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Peripheral artery disease is a progressive, devastating disease that leads to critical limb ischemia (CLI). Therapeutic angiogenesis using stem cell therapy has emerged as a promising approach for its treatment; however, adapting cell-based therapy has been limited by poor cell survival and low treatment efficiency. To overcome unmet clinical needs, we developed a fibroblast growth factor 2 (FGF2)-immobilized matrix that enabled control of cell adhesion to the surface and exerted a priming effect on the cell. Human adipose-derived stem cells (hASCs) grown in this matrix formed a functionally enhanced cells spheroid (FECS-Ad) that secreted various angiogenic factors including interleukin-8 (IL-8). We demonstrated that IL-8 was upregulated by the FGF2-mediated priming effect during FECS-Ad formation. Immobilized FGF2 substrate induced stronger IL-8 expression than soluble FGF2 ligands, presumably through the FGFR1/JNK/NF-κB signaling cascade. In IL-8-silenced FECS-Ad, vascular endothelial growth factor (VEGF) expression was decreased and angiogenic potential was reduced. Intramuscular injection of FECS-Ad promoted angiogenesis and muscle regeneration in mouse ischemic tissue, while IL-8 silencing in FECS-Ad inhibited these effects. Taken together, our data demonstrate that IL-8 contributes to therapeutic angiogenesis and suggest that FECS-Ad generated using the MBP-FGF2 matrix might provide a reliable platform for developing therapeutic agents to treat CLI.

A significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Cell therapies for the treatment of skin disorders could benefit from simple, safe and efficient technology for the transdermal delivery of therapeutic cells. Conventional cell delivery by hypodermic-needle injection is associated with poor patient compliance, requires trained personnel, generates waste and has non-negligible risks of injury and infection. Here, we report the design and proof-of-concept application of cryogenic microneedle patches for the transdermal delivery of living cells. The microneedles are fabricated by stepwise cryogenic micromoulding of cryogenic medium with pre-suspended cells, and can be easily inserted into porcine skin and dissolve after deployment of the cells. In mice, cells delivered by the cryomicroneedles retained their viability and proliferative capability. In mice with subcutaneous melanoma tumours, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumour growth than intravenous and subcutaneous injections of the cells. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies. Cryogenic microneedle patches can deliver mammalian cells before dissolving into the skin, as shown with the transdermal delivery of ovalbumin-pulsed dendritic cells in mice with subcutaneous tumours.

Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

Neural precursor/stem cell transplantation therapies promote regeneration in neurological injuries, but current cell delivery methods have drawbacks. These include risks with surgical microinjection (e.g., hemorrhage, embolism) and high cell loss with systemic delivery/passage through fine gauge needles. Aerosolized cell delivery offers significant benefits including rapid and minimally invasive cell delivery, and ease of delivery to end users. To develop this approach, it is necessary to prove that 1) aerosolization does not have detrimental effects on transplant cells and 2) suitable media can be identified to support cell delivery. To achieve these aims, cells are sprayed using a commercial spray device or stored in Hibernate‐A, a CO2‐independent nutrient solution. Histological assessments consist of cell viability analysis, immunocytochemistry, and EdU labeling. It is shown that a major neural precursor transplant population—oligodendrocyte precursor cells (OPCs)—survive following aerosolized delivery and retain their capacity for proliferation and differentiation (key to their repair function). Hibernate‐A can support OPCs’ survival without specialized maintenance conditions, with no detrimental impact on cell fate. It is considered that this data supports the concept of a novel class of advanced medical spray devices to facilitate transport and delivery of transplant populations in neural cell therapy.

Soft materials that facilitate the three-dimensional (3D) encapsulation, proliferation, and facile local delivery of cells to targeted tissues will aid cell-based therapies, especially those that depend on the local engraftment of implanted cells. Herein, we develop a negatively charged fibrillar hydrogel based on the de novo-designed self-assembling peptide AcVES3-RGDV. Cells are easily encapsulated during the triggered self-assembly of the peptide leading to gel formation. Self-assembly is induced by adjusting the ionic strength and/or temperature of the solution, while avoiding large changes in pH. The AcVES3-RGDV gel allows cell–material attachment enabling both two-dimensional and 3D cell culture of adherent cells. Gel–cell constructs display shear-thin/recovery rheological properties enabling their syringe-based delivery. In vivo cellular fluorescence as well as tissue resection experiments show that the gel supports the long-term engraftment of cells delivered subcutaneously into mice.

