na swój szeroki potencjał różnicowania, aktywność parakrynną, niską immunogenność i zdolności immunomodulacyjne od lat pozostają przedmiotem zainteresowania medycyny regeneracyjnej. Jednakże, zastosowanie terapii komórkowych budzi obawy o długofalowe skutki przeczepienia komórek, w tym możliwość transformacji nowotworowych. W tym kontekście, nowe perspektywy terapii otwiera możliwość wykorzystania bioaktywnych mikropęcherzyków ( z ang. microvesicles; MV), złuszczanych z powierzchni komórek, w tym komórek macierzystych, oraz zdolnych do modulowania fenotypu i funkcji komórek docelowych poprzez transfer receptorów powierzchniowych, białek, lipidów i kwasów nukleinowych. Dotychczasowe doniesienia sugerują, że proces złuszczania mikrofragmentów oraz ich biologicznie aktywna zawartość zależy od stanu fizjologicznego komórki, stąd istnieje potrzeba optymalizacji warunków hodowli komórek donorowych in vitro w celu pozyskania z nich MV o możliwie najwyższym potencjale terapeutycznym. W niniejszej pracy porównano dwa protokoły hodowli mysich szpikowych MSC: 1) hodowlę w standardowych warunkach, tj. medium DMEM/F12 z dodatkiem 10% płodowej surowicy bydlęcej (z ang. fetal bovine serum; FBS) oraz 2) hodowlę w warunkach bezsurowiczych, tj. medium DMEM/F12 z dodatkiem albuminy wołowej (z ang. bovine serum albumin; BSA). Analiza cytometryczna profilu antygenów powierzchniowych oraz cyklu komórkowego nie wykazała różnic pomiędzy porównywanymi grupami MSC w obu zastosowanych pożywkach. Natomiast, zaobserwowano zmienioną morfologię, zahamowanie aktywności proliferacyjnej i metabolicznej oraz obniżoną żywotność komórek w DMEM/F12+0,5% BSA, w porównaniu do MSC w DMEM/F12+10% FBS. Mikrofragmenty pozyskane poprzez ultrawirowanie medium kondycjonowanego zebranego z obu hodowli MSC wykazywały ekspresję niektórych antygenów powierzchniowych komórek donorowych, tj. CD49e, CD105, Sca-1, CD29, CD81 i CD90. Nie stwierdzono istotnych różnic w fenotypie i wielkości MV pomiędzy analizowanymi grupami. Analiza genetyczna ekspresji wybranych genów metodą PCR w czasie rzeczywistym wykazała, że jedynie w przypadku MV pozyskanych z MSC hodowanych w medium z surowicą nastąpił transfer transkryptów dla ważnych regulatorów procesów kardiomiogenezy i angiogenezy, takich jak Mesp-1, Gata-4, Gata-2 i VE-kadheryna, czego nie obserwowano dla MV pozyskanych z MSC hodowanych w medium bezsurowiczym.W świetle powyższych wyników, wydaje się, że utrzymanie standardowych warunków hodowli, tj. obecność surowicy w składzie medium hodowlanego warunkuje optymalny fenotyp i funkcje komórek MSC in vitro. Zaobserwowana obecność transkryptów dla genów związanych z procesem kardiomiogenezy i angiogenezy, jedynie w MV pochodzących z komórek MSC hodowanych w medium z surowicą sugeruje zasadność wykorzystania tego protokołu hodowli MSC w celu pozyskania bioaktywnych MV, których potencjał terapeutyczny zostanie dalej zbadany w modelach zawału mięśnia sercowego in vivo.Mesenchymal stem cells (MSC) due to their wide differentiation potential, paracrine activity, low immunogenicity and immunomodulatory properties have been for years in the limelight of regenerative medicine. However, many concerns related to long-term outcomes of cell therapy, including possible tumor formation, still remain to be resolved. In this context, bioactive microvesicles (MV) may be an alternative for use of whole cells for therapy. MV are shed from cell surface and capable of modulating phenotype and function of target cells via transfer of surface receptors, proteins, lipids and nucleic acids. According to previous reports, it has been suggested that both MV shedding process and their bioactive cargo composition were affected by cell physiological state. Thus, there is a need for optimization of in vitro cell culture procedure to obtain MV with possibly greatest therapeutic potential.In this study, two protocols for in vitro culture of murine bone marrow-derived MSC were compared, namely, 1) standard culture in medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS) or 2) culture in serum-free conditions with employing medium DMEM/F12 with 0,5% bovine serum albumin (BSA).Cell surface antigen profile and cell cycle analysis by flow cytometry did not reveal any significant differences between the two MSC groups. However, some adverse changes of cell morphology, decreased proliferation and metabolic activity, as well as impaired viability of MSC cultured in medium DMEM/F12+0,5% BSA were observed, when compared to MSC cultivated in medium DMEM/F12+10% FBS.Microvesicles isolated by ultracentrifugation of conditioned media collected from both MSC cultures, expressed some of MSC surface markers, such as CD49e, CD105, Sca-1, CD29, CD81 and CD90. No significant differences in phenotype or size were observed between the two MV groups. Expression of selected genes was studied by real time PCR. Transfer of transcripts for some crucial regulators of angiogenesis and cardiomyogenesis, such as MESP-1, Gata-4, Gata-2 and VE-cadherin was observed for MV derived from MSC cultured in medium DMEM/F12+10%FBS. Importantly, none of these transcripts were detected in the MV fraction derived from MSC cultured in serum-free conditions.According to these results, it seems that standard culture conditions, where serum is present, are still optimal for normal phenotype and function of MSC in vitro. Based on the observation about the presence of transcripts for vast regulators of cardiomyogenesis and angiogenesis only in MV derived from MSC cultured in presence of serum, we conclude that use of this protocol may be optimal for harvesting of MV with greatest therapeutic potential, which will be further evaluated in in vivo model of acute myocardial infarction

