Strategies to Overcome the Barrier of Ischemic Microenvironment in Cell Therapy of Cardiovascular Disease.

The transplantation of various immune cell types are promising approaches for the treatment of ischemic cardiovascular disease including myocardial infarction (MI) and peripheral arterial disease (PAD). Major limitation of these so-called Advanced Therapy Medicinal Products (ATMPs) is the ischemic microenvironment affecting cell homeostasis and limiting the demanded effect of the transplanted cell products. Accordingly, different clinical and experimental strategies have been evolved to overcome these obstacles. Here, we give a short review of the different experimental and clinical strategies to solve these issues due to ischemic cardiovascular disease.

Human adipose-derived mesenchymal stem cell-based medical microrobot system for knee cartilage regeneration in vivo.

Targeted cell delivery by a magnetically actuated microrobot with a porous structure is a promising technique to enhance the low targeting efficiency of mesenchymal stem cell (MSC) in tissue regeneration. However, the relevant research performed to date is only in its proof-of-concept stage. To use the microrobot in a clinical stage, biocompatibility and biodegradation materials should be considered in the microrobot, and its efficacy needs to be verified using an in vivo model. In this study, we propose a human adipose-derived MSC-based medical microrobot system for knee cartilage regeneration and present an in vivo trial to verify the efficacy of the microrobot using the cartilage defect model. The microrobot system consists of a microrobot body capable of supporting MSCs, an electromagnetic actuation system for three-dimensional targeting of the microrobot, and a magnet for fixation of the microrobot to the damaged cartilage. Each component was designed and fabricated considering the accessibility of the patient and medical staff, as well as clinical safety. The efficacy of the microrobot system was then assessed in the cartilage defect model of rabbit knee with the aim to obtain clinical trial approval.

An Atmosphere-Breathing Refillable Biphasic Device for Cell Replacement Therapy.

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 unfeasible and, where possible, generally requires invasive surgical procedures. Here, the design and testing of a modular transcutaneous biphasic (BP) cell delivery device that provides enhanced and unlimited oxygen supply by direct contact with the atmosphere is presented. Critically, the cell delivery unit is demountable from the fixed components of the device, allowing for surgery-free refilling of the therapeutic cells. Mass transfer studies show significantly improved performance of the BP device in comparison to subcutaneous controls. The device is also tested for islet encapsulation in an immunocompetent diabetes rodent model. Robust cell survival and diabetes correction is observed following a rat-to-mouse xenograft. Lastly, nonsurgical cell refilling is demonstrated in dogs. These studies show the feasibility of this novel device for cell replacement therapies.

Smart polymers for cell therapy and precision medicine.

Soft materials have been developed very rapidly in the biomedical field over the past 10 years because of advances in medical devices, cell therapy, and 3D printing for precision medicine. Smart polymers are one category of soft materials that respond to environmental changes. One typical example is the thermally-responsive polymers, which are widely used as cell carriers and in 3D printing. Self-healing polymers are one type of smart polymers that have the capacity to recover the structure after repeated damages and are often injectable through needles. Shape memory polymers are another type with the ability to memorize their original shape. These smart polymers can be used as cell/drug/protein carriers. Their injectability and shape memory performance allow them to be applied in bioprinting, minimally invasive surgery, and precision medicine. This review will describe the general materials design, characterization, as well as the current progresses and challenges of these smart polymers.

A bioresorbable biomaterial carrier and passive stabilization device to improve heart function post-myocardial infarction.

The limited regenerative capacity of the heart after a myocardial infarct results in remodeling processes that can progress to congestive heart failure (CHF). Several strategies including mechanical stabilization of the weakened myocardium and regenerative approaches (specifically stem cell technologies) have evolved which aim to prevent CHF. However, their final performance remains limited motivating the need for an advanced strategy with enhanced efficacy and reduced deleterious effects. An epicardial carrier device enabling a targeted application of a biomaterial-based therapy to the infarcted ventricle wall could potentially overcome the therapy and application related issues. Such a device could play a synergistic role in heart regeneration, including the provision of mechanical support to the remodeling heart wall, as well as providing a suitable environment for in situ stem cell delivery potentially promoting heart regeneration. In this study, we have developed a novel, single-stage concept to support the weakened myocardial region post-MI by applying an elastic, biodegradable patch (SPREADS) via a minimal-invasive, closed chest intervention to the epicardial heart surface. We show a significant increase in %LVEF 14 days post-treatment when GS (clinical gold standard treatment) was compared to GS + SPREADS + Gel with and without cells (p ≤ 0.001). Furthermore, we did not find a significant difference in infarct quality or blood vessel density between any of the groups which suggests that neither infarct quality nor vascularization is the mechanism of action of SPREADS. The SPREADS device could potentially be used to deliver a range of new or previously developed biomaterial hydrogels, a remarkable potential to overcome the translational hurdles associated with hydrogel delivery to the heart.