Hydrogels have been widely used for cell delivery to enhance cell-based therapies for cartilage tissue regeneration. To better support cartilage deposition, it is imperative to determine hydrogel formulation with physical and biochemical cues that are optimized for different cell populations. Previous attempts to identify optimized hydrogels rely mostly on testing hydrogel formulations with discrete properties, which are time-consuming and require large amounts of cells and materials. Gradient hydrogels encompass a range of continuous changes in niche properties, therefore offering a promising solution for screening a wide range of cell-niche interactions using less materials and time. However, harnessing gradient hydrogels to assess how matrix stiffness modulates cartilage formation by different cell types in vivo have never been investigated before. The goal of this study is to fabricate gradient hydrogels for screening the effects of varying hydrogel stiffness on cartilage formation by mesenchymal stem cells (MSCs) and chondrocytes, respectively, the two most commonly used cell populations for cartilage regeneration. We fabricated stiffness gradient hydrogels with tunable dimensions that support homogeneous cell encapsulation. Using gradient hydrogels with tunable stiffness range, we found MSCs and chondrocytes exhibit opposite trend in cartilage deposition in response to stiffness changes in vitro. Specifically, MSCs require soft hydrogels with Young's modulus less than 5 kPa to support faster cartilage deposition, as shown by type II collagen and sulfated glycosaminoglycan staining. In contrast, chondrocytes produce cartilage more effectively in stiffer matrix (>20 kPa). We chose optimal ranges of stiffness for each cell population for further testing in vivo using a mouse subcutaneous model. Our results further validated that soft matrix (Young's modulus 20 kPa) is more desirable for supporting chondrocyte-based cartilage deposition. Our results show the importance of optimizing niche cues in a cell-type-specific manner and validate the potential of using gradient hydrogels for optimizing niche cues to support cartilage regeneration in vitro and in vivo. IMPACT STATEMENT: The present study validates the utility of gradient hydrogels for determining optimal hydrogel stiffness for supporting cartilage regeneration using both chondrocytes and stem cells. We demonstrate that such gradient hydrogels can be used for fast optimizing matrix stiffness for specific cell type to support optimal cartilage regeneration. To our knowledge, this is the first demonstration of applying gradient hydrogels for assessing optimal niche cues that support tissue regeneration in vivo and may be used for assessing optimal niche cues for different cell types to regeneration of different tissues.

In the past decades, numerous preclinical studies and several clinical trials have evidenced the feasibility of cell transplantation in treating heart diseases. Over the years, different delivery routes of cell therapy have emerged and broadened the width of the field. However, a common hurdle is shared by all current delivery routes: low cell retention. A myriad of studies confirm that cell retention plays a crucial role in the success of cell‐mediated cardiac repair. It is important for any delivery route to maintain donor cells in the recipient heart for enough time to not only proliferate by themselves, but also to send paracrine signals to surrounding damaged heart cells and repair them. In this review, we first undertake an in‐depth study of primary theories of cell loss, including low efficiency in cell injection, “washout” effects, and cell death, and then organize the literature from the past decade that focuses on cell transplantation to the heart using various cell delivery routes, including intracoronary injection, systemic intravenous injection, retrograde coronary venous injection, and intramyocardial injection. In addition to a recapitulation of these approaches, we also clearly evaluate their strengths and weaknesses. Furthermore, we conduct comparative research on the cell retention rate and functional outcomes of these delivery routes. Finally, we extend our discussion to state‐of‐the‐art bioengineering techniques that enhance cell retention, as well as alternative delivery routes, such as intrapericardial delivery. A combination of these novel strategies and more accurate assessment methods will help to address the hurdle of low cell retention and boost the efficacy of cell transplantation to the heart.

Background: Long-term exposure to inhalable silica particles may lead to a serious systemic pulmonary disease called silicosis. However, the role and mechanisms of exosomes in silicosis are not well understood. We previously reported that serum exosomal micro (mi) RNA profile was altered in pneumoconiosis patients and silica-exposed macrophages.This study was aimed to explore and verify the role of the exosomal miRNA in lung fibrosis when exposed to silica particles. Results: The RT-qPCR result revealed that the levels of the miR-107, miR-122-5p, miR-125a-5p, miR-126-5p, and miR-335-5p were elevated in serous exosomes of silicosis patients. A bioinformatics analysis predicted 5 potential interactions involving these miRNAs, with miR-107–cyclin-dependent kinase (CDK) 6 having the highest score. In a mouse model of silica particle-induced silicosis, miR-107 level in serum exosomes and lung tissue was increased during the development of fibrosis, while inhibition of miR-107 reduced pulmonary fibrosis. The number of exosomes secreted by macrophages exposed to silica particles was also increased and showed altered cargo composition, and showed a capacity to promote lung fibroblast transdifferentiation through a possible mechanism involving the delivery of miR-107 by macrophages to lung fibroblasts via exosomes, resulting in targeted inhibition of CDK6, reduced retinoblastoma protein phosphorylation, and inhibition of E2F1 and cell cycle progression. Conclusion: In summary, exosomal miR-107 derived from macrophages exposed to silica particles were transferred to pulmonary fibroblasts to trigger their transdifferentiation by targeting CDK6 and arresting cell cycle. These findings provide insight into the pathogenesis of silicosis and potential targets for intervention.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution., The type III secretion system (T3SS) of bacteria can be used to inject cargo into eukaryotic cells but its lack of target specificity is a disadvantage. Here the authors place the T3SS under the regulation of light by engineering optogenetic switches into the dynamic cytosolic T3SS component SctQ.