Non-viral delivery of RNA for therapeutic T cell engineering

Adoptive T cell transfer has shown great success in treating blood cancers, resulting in a growing number of FDAapproved therapies using chimeric antigen receptor (CAR) -engineered T cells. However, the effectiveness of this treatment for solid tumors is still not satisfactory, emphasizing the need for improved T cell engineering strategies and combination approaches. Currently, CAR T cells are mainly manufactured using gammaretroviral and lentiviral vectors due to their high transduction efficiency. However, there are concerns about their safety, the high cost of producing them in compliance with current Good Manufacturing Practices (cGMP), regulatory obstacles, and limited cargo capacity, which limit the broader use of engineered T cell therapies. To overcome these limitations, researchers have explored non -viral approaches, such as membrane permeabilization and carrier -mediated methods, as more versatile and sustainable alternatives for next -generation T cell engineering. Non -viral delivery methods can be designed to transport a wide range of molecules, including RNA, which allows for more controlled and safe modulation of T cell phenotype and function. In this review, we provide an overview of non -viral RNA delivery in adoptive T cell therapy. We first define the different types of RNA therapeutics, highlighting recent advancements in manufacturing for their therapeutic use. We then discuss the challenges associated with achieving effective RNA delivery in T cells. Next, we provide an overview of current and emerging technologies for delivering RNA into T cells. Finally, we discuss ongoing preclinical and clinical studies involving RNA -modified T cells

Tendon explant cultures to study the communication between adipose stem cells and native tendon niche