Bringing Stem Cell-Based Therapies for Type 1 Diabetes to the Clinic: Early Insights from Bioprocess Economics and Cost-Effectiveness Analysis.

Differentiation of pluripotent stem cells (PSCs) into ß cells could provide insulin independence for type 1 diabetes (T1D) patients. This approach would reduce the clinical complications that most patients managed on intensive insulin therapy (IIT) face. However, bottlenecks of PSC manufacturing and limited engraftment of encapsulated cells hinder the long-term effectiveness of these therapies. A bioprocess decision-support tool is combined with a disease state-transition model to evaluate the cost-effectiveness of the stem cell-based therapy against IIT. Clinical effectiveness is assessed in quality-adjusted life years (QALYs). Manufacturing costs per patient reduce from $430 000 to $160 000 with optimization of batch size and annual demand. For 96% of the patients, cell therapy improves the quality of life compared to IIT. Cost savings are achieved for 2% of the population through prevention of renal disease. The therapy is cost-effective for 3.4% of patients when a willingness to pay (WTP) of up to $150 000 per QALY is considered. A 75% cost reduction in the cell therapy price increases cost-effectiveness likelihood to 51% at $100 000 per QALY. This study highlights the need for scalable manufacturing platforms for stem cell therapies, as well as to prioritizing access to the therapy to patients with an increased likelihood of costly complications.

Modeling biological and economic uncertainty on cell therapy manufacturing: the choice of culture media supplementation.

AIM: To evaluate the cost-effectiveness of autologous cell therapy manufacturing in xeno-free conditions. MATERIALS & METHODS: Published data on the isolation and expansion of mesenchymal stem/stromal cells introduced donor, multipassage and culture media variability on cell yields and process times on adherent culture flasks to drive cost simulation of a scale-out campaign of 1000 doses of 75 million cells each in a 400 square meter Good Manufacturing Practices facility. RESULTS & CONCLUSION: Passage numbers in the expansion step are strongly associated with isolation cell yield and drive cost increases per donor of $1970 and 2802 for fetal bovine serum and human platelet lysate. Human platelet lysate decreases passage numbers and process costs in 94.5 and 97% of donors through lower facility and labor costs. Cost savings are maintained with full equipment depreciation and higher numbers of cells per dose, highlighting the number of cells per passage step as the key cost driver.

An immune cell spray (ICS) formulation allows for the delivery of functional monocyte/macrophages.

Macrophages are key cells of the innate immune system and act as tissue resident macrophages (TRMs) in the homeostasis of various tissues. Given their unique functions and therapeutic use as well as the feasibility to derive macrophages in vitro from hematopoietic stem cell (HSC) sources, we propose an "easy-to-use" immune cell spray (ICS) formulation to effectively deliver HSC-derived macrophages. To achieve this aim, we used classical pump spray devices to spray either the human myeloid cell line U937 or primary murine HSC-derived macrophages. For both cell types used, one puff could deliver cells with maintained morphology and functionality. Of note, cells tolerated the spraying process very well with a recovery of more than 90%. In addition, we used osmotic preconditioning to reduce the overall cell size of macrophages. While a 800 mosm hyperosmolar sucrose solution was able to reduce the cell size by 27%, we identified 600 mosm to be effective to reduce the cell size by 15% while maintaining macrophage morphology and functionality. Using an isolated perfused rat lung preparation, the combinatorial use of the ICS with preconditioned and genetically labeled U937 cells allowed the intra-pulmonary delivery of cells, thus paving the way for a new cell delivery platform.

Versatile graphene biosensors for enhancing human cell therapy.

