Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion.

Mesenchymal stromal cells (MSCs) are a candidate for cell immunotherapy due to potent immunomodulatory activity found in their secretome. Though studies on their secreted substances have been reported, the time dynamics of MSC potency remain unclear. Herein, we report on the dynamics of MSC secretome potency in an ex vivo hollow fiber bioreactor using a continuous perfusion cell culture system that fractionated MSC-secreted factors over time. Time-resolved fractions of MSC-conditioned media were evaluated for potency by incubation with activated immune cells. Three studies were designed to characterize MSC potency under: (1) basal conditions, (2) in situ activation, and (3) pre-licensing. Results indicate that the MSC secretome is most potent in suppressing lymphocyte proliferation during the first 24 h and is further stabilized when MSCs are prelicensed with a cocktail of pro-inflammatory cytokines, IFNγ, TNFα, and IL-1ß. The evaluation of temporal cell potency using this integrated bioreactor system can be useful in informing strategies to maximize MSC potency, minimize side effects, and allow greater control for the duration of ex vivo administration approaches.

STAT4 increases the phenotypic and functional heterogeneity of intestinal tissue-resident memory T cells.

Tissue-resident memory T cells (Trms) are an important subset of lymphocytes that are lodged within non-lymphoid tissues and carry out diverse functions to control local pathogen replication. CD103 has been used to broadly define subsets of Trms within the intestine, with CD103+ and CD103- subsets having unique transcriptional profiles and effector functions. Here we identify signal transducer and activator of transcription 4 (STAT4) as an important regulator of CD103- Trm differentiation. STAT4-deficient cells trafficked to the intestine and localized to areas of infection but displayed impaired Trm differentiation with fewer CD103- Trms. Single-cell RNA-sequencing demonstrated that STAT4-deficiency led to a reduction in CD103- Trm subsets and expansion of a single population of CD103+ cells. Alterations in Trm populations were due, in part, to STAT4-mediated inhibition of transforming growth factor (TGF)-ß-driven expression of Trm signature genes. STAT4-dependent Trm populations expressed genes associated with cytokine production and cell migration, and STAT4-deficient Trm cells had altered localization within the tissue and reduced effector function after reactivation in vivo. Overall, our data indicate that STAT4 leads to increased differentiation of CD103- Trms, in part by modulating the expression of TGF-ß-regulated genes, and results in increased Trm heterogeneity and function within the intestinal tissue.

3D Microcapsules for Human Bone Marrow Derived Mesenchymal Stem Cell Biomanufacturing in a Vertical-Wheel Bioreactor.

Microencapsulation of human mesenchymal stromal cells (MSCs) via electrospraying has been well documented in tissue engineering and regenerative medicine. Herein, we report the use of microencapsulation, via electrospraying, for MSC expansion using a commercially available hydrogel that is durable, optimized to MSC culture, and enzymatically degradable for cell recovery. Critical parameters of the electrospraying encapsulation process such as seeding density, correlation of microcapsule output with hydrogel volume, and applied voltage were characterized to consistently fabricate cell-laden microcapsules of uniform size. Upon encapsulation, we then verified ~ 10x expansion of encapsulated MSCs within a vertical-wheel bioreactor and the preservation of critical quality attributes such as immunophenotype and multipotency after expansion and cell recovery. Finally, we highlight the genetic manipulation of encapsulated MSCs as an example of incorporating bioactive agents in the capsule material to create new compositions of MSCs with altered phenotypes.

CD103 fate mapping reveals that intestinal CD103- tissue-resident memory T cells are the primary responders to secondary infection.