Extracellular vesicles (EVs) contain multiple factors that regulate cell and tissue function. However, understanding of their influence on gametes, including communication with the oocyte, remains limited. In the present study, we characterized the proteome of domestic cat (Felis catus) follicular fluid EVs (ffEV). To determine the influence of follicular fluid EVs on gamete cryosurvival and the ability to undergo in vitro maturation, cat oocytes were vitrified using the Cryotop method in the presence or absence of ffEV. Vitrified oocytes were thawed with or without ffEVs, assessed for survival, in vitro cultured for 26 hours and then evaluated for viability and meiotic status. Cat ffEVs had an average size of 129.3 ± 61.7 nm (mean ± SD) and characteristic doughnut shaped circular vesicles in transmission electron microscopy. Proteomic analyses of the ffEVs identified a total of 674 protein groups out of 1,974 proteins, which were classified as being involved in regulation of oxidative phosphorylation, extracellular matrix formation, oocyte meiosis, cholesterol metabolism, glycolysis/gluconeogenesis, and MAPK, PI3K-AKT, HIPPO and calcium signaling pathways. Furthermore, several chaperone proteins associated with the responses to osmotic and thermal stresses were also identified. There were no differences in the oocyte survival among fresh and vitrified oocyte; however, the addition of ffEVs to vitrification and/or thawing media enhanced the ability of frozen-thawed oocytes to resume meiosis. In summary, this study is the first to characterize protein content of cat ffEVs and their potential roles in sustaining meiotic competence of cryopreserved oocytes.

BackgroundIn the field of cell therapy for heart disease, a new paradigm of repeated dosing of cells has recently emerged. However, the lack of a repeatable cell delivery method in preclinical studies in rodents is a major obstacle to investigating this paradigm.Methods and ResultsWe have established and standardized a method of echocardiography-guided percutaneous left ventricular intracavitary injection (echo-guided LV injection) as a cell delivery approach in infarcted mice. Here, we describe the method in detail and address several important issues regarding it. First, by integrating anatomical and echocardiographic considerations, we have established strategies to determine a safe anatomical window for injection in infarcted mice. Second, we summarize our experience with this method (734 injections). The overall survival rate was 91.4%. Previous studies results suggest that 1×106 cells delivered via this method yielded similar retention in the heart at 24 h as 1×105 cells delivered via intracoronary or intramyocardial injection. Lastly, we examined the efficacy of this cell delivery approach. Compared with vehicle treatment, cardiac mesenchymal cells (CMCs) delivered via this method improved cardiac function assessed both echocardiographically and hemodynamically. Furthermore, repeated injections of CMCs via this method yielded greater cardiac function improvement than single dose administration.ConclusionEcho-guided LV injection is a feasible, reproducible, relatively less invasive and effective delivery method for cell therapy in heart disease. It is an important approach that could move the field of cell therapy forward, especially with regard to repeated cell administrations.

Cell transplantation is an ambitious, but arguably realistic, therapy for repair of the nervous system. Cell delivery is a major challenge for clinical translation, especially given the apparently inhibitory astrogliotic environment in degenerated tissue. However, astrogliotic tissue also contains endogenous structural and biochemical cues that can be harnessed for functional repair. Minimizing damage to these cues during cell delivery could enhance cell integration. This theory is supported by studies with an auditory astrocyte scar model, in which cells delivered onto the surface of the damaged nerve were more successfully integrated in the host than those injected into the tissue. We consider the application of this less invasive approach for nerve injury and its potential application to some neurodegenerative disorders., ALSの治療に新しい細胞移植法を提唱 --移植細胞を「表面に置くだけ」で中枢神経と筋肉をつなぐ--. 京都大学プレスリリース. 2018-07-25.

Mounting evidence suggests that therapeutic cell and drug delivery strategies designed to actively harness the regenerative potential of the inflammatory response have great potential in regenerative medicine. In particular, macrophages have emerged as a primary target because of their critical roles in regulating multiple phases of tissue repair through their unique ability to rapidly shift phenotypes. Herein, we review macrophage-based therapies, focusing on the translational potential for cell delivery of ex vivo-activated macrophages and delivery of molecules and biomaterials to modulate accumulation and phenotype of endogenous macrophages. We also review current obstacles to progress in translating basic findings to therapeutic applications, including the need for improved understanding of context-dependent macrophage functions and the myriad factors that regulate macrophage phenotype; potential species-specific differences (e.g. humans versus mice); quality control issues; and the lack of standardized procedures and nomenclature for characterizing macrophages. Looking forward, the inherent plasticity of macrophages represents a daunting challenge for harnessing these cells in regenerative medicine therapies but also great opportunity for improving patient outcomes in a variety of pathological conditions.