Technological advances in engineering and cell biology stimulate novel approaches for medical treatment, in particular cell-based therapy. The first cell-based gene therapy against cancer was recently approved by the US Food and Drug Administration. Progress in cancer diagnosis includes a blood test detecting five cancer types. Numerous stem cell phase I/II clinical trials showing safety and efficacy will soon pursue qualifying criteria for advanced therapy medicinal products (ATMP), aspiring to join the first stem-cell therapy approved by the European Medicines Agency. Cell based therapy requires extensive preclinical characterisation of biomarkers indicating mechanisms of action crucial to the desired therapeutic effect. Quantitative analyses monitoring critical functions for the manufacture of optimal cell and tissue-based clinical products include successful potency assays for implementation. The challenge to achieve high quality measurement is increasingly met by progress in biosensor design. We adopt a cell therapy perspective to highlight recent examples of graphene-enhanced biointerfaces for measurement of biomarkers relevant to cancer treatment, diagnosis and tissue regeneration. Graphene based biosensor design problems can thwart their use for health care transformative point of care testing and real-time applications. We discuss concerns to be addressed and emerging solutions for establishing clinical grade biosensors to accelerate human cell therapy.

An oxygen plasma treated poly(dimethylsiloxane) bioscaffold coated with polydopamine for stem cell therapy.

In this study, 3D macroporous bioscaffolds were developed from poly(dimethylsiloxane) (PDMS) which is inert, biocompatible, non-biodegradable, retrievable and easily manufactured at low cost. PDMS bioscaffolds were synthesized using a solvent casting and particulate leaching (SCPL) technique and exhibited a macroporous interconnected architecture with 86 ± 3% porosity and 300 ± 100 µm pore size. As PDMS intrinsically has a hydrophobic surface, mainly due to the existence of methyl groups, its surface was modified by oxygen plasma treatment which, in turn, enabled us to apply a novel polydopamine coating onto the surface of the bioscaffold. The addition of a polydopamine coating to bioscaffolds was confirmed using composition analysis. Characterization of oxygen plasma treated-PDMS bioscaffolds coated with polydopamine (polydopamine coated-PDMS bioscaffolds) showed the presence of hydroxyl and secondary amines on their surface which resulted in a significant decrease in water contact angle when compared to uncoated-PDMS bioscaffolds (35 ± 3%, P < 0.05). Seeding adipose tissue-derived mesenchymal stem cells (AD-MSCs) into polydopamine coated-PDMS bioscaffolds resulted in cells demonstrating a 70 ± 6% increase in viability and 40 ± 5% increase in proliferation when compared to AD-MSCs seeded into uncoated-PDMS bioscaffolds (P < 0.05). In summary, this two-step method of oxygen plasma treatment followed by polydopamine coating improves the biocompatibility of PDMS bioscaffolds and only requires the use of simple reagents and mild reaction conditions. Hence, our novel polydopamine coated-PDMS bioscaffolds can represent an efficient and low-cost bioscaffold platform to support MSC therapies.

Survival of Mesenchymal Stem Cells in Different Methods of Nebulization.

We compared survival of bone marrow mesenchymal stem cells after compressor, ultrasound, and mesh nebulization of the cell suspension over 10 min. Viability of stromal cells was best preserved after compressor nebulization (72%). Cell survival after ultrasonic nebulization was significantly lower (20%). After mesh nebulization, no live cells were found. Thus, compressor nebulization is the most preferable method of the production of cell aerosol for their delivery to the lower respiratory tract.

Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges.

Human mesenchymal stem cell (hMSC)-based therapies are of increasing interest in the field of regenerative medicine. As economic considerations have shown, allogeneic therapy seems to be the most cost-effective method. Standardized procedures based on instrumented single-use bioreactors have been shown to provide billion of cells with consistent product quality and to be superior to traditional expansions in planar cultivation systems. Furthermore, under consideration of the complex nature and requirements of allogeneic hMSC-therapeutics, a new equipment for downstream processing (DSP) was successfully evaluated. This mini-review summarizes both the current state of the hMSC production process and the challenges which have to be taken into account when efficiently producing hMSCs for the clinical scale. Special emphasis is placed on the upstream processing (USP) and DSP operations which cover expansion, harvesting, detachment, separation, washing and concentration steps, and the regulatory demands.

TGF-ß1/CD105 signaling controls vascular network formation within growth factor sequestering hyaluronic acid hydrogels.