Tissue-resident memory T (TRM) cells remain poised in the tissue and mediate robust protection from secondary infection. TRM cells within the intestine and other tissues are heterogeneous in their phenotype and function; however, the contributions of these TRM subsets to secondary infection remain poorly defined. To address the plasticity of intestinal TRM subsets and their role in local and systemic immunity, we generated mice to fate map intestinal CD103+ TRM cells and track their location and function during secondary infection with Yersinia pseudotuberculosis. We found that CD103+ TRM cells remained lodged in the tissue and were poorly reactivated during secondary challenge. CD103- TRM cells were the primary responders to secondary infection and expanded within the tissue, with limited contribution from circulating memory T cells. The transcriptional profile of CD103- TRM cells demonstrated maintenance of a gene signature similar to circulating T cells along with increased cytokine production and migratory potential. CD103- TRM cells also expressed genes associated with T cell receptor (TCR) activation and displayed enhanced TCR-mediated reactivation both in vitro and in vivo compared with their CD103+ counterparts. These studies reveal the limited recall potential of CD103+ TRM subsets and the role of CD103- TRM cells as central memory-like T cells within peripheral tissues.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Selecting a Cell Engineering Methodology During Cell Therapy Product Development.

When considering the development pathway for a genetically modified cell therapy product, it is critically important that the product is engineered consistent with its intended human use. For scientists looking to develop and commercialize a new technology, the decision to select a genetic modification method depends on several practical considerations. Whichever path is chosen, the developer must understand the key risks and potential mitigations of the cell engineering approach. The developer should also understand the clinical implications: permanent/memory establishment versus transient expression, and clinical manufacturing considerations when dealing with transplantation of genetically engineered cells. This review covers important topics for mapping out a strategy for developers of new cell-based therapeutics. Biological, technological, manufacturing, and clinical considerations are all presented to map out development lanes for the initiation and risk management of new gene-based cell therapeutic products for human use.

A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion.

Cells exchange substances with their surroundings during metabolism, signaling, and other functions. These fluxes are dynamic, changing in response to external cues and internal programs. Static cultures are inadequate for measuring these dynamics because the environments of the cells change as substances accumulate or deplete from medium, unintentionally affecting cell behavior. Static cultures offer limited time resolution due to the impracticality of frequent or prolonged manual sampling, and cannot expose cells to smooth, transient changes in stimulus concentrations. In contrast, perfusion cultures constantly maintain cellular environments and continuously sample the effluent stream. Existing perfusion culture systems are either microfluidic, which are difficult to make and use, or macrofluidic devices built from custom parts that neglect solute dispersion. In this study, a multiplexed macrofluidic perfusion culture platform was developed to measure secretion and absorption rates of substances by cells in a temporally controlled environment. The modular platform handles up to 31 streams with automated fraction collection. This paper presents the assembly of this dynamic bioreactor from commercially available parts, and a method for quantitatively handling the effects of dispersion using residence time distributions. The system is then applied to monitor the secretion of a circadian clock gene-driven reporter from engineered cells.

Non-invasive image-based cytometry for high throughput NK cell cytolysis analysis.

Natural Killer (NK) cells are lymphocytes that are the first line of defense against malignantly transformed cells, virally infected cells and other stressed cell types. To study the cytolytic function of NK cells in vitro, a cytotoxicity assay is normally conducted against a target cancerous cell line. Current assay methods are typically performed in mixed 2D cocultures with destructive endpoints and low throughput, thereby limiting the scale, time-resolution, and relevance of the assay to in vivo conditions. Here, we evaluated a novel, non-invasive, quantitative image-based cytometry (qIBC) assay for detection of NK-mediated killing of target cells in 2D and 3D environments in vitro and compared its performance to two common flow cytometry- and fluorescence-based cytotoxicity assays. Similar to the other methods evaluated, the qIBC assay allowed for reproducible detection of target cell killing across a range of effector-to-target ratios with reduced variability. The qIBC assay also allowed for detection of NK cytolysis in 3D spheroids, which enabled scalable measurements of cell cytotoxicity in 3D models. Our findings suggest that quantitative image-based cytometry would be suitable for rapid, high-throughput screening of NK cytolysis in vitro, including in quasi-3D structures that model tissue environments in vivo.

Coencapsulation of ISCs and MSCs Enhances Viability and Function of both Cell Types for Improved Wound Healing.

INTRODUCTION: We previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs. MATERIALS AND METHODS: MSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-ß1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis. RESULTS: ISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar. CONCLUSIONS: Combining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.

Real-time transfer of lentiviral particles by producer cells using an engineered coculture system.

Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6 days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.