Mesenchymal stem/progenitor cells (MSCs) are key players in regenerative medicine, relying principally on their differentiation/regeneration potential, immunomodulatory properties, paracrine effects, and potent homing ability with minimal if any ethical concerns. Even though multiple preclinical and clinical studies have demonstrated remarkable properties for MSCs, the clinical applicability of MSC-based therapies is still questionable. Several challenges exist that critically hinder a successful clinical translation of MSC-based therapies, including but not limited to heterogeneity of their populations, variability in their quality and quantity, donor-related factors, discrepancies in protocols for isolation, in vitro expansion and premodification, and variability in methods of cell delivery, dosing, and cell homing. Alterations of MSC viability, proliferation, properties, and/or function are also affected by various drugs and chemicals. Moreover, significant safety concerns exist due to possible teratogenic/neoplastic potential and transmission of infectious diseases. Through the current review, we aim to highlight the major challenges facing MSCs’ human clinical translation and shed light on the undergoing strategies to overcome them.

It is important to create new, specifically designed and controlled nanomaterials that can be used as molecular delivery systems for cells. Here, we describe a method for creating a nanosized DNA capsule (NC) using a photocaged unlocking system as a carrier for cell delivery. The photocaged NC (caged-NC) was designed and constructed to control the opening of the closed NC by photoirradiation. The opening of the NC was observed by atomic force microscopy, and the dynamic opening of the caged-NC was characterized by fluorescence quenching and recovery processes. The caged-NC was then introduced into the cytoplasm of a cell, where the photoinduced opening of the caged-NC was observed. The selective opening of the caged-NC in a single cell was successfully achieved by laser irradiation of individual cells. The caged-NC system could be used as a delivery system for relatively large nanomaterials in cells, similar to a native virus system.

Stem cell therapies have emerged as promising treatments for injuries and diseases in regenerative medicine. Yet, delivering stem cells therapeutically can be complicated by invasive administration techniques, heterogeneity in the injection media, and/or poor cell retention at the injection site. Despite these issues, traditional administration protocols using bolus injections in a saline solution or surgical implants of cell‐laden hydrogels have highlighted the promise of cell administration as a treatment strategy. To address these limitations, we have designed an injectable polymer–nanoparticle (PNP) hydrogel platform exploiting multivalent, noncovalent interactions between modified biopolymers and biodegradable nanoparticles for encapsulation and delivery of human mesenchymal stem cells (hMSCs). hMSC‐based therapies have shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery.

This chapter mainly describes a cell-based treatment focused on a cultured corneal endothelial cell (CEC)-injection therapy, and it is devided into three parts; i.e., Background, Current State, and Future Directions. The background involves the successful culture of CECs in primates, animal-model experiments in which a cultured CEC sheet is used, and modification of the cultivation protocol and its use for CEC-injection therapy in an animal-model experiment. The current state involves cultured human CECs (HCECs) for clinical use, the clinical trials of cultured HCEC-injection therapy, the evaluation of the antigenecity of allogeneic cells and the fate of escaped cells from the eye post injection. As for the future directions, the ‘seeds and soil’ theory in the clinical setting, the combination therapy ewith ROCK-inhibitor eye drops, and the advancement of cell delivery and cell storage are all discussed.

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such ‘next generation’ implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage., Despite recent progress to advance cardiac cell-based therapy for patients, heart failure mortality rivals most cancers. Here, the authors describe an approach to control and pattern 3 distinct human cardiac cell populations to promote superior repair and regeneration after myocardial infarction.

Osteoarthritis (OA) is one of the main causes of pain and disability worldwide. In recent years, numerous efforts have been made in pharmaceutical and surgical therapies for OA management. The therapeutic features of mesenchymal stem cells (MSCs), have led to numerous preclinical and clinical trials that confirmed the efficacy of cell therapy as treatment for OA. Recent works have attempted to customize cell participation in tissue regeneration using site specific targeting approaches. Targeting approaches are based on direct modifications to the surface of MSCs or indirect modifications on an engineered nanomediator. Here, we provide a comprehensive review of the advances in targeted cell delivery and define the priorities for future work in terms of OA and cartilage regeneration.

Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.