Poor clinical outcomes of tendon repair, together with limited regenerative capacity of the tissue, have triggered the search for alternative regenerative medicine strategies. Human adipose-derived stem cells (hASCs) are being investigated as a promising cell source in contributing for tendon repopulation and reconstruction. However, the mechanisms involved in a potential beneficial effect in tendon regeneration are still to be uncovered. To gain further insights on the bi-directional crosstalk occurring between stem cells and the native tendon niche, it was used an indirect (trans-well) system for co-culturing human tendon explants and hASCs. The maintenance of tissue architecture was studied up to 14 days by histological techniques. The secretion of MMPs was evaluated at day 3. The behavior of hASCs was assessed regarding cell elongation and extracellular matrix (ECM) production. The paracrine communication enhanced collagenolytic activity of MMPs in co-cultures at day 3, in comparison to hASCs alone or tendon explants alone, suggesting that ECM remodeling is triggered early in culture. Moreover, hASCs were spontaneously more elongated in co-cultures and the deposition of collagen type III and tenascin-C by hASCs in co-culture was observed at a lower extent after 7 days, in comparison to hASCs alone, being lately recovered at day 14. Overall, explant co-cultures established herein may constitute a tool for replicating the first steps in tendon healing and help uncovering the bi-directional communication occurring between hASCs and the native tendon niche.Authors acknowledge Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of FCT-POPH-FSE, the PhD grant SFRH/BD/96593/2013 of R.C-A and the consolidator grant IF/00593/2015 of M.E.G. and to the project RL3-TECT-NORTE-07-0124-FEDER-000020 cofinanced by ON.2 under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Photothermal nanofibers enable macromolecule delivery in unstimulated human T cells

Cell therapies such as adoptive T cell transfer require ex vivo modification of cells with exogenous cargo to modulate their phenotype (e.g., to express a synthetic antigen receptor) for optimal therapeutic efficacy upon reinfusion in a patient. Several studies have shown superior anti-tumor activity of minimally differentiated T cell subsets over their activated counterparts. Therefore, developing techniques for safe and efficient manipulation of these quiescent cells is important for both clinical applications and fundamental studies of T cell biology. Photoporation with photothermal electrospun nanofibers (PEN) is an efficient and minimally perturbing nonviral intracellular delivery technique for activated and expanded T cells. However, the technique has not yet been applied to unstimulated T cells. Here, we investigated the potential of PEN photoporation for delivery of macromolecules into these cells. First, we confirmed with inductively coupled plasma tandem mass spectrometry that there was no significant iron release from fibers after laser activation of PEN substrates for laser fluences up to 0.36 J/cm2. Next, we demonstrated successful intracellular delivery of 150 kDa FITC-dextran as model macromolecule in resting and pre-activated lymphocytes with 55-60 % delivery efficiency. By analyzing metabolic activity, activation surface marker presentation and extracellular cytokine release, we found that PEN treatment had no effect on cell proliferation and limited impact on T cell activation propensity for all tested irradiation energies. Thus, our findings show that PEN photoporation holds promise as a safe and efficient delivery strategy, paving the way for its use in genetic modification of minimally differentiated T cells

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage

Photothermal nanofibers enable macromolecule delivery in unstimulated human T cells

Abstract: Cell therapies such as adoptive T cell transfer require ex vivo modification of cells with exogenous cargo to modulate their phenotype (e.g., to express a synthetic antigen receptor) for optimal therapeutic efficacy upon reinfusion in a patient. Several studies have shown superior anti-tumor activity of minimally differentiated T cell subsets over their activated counterparts. Therefore, developing techniques for safe and efficient manipulation of these quiescent cells is important for both clinical applications and fundamental studies of T cell biology. Photoporation with photothermal electrospun nanofibers (PEN) is an efficient and minimally perturbing nonviral intracellular delivery technique for activated and expanded T cells. However, the technique has not yet been applied to unstimulated T cells. Here, we investigated the potential of PEN photoporation for delivery of macromolecules into these cells. First, we confirmed with inductively coupled plasma tandem mass spectrometry that there was no significant iron release from fibers after laser activation of PEN substrates for laser fluences up to 0.36 J/cm2. Next, we demonstrated successful intracellular delivery of 150 kDa FITC-dextran as model macromolecule in resting and pre-activated lymphocytes with 55-60 % delivery efficiency. By analyzing metabolic activity, activation surface marker presentation and extracellular cytokine release, we found that PEN treatment had no effect on cell proliferation and limited impact on T cell activation propensity for all tested irradiation energies. Thus, our findings show that PEN photoporation holds promise as a safe and efficient delivery strategy, paving the way for its use in genetic modification of minimally differentiated T cells