Cell-based strategies for the treatment of ischemic diseases are at the forefront of tissue engineering and regenerative medicine. Cell therapies purportedly can play a key role in the neovascularization of ischemic tissue; however, low survival and poor cell engraftment with the host vasculature following implantation limits their potential to treat ischemic diseases. To overcome these limitations, we previously developed a growth factor sequestering hyaluronic acid (HyA)-based hydrogel that enhanced transplanted mouse cardiosphere-derived cell survival and formation of vasculature that anastomosed with host vessels. In this work, we examined the mechanism by which HyA hydrogels presenting transforming growth factor beta-1 (TGF-ß1) promoted proliferation of more clinically relevant human cardiosphere-derived cells (hCDC), and their formation of vascular-like networks in vitro. We observed hCDC proliferation and enhanced formation of vascular-like networks occurred in the presence of TGF-ß1. Furthermore, production of nitric oxide (NO), VEGF, and a host of angiogenic factors were increased in the presence of TGF-ß1. This response was dependent on the co-activity of CD105 (Endoglin) with the TGF-ßR2 receptor, demonstrating its role in the process of angiogenic differentiation and vascular organization of hCDC. These results demonstrated that hCDC form vascular-like networks in vitro, and that the induction of vascular networks by hCDC within growth factor sequestering HyA hydrogels was mediated by TGF-ß1/CD105 signaling.

Minimally invasive and targeted therapeutic cell delivery to the skin using microneedle devices.

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 are maintained after injection through hollow microneedles with a bore size ≥ 75 µm. Healthy cells are delivered into the 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 repigmentation, wound repair, scar and burn remodelling, immune therapies and cancer vaccines.

Improvement of Local Cell Delivery Using Helix Transendocardial Delivery Catheter in a Porcine Heart.

Cardiac regeneration strategies using stem cells have shown variable and inconsistent results with respect to patient cardiac function and clinical outcomes. There has been increasing consensus that improving the efficiency of delivery may improve results. The Helix transendocardial delivery system (BioCardia Inc.) has been developed to enable percutaneous transendocardial biotherapeutic delivery. Therefore, we evaluated cell retention using this unique system compared with direct transepicardial injection and intracoronary infusion in an animal model.Twelve healthy swine were used in this study. 18Fluorodeoxyglucose (FDG)-labeled bone marrow mononuclear cells were delivered via percutaneous transendocardial route using the Helix system (TE group, n = 5), via direct transepicardial injection using a straight 27-gauge needle in an open chest procedure (TP group, n = 4), or via percutaneous intracoronary (IC) infusion (IC group, n = 3). One hour after cell delivery, the distribution of injected cells within the myocardium was assessed by PET-CT. Regions of interest were defined and their signals were compared in each group. Retention rates were calculated as a percentage of the comparing signal.The distribution of injected cells in the myocardium was higher in the TE group (17.9%) than in the TP group (6.0%, versus TE, P < 0.001) and the IC group (1.0%, versus TE, P < 0.001). Consistent with previous reports, there were signal distributions in the lungs, liver, and kidneys in qualitative whole body PET assessment.TE cell delivery using a helical infusion catheter is more efficient in cell retention than either TP delivery or IC delivery using PET-CT analysis.

A novel filtration system for point of care washing of cellular therapy products.

The cell therapy industry would greatly benefit from a simple point of care solution to remove dimethylsulphoxide (DMSO) from small-volume thawed cell suspensions before injection. A novel dead-end filtration device has been designed and validated, which takes advantage of the higher density of thawed cell suspensions to remove the DMSO and protein impurities from the cell suspension without fouling the filter membrane. The filter was designed to avoid fluid circuits and minimize the surface area that is contacted by the cell suspension, thus reducing cell losses by design. The filtration process was established through optimization of the fluid flow configuration, backflush cycles and filter geometry. Overall, this novel filtration device allows for a 1 ml of thawed cryopreserved cell suspensions, containing 107 cells of a fetal lung fibroblast cell line (MRC-5), to be washed in less than 30 min. More than 95% of the DMSO and up to 94% of the albumin-fluorescein-isothiocyanate content can be removed while the viable cell recovery is higher than 80%. It is also demonstrated that this system can be used for bone marrow-derived human mesenchymal stem cells with more than 73% cell recovery and 85% DMSO reduction. This is the first time that a dead end (normal) filtration process has been used to successfully wash high-density human cell suspensions. In practice, this novel solid-liquid separation technology fills the need for small-volume washing in closed processing systems for cellular therapies. Copyright © 2016 John Wiley & Sons, Ltd.