Microrobots with simultaneously improved degradability and mechanical strength are highly demanded in performing in vivo delivery tasks in clinical applications. The properties of degradability and mechanical strength are contradictory for many materials used to make microrobots. This article proposes a new design that can result in 3D cell culture microrobots with improved degradability and mechanical strength from the following perspectives. First, the mechanical strength of a microrobot is improved using triangle patterns to replace hexagon pattern in the microrobot structure, which can provide more supporting grids to obtain increased mechanical strength. Second, the relationship between structural design and material composition in relation to the mechanical strength of microrobot is investigated. The study reveals that triangle‐patterned microrobots have increased mechanical strength compared with hexagon‐patterned microrobots, thereby allowing high composition of degradable material that leads to the fast degradation of the microrobot. It is also shown that the triangle‐patterned microrobots can maintain the same structural integrity and cell capacity as hexagon‐patterned microrobots. Finally, the demonstration shows that the triangle‐patterned microrobot can be precisely navigated in microfluidic channels. This article successfully demonstrates that the degradability and mechanical strength can be improved simultaneously through the microrobot structural design.

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.

For many cellular therapies being evaluated in preclinical and clinical trials, the mechanisms behind their therapeutic effects appear to be the secretion of growth factors and cytokines, also known as paracrine activity. Often, delivered cells are transient, and half-lives of the growth factors that they secrete are short, limiting their long-term effectiveness. The goal of this study was to optimize a hydrogel system capable of in situ cell delivery that could sequester and release growth factors secreted from those cells after the cells were no longer present. Here, we demonstrate the use of a fast photocross-linkable heparin-conjugated hyaluronic acid (HA-HP) hydrogel as a cell delivery vehicle for sustained growth factor release, which extends paracrine activity. The hydrogel could be modulated through cross-linking geometries and heparinization to support sustained release proteins and heparin-binding growth factors. To test the hydrogel in vivo, we used it to deliver amniotic fluid-derived stem (AFS) cells, which are known to secrete cytokines and growth factors, in full thickness skin wounds in a nu/nu murine model. Despite transience of the AFS cells in vivo, the HA-HP hydrogel with AFS cells improved wound closure and reepithelialization and increased vascularization and production of extracellular matrix in vivo. These results suggest that HA-HP hydrogel has the potential to prolong the paracrine activity of cells, thereby increasing their therapeutic effectiveness in wound healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1986-2000, 2017.

Despite decades of intensive attention directed to creation of genetically altered cells (e.g., as in development of chimeric antigen receptor (CAR) T-cells) and/or to achieve requisite in vitro accumulation of desired immunologic effectors (e.g., elaboration of virus-specific T cells, expansion of NK cells, differentiation of dendritic cells, isolation, and propagation of Tregs, etc.), there has been essentially no interest in the most fundamental of all hurdles: assuring tissue-specific delivery of administered therapeutic cells to sites where they are needed. With regards to use of CAR T-cells, the absence of information on the efficacy of cell delivery is striking, especially in light of the clear association between administered cell dose and adverse events, and the obvious fact that pertinent cell acquisition/expansion costs would be dramatically curtailed with more efficient delivery of the administered cell bolus. Herein, based on information garnered from studies of human leukocytes and adult stem cells, the logic underlying the use of cell surface glycoengineering to enforce E-selectin ligand expression will be conveyed in the context of how this approach offers strategies to enhance delivery of CAR T-cells to marrow and to tumor beds. This application of glycoscience principles and techniques with intention to optimize cell therapeutics is a prime example of the emerging field of “translational glycobiology.”

Bacteria have been extensively utilized for bioimaging, diagnosis and therapy given their unique characteristics including genetic manipulation, rapid proliferation and disease site targeting specificity. However, clinical translation of bacteria for these applications has been largely restricted by their unavoidable side effects and low treatment efficacies. Engineered bacteria for biomedical applications ideally need to generate only a low inflammatory response, show slow elimination by macrophages, low accumulation in normal organs, and almost unchanged inherent bioactivities. Here we describe a set of stealth bacteria, cell membrane coated bacteria (CMCB), meeting these requirement. Our findings are supported by evaluation in multiple mice models and ultimately demonstrate the potential of CMCB to serve as efficient tumor imaging agents. Stealth bacteria wrapped up with cell membranes have the potential for a myriad of bacterial-mediated biomedical applications., The use of engineered bacteria for biomedical applications is limited by side effects such as inflammatory response. Here the authors engineer cell membrane coated bacteria as in vivo tumor imaging agents, and show that these generate a lower inflammatory response and reduced macrophage clearance.

Despite huge progress in interrogating the benefits of stem cells for lung diseases, more studies on optimal routes of cell delivery are required. The ability of intravenously injected stem cells to reach all parts of the lung is limited, particularly when the extracellular matrix and vasculature are damaged in various lung diseases. Hence, an alternative route of stem cell administration that delivers cells directly to the lung through the airway may provide a more adequate supply of stem cells at the site of injury. In many lung injuries and diseases, much of the damage occurs in the airway epithelium, and thus intratracheal administration of the stem cells directly delivered to the injury site may be beneficial. For optimal therapeutic benefits, it is important that cells are uniformly distributed across the whole lung. The exogenous cells are required to be in a healthy state when delivered to the injured area. Therefore, there is a need to optimise the technology to deliver cells directly to airways. This chapter presents an overview of the currently available technologies for cell spraying and discusses other important parameters for successful delivery of stem cells to the lung.