Inexpensive Attachment Device for Cell Therapy Administration into Injured Spinal Cord.

BACKGROUND: Cell therapy is configured as a promising strategy for the treatment of spinal cord injury (SCI), but it requires reliable systems to achieve microinjections with different rates and volumes, according to the different characteristics of the injured spinal cord tissue and the targets previously selected. OBJECTIVE: We sought to describe an original and inexpensive device for support of microinjection systems in the course of spinal cord surgery. METHODS: Our attachment device consists of an arch and a system of bars that can be fixed to the operating table and on which a microinjection pump can be displaced and fixed in the course of surgery. RESULTS: This device has been used for therapy administration into injured spinal cords. It is easy to use and permits reproducible results. CONCLUSION: We have described an original attachment device for the support of a microinjection pump. It is applicable to spinal cord surgery and should be considered as a cheap solution for intralesional administration of cell therapy after spinal cord injury.

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products.

The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is needed. This forum concluded that the continued collective actions are expected to promote international convergence of regulatory approaches of the products. The Pharmaceuticals and Medical Devices Agency and Japanese Society for Regenerative Medicine jointly convened the forum with support from the National Institutes of Biomedical Innovation, Health and Nutrition. Participants at the forum include 300 experts in and outside of Japan.

Fabrication of silk mesh with enhanced cytocompatibility: preliminary in vitro investigation toward cell-based therapy for hernia repair.

Recent studies have demonstrated that combining cells with meshes prior to implantation successfully enhanced hernia repair. The idea is to create a biologic coating surrounding the mesh with autologous cells, before transplantation into the patient. However, due to the lack of a prompt and robust cell adhesion to the meshes, extensive in vitro cultivation is required to obtain a homogenous cell layer covering the mesh. In this context, the objective of this publication is to manufacture meshes made of silk fibres and to enhance the cytoadhesion and cytocompatibility of the biomaterial by surface immobilization of a pro-adhesive wheat germ agglutinin (lectin WGA). We first investigated the affinity between the glycoprotein WGA and cells, in solution and then after covalent immobilization of WGA on silk films. Then, we manufactured meshes made of silk fibres, tailored them with WGA grafting and finally evaluated the cytocompatibility and the inflammatory response of silk and silk-lectin meshes compared to common polypropylene mesh, using fibroblasts and peripheral blood mononuclear cells, respectively. The in vitro experiments revealed that the cytocompatibility of silk can be enhanced by surface immobilization with lectin WGA without exhibiting negative response in terms of pro-inflammatory reaction. Grafting lectin to silk meshes could bring advantages to facilitate cell-coating of meshes prior to implantation, which is an imperative prerequisite for abdominal wall tissue regeneration using cell-based therapy.

Developing robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) for cell therapies.

Cell encapsulation holds enormous potential to treat a number of hormone deficient diseases and endocrine disorders. We report a simple and universal approach to fabricate robust, hydrogel-based, nanofiber-enabled encapsulation devices (NEEDs) with macroscopic dimensions. In this design, we take advantage of the well-known capillary action that holds wetting liquid in porous media. By impregnating the highly porous electrospun nanofiber membranes of pre-made tubular or planar devices with hydrogel precursor solutions and subsequent crosslinking, we obtained various nanofiber-enabled hydrogel devices. This approach is broadly applicable and does not alter the water content or the intrinsic chemistry of the hydrogels. The devices retained the properties of both the hydrogel (e.g. the biocompatibility) and the nanofibers (e.g. the mechanical robustness). The facile mass transfer was confirmed by encapsulation and culture of different types of cells. Additional compartmentalization of the devices enabled paracrine cell co-cultures in single implantable devices. Lastly, we provided a proof-of-concept study on potential therapeutic applications of the devices by encapsulating and delivering rat pancreatic islets into chemically-induced diabetic mice. The diabetes was corrected for the duration of the experiment (8 weeks) before the implants were retrieved. The retrieved devices showed minimal fibrosis and as expected, live and functional islets were observed within the devices. This study suggests that the design concept of NEEDs may potentially help to overcome some of the challenges in the cell encapsulation field and therefore contribute to the development of cell therapies in future.