Cellular-based therapies have drawn a great deal of attention thanks to their regenerative medicine approaches to treat incurable diseases and specific injuries. In this regard, injectable cell delivery systems could actualize the therapeutically beneficial outcomes of cell-based therapeutic products. These systems have found considerable clinical uses. Hence, the recent studies have focused on developing injectable bio-constructs to protect transplanted cells during delivery and stimulating endogenous regeneration through interactions of these cells and host tissue. This paper introduces a framework, as a general concept, to improve cell delivery systems for cell-based therapeutic products. Studies on stable injectable carriers can enhance cell homing, proliferation, viability, dressing of irregular shape of target sites, and subsequently support transplanted cell functionality. However, more studies should be conducted on new technologies for the injectable cell-based product for cell delivery and the clinical applications.

Restoration of vision due to corneal blindness from corneal endothelial dysfunction can be achieved via a corneal transplantation. However, global shortage of donor tissues has driven the development cell-based therapeutics. With the capacity to propagate regulatory compliant human corneal endothelial cells (CEnCs), this study evaluated the functionality of propagated CEnCs delivered via tissue-engineered endothelial keratoplasty (TE-EK) or corneal endothelial cell injection (CE-CI) within a rabbit model of bullous keratopathy. For animals with TE-EK grafts, central corneal thickness (CCT) increased to >1000 μm post-operatively. Gradual thinning with improvements in corneal clarity was observed from week 1. CCT at week 3 was 484.3 ± 73.7 μm. In rabbits with CE-CI, corneal clarity was maintained throughout, and CCT at week 3 was 582.5 ± 171.5 μm. Control corneas remained significantly edematous throughout the study period compared to their respective experimental groups (p

The bovine lactoferricin L6 (RRWQWR) has been previously identified as a novel cell-penetrating peptide (CPP) that is able to efficiently internalize into human cells. L6 interacts with quantum dots (QDs) noncovalently to generate stable L6/QD complexes that enter cells by endocytosis. In this study, we demonstrate a modified L6 (HL6; CHHHHHRRWQWRHHHHHC), in which short polyhistidine peptides are introduced into both flanks of L6, has enhanced cell-penetrating ability in human bronchoalveolar carcinoma A549 cells. The mechanism of cellular uptake of HL6/QD complexes is primarily direct membrane translocation rather than endocytosis. Dimethyl sulfoxide (DMSO), but not pyrenebutyrate (PB), ethanol, oleic acid, or 1,2-benzisothiazol-3(2 H)-one (BIT), slightly enhances HL6-mediated protein transduction efficiency. Neither HL6 nor HL6/QD complexes are cytotoxic to A549 or HeLa cells. These results indicate that HL6 could be a more efficient drug carrier than L6 for biomedical as well as biotechnological applications, and that the function of polyhistidine peptides is critical to CPP-mediated protein transduction.

Current approaches for precision deposition of cells are not optimized for moist environments or for substrates with complex surface topographic features, for example, the surface of dermal matrices and other biomaterials. To overcome these challenges, an approach is presented that utilizes cell confinement in phase-separating polymer solutions of polyethylene glycol and dextran to precisely deliver keratinocytes in well-defined colonies. Using this approach, keratinocyte colonies are produced with superior viability, proliferative capacity, and barrier formation compared with the same number of cells dispersedly seeded across substrate surfaces. It is further demonstrated that keratinocytes delivered in colonies to the surface of acellular dermal matrices form an intact epidermal basal layer more rapidly and more completely than cells delivered by conventional dispersed seeding. These findings demonstrate that delivery of keratinocytes in phase-separating polymer solutions holds potential for enhancing growth of keratinocytes in culture and production of functional skin equivalents.

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Background Immature neurons can extend processes after transplantation in adult animals. Neuronal relays can form between injected neural stem cells (NSCs) and surviving neurons, possibly improving recovery after spinal cord injury (SCI). Cell delivery methods of single or multiple bolus injections of concentrated cell suspensions thus far tested in preclinical and clinical experiments are suboptimal for new tract formation. Nonuniform injectate dispersal is often seen due to gravitational cell settling and clumping. Multiple injections have additive risks of hemorrhage, parenchymal damage, and cellular reflux and require additional surgical exposure. The deposition of multiply delivered cells boluses may be uneven and discontinuous. Objective To develop an injection apparatus and methodology to deliver continuous cellular trails bridging spinal cord lesions. Methods We improved the uniformity of cellular trails by formulating NSCs in hyaluronic acid. The TrailmakerTM stereotaxic injection device was automatized to extend a shape memory needle from a single-entry point in the spinal cord longitudinal axis to "pioneer" a new trail space and then retract while depositing an hyaluronic acid-NSC suspension. We conducted testing in a collagen spinal models, and animal testing using human NSCs (hNSCs) in rats and minipigs. Results Continuous surviving trails of hNSCs within rat and minipig naive spinal cords were 12 and 40 mm in length. hNSC trails were delivered across semi-acute contusion injuries in rats. Transplanted hNSCs survived and were able to differentiate into neural lineage cells and astrocytes. Conclusion The TrailmakerTM creates longitudinal cellular trails spanning multiple levels from a single-entry point. This may enhance the ability of therapeutics to promote functional relays after SCI.

Cell separation and delivery have recently gained significant attention in biological and biochemical studies. In thiswork, an optical method for separation and controllable delivery of cells by using an abruptly tapered fiber probe is reported. By launching a laser beam at the wavelength of 980 nm into the fiber, a mixture of cells with sizes of ~5 and ~3 μm and poly(methyl methacrylate) particles with size of 5 μm are separated into three chains along the direction of propagation of light. The cell and particle chains are delivered in three dimensions over 600 μm distance. Experimental results are interpreted by numerical simulations. Optical forces and forward migration velocities of different particles and cells are calculated and discussed.

Microsphere technology serves as an efficient and effective platform for cell applications (in vitro cell culture and in vivo cell delivery) due to its mimicry of the 3D native environment, high surface area:volume ratio, and ability to isolate the entrapped cells from the environment. Properties of cell-laden microspheres are determined by the type of application and the cell. While high cell densities are preferable for large-scale therapeutic biomolecule production in vitro, an immunoprotective barrier is most important for allogeneic pancreatic islet transplantation into patients. Furthermore, the biological cells require a suitable microenvironment in terms of its physical and biochemical properties. Here, we discuss applications of cell-laden microspheres and their corresponding design parameters.

The effectiveness of stem-cell therapies has been hampered by cell death and limited control over fate1. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype2–4. Stem cell behavior can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials5–7, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem-cell behaviors in situ.

Cell therapy is one of the most promising areas within regenerative medicine. However, its full potential is limited by the rapid loss of introduced therapeutic cells before their full effects can be exploited, due in part to anoikis, and in part to the adverse environments often found within the pathologic tissues that the cells have been grafted into. Encapsulation of individual cells has been proposed as a means of increasing cell viability. In this study, we developed a facile, high throughput method for creating temperature responsive microcapsules comprising agarose, gelatin and fibrinogen for delivery and subsequent controlled release of cells. We verified the hypothesis that composite capsules combining agarose and gelatin, which possess different phase transition temperatures from solid to liquid, facilitated the destabilization of the capsules for cell release. Cell encapsulation and controlled release was demonstrated using human fibroblasts as model cells, as well as a therapeutically relevant cell line—human umbilical vein endothelial cells (HUVECs). While such temperature responsive cell microcapsules promise effective, controlled release of potential therapeutic cells at physiological temperatures, further work will be needed to augment the composition of the microcapsules and optimize the numbers of cells per capsule prior to clinical evaluation.

Cell encapsulation and cell delivery have been proposed as an alternative approach to treat various diseases since they allow localized and controlled delivery of therapeutic cells to specific physiological sites to restore the lost functions, which can overcome many current limitations in the present therapeutic technologies. In particular, great interests have been attracted in utilizing stem cell as the cell source for cell encapsulation and therapy in the recent years. This chapter provides an overview of cell encapsulation technology based on hydrogel biomaterials, with a focus on stem cell encapsulation and some recent developments of such strategy for its use in treatment of diabetes. It is well established that stem cell encapsulation can be a promising approach for therapy of diabetes, especially in cases where limited cells are available for differentiation and expansion. Several potential stem cell candidates and their differentiation protocols have been examined and developed. The authors believe that stem cell encapsulation may see exciting improvement in the next few decades. However, a few challenges have to be addressed such as safety and efficacy of this approach for treatment of diabetes and scale-up of manufacturing process under Good Manufacturing Practice conditions before the technology can enter human clinical trials and become a real clinical therapeutic strategy.

Background Translation of cell therapies to the clinic is accompanied by numerous challenges, including controlled and targeted delivery of the cells to their site of action, without compromising cell viability and functionality. Objectives To explore the use of hollow microneedle devices (to date only used for the delivery of drugs and vaccines into the skin and for the extraction of biological fluids) to deliver cells into skin in a minimally-invasive, user-friendly and targeted fashion. Methods Melanocyte, keratinocyte and mixed epidermal cell suspensions were passed through various types of microneedles and subsequently delivered into the skin. Results Cell viability and functionality is maintained after injection through hollow microneedles with a bore size ≥75 μm. Healthy cells are delivered into skin at clinically relevant depths. Conclusions Hollow microneedles provide an innovative and minimally-invasive method for delivering functional cells into the skin. Microneedle cell delivery represents a potential new treatment option for cell therapy approaches including skin re-pigmentation, wound repair, scar and burn remodelling, immune therapies, and cancer vaccines.

Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted exosomes. Here, we show the first demonstration of a simple and rapid production method of CDNs using spin cups via a cell shearing approach, which offers clear advantages in terms of yield and cost-effectiveness over both traditional exosomes isolation, and also existing CDNs fabrication techniques. The CDNs obtained were of a higher protein yield and showed similarities in terms of physical characterization, protein and lipid analysis to both exosomes and CDNs previously reported in the literature. In addition, we investigated the mechanisms of cellular uptake of CDNs in vitro and their biodistribution in an in vivo mouse tumour model. Colocalization of the CDNs at the tumour site in a cancer mouse model was demonstrated, highlighting the potential for CDNs as anti-cancer strategy. Taken together, the results suggest that CDNs could provide a cost-effective alternative to exosomes as an ideal drug nanocarrier.

In more than one decade, cell transplantation has created an important strategy to treat a wide variety of diseases characterized by tissue and cell dysfunctions. In this course of action, cell delivery to target site has been always one of the most important constraints and complications, as only a small proportion of the cells are housed in the target sites. Nanotechnology and nanoscale biomaterials have been helpful for cell transplantation in various fields of regenerative medicine including diagnosis, delivery systems for the cell, drug or gene, and cells protection system. In this study, the basic concepts and recently studied aspects of cell delivery systems based on nanoscale biomaterials for transplantation and clinical applications are highlighted. Nanomaterials may be used in combination with cell therapy to control the release of drugs or special factors of engineered cells after transplantation.

Spinal cord injury (SCI) is a complex pathology that evolves after primary acute mechanical injury, causing further damage to the spinal cord tissue that exacerbates clinical outcomes. Based on encouraging results from preclinical experiments, some cell treatments being translated into clinical practice demonstrate promising and effective improvement in sensory/motor function. Combinatorial treatments of cell and drug/biological factors have been demonstrated to be more effective than cell treatments alone. Recent advances have led to the development of biomaterials aiming to promote in situ cell delivery for SCI, together with combinatorial strategies using drugs/biomolecules to achieve a maximized multitarget approach. This review provides an overview of single and combinatorial regenerative cell treatments as well as potential delivery options to treat SCI.

Traumatic, ischemic and neurodegenerative central nervous system (CNS) diseases and injuries result in pathological processes that cause irreversible destruction of neural tissue. Currently, there are limited surgical or pharmacological interventions in clinical use that can restore meaningful function in these disorders. Transplantation of neural cells and neural tissue engineering represent potential therapeutic strategies to repopulate CNS lesions with new viable neural tissue that may be capable of recapitulating this lost function. However, despite their considerable promise, CNS cell therapies still face a number of persistent challenges that hamper their performance and diminish their clinical translation potential. To improve prospects for clinical CNS cell transplantation, the development of innovative bioengineered smart materials will be required that are capable of facilitating enhanced local survival of transplanted cells, preventing undesired cell migration away from diseased tissue, directing differentiation outcomes in a temporally controlled manner, improving host–graft integration and mitigating immune rejection. Within this chapter, we outline the interwoven history of the development of biomaterials and cell therapies for various CNS injuries and diseases as well as how biomaterials are being applied to address CNS cell transplantation challenges. Furthermore, we highlight emerging smart biomaterial strategies that may resolve the persistent challenges facing CNS cell transplantation.

Cell replacement therapy is emerging as a promising treatment platform for many endocrine disorders and hormone deficiency diseases. The survival of cells within delivery devices is, however, often limited due to low oxygen levels in common transplantation sites. Additionally, replacing implanted devices at the end of the graft lifetime is often infeasible and, if possible, generally requires invasive surgical procedures. This report presents the design and testing of a modular transcutaneous biphasic cell delivery device which provides enhanced and unlimited oxygen supply by direct contact with the atmosphere. Critically, the cell delivery unit was demountable from the fixed components of the device, allowing for surgery-free refilling of the therapeutic cells. Mass transfer studies showed significantly improved performance of the biphasic device in comparison to subcutaneous controls. The device was also tested for islet encapsulation in an immunocompetent diabetes rodent model. Robust cell survival and diabetes correction was observed following a rat-to-mouse xenograft. Lastly, non-surgical cell refilling was demonstrated in dogs. These studies show the feasibility of this novel device for cell replacement therapies

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell-based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron-scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell-loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod-shaped mini-tissues that further assemble into organized macroscopic and macroporous structures post-injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.