In Vivo Activity of Genetically Modified Cells Preseeded in Rat Vascularized Composite Allografts.

OBJECTIVE: Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model. RESULTS: Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P = .02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs-an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes.

Ex vivo perfusion-based engraftment of genetically engineered cell sensors into transplantable organs.

Cellular rejection of liver transplant allografts remains a concern despite immunosuppressant use. Existing transplant biomarkers are often not sensitive enough to detect acute or chronic rejection at an early enough stage to allow successful clinical intervention. We herein developed a cell-based sensor that can potentially be used for monitoring local events following liver transplantation. Utilizing a machine perfusion system as a platform to engraft the cells into a donor liver, we effectively established the biocompatibility of the biosensor cells and confirmed efficient delivery of cells distributed throughout the organ. This work proves an innovative concept of integrating synthetic reporter cells ex vivo into organs as a transplant-within-a-transplant during functional organ preservation with a vision to use cell biosensors as a broad way to monitor and treat tissue transplants.

Effects of intermittent T-cell cluster disaggregation on proliferative capacity and checkpoint marker expression.

Background/aim: T-cell immunotherapies are rapidly gaining grounds in clinical success. Presently, there is first-to-market knowledge on the translation of research scale methods to clinical and commercial scales. Improved understanding can lead to more consistent and efficient production, scaling, and eventual potency. T-cell checkpoint markers, proliferation, and T-cell cluster size and disaggregation are one set of parameters that have yet to be explored. Methods: We herein activated T-cells and assessed various mechanical dissociation frequencies in relation to expression of checkpoint markers (measured by flow cytometry). Results: We herein find increased T-cell proliferation capacity with increased dissociation frequency. We also find that with increased cluster size and duration, lower proliferation, and increased expression of checkpoint markers. Conclusions: These findings reveal new translation findings with respect to T-cell handling and production and suggest that T-cell disaggregation may be important to improved cell yields and phenotype.

Closed loop bioreactor system for the ex vivo expansion of human T cells.

BACKGROUND AIM: Translation of therapeutic cell therapies to clinical-scale products is critical to realizing widespread success. Currently, however, there are limited tools that are accessible at the research level and readily scalable to clinical-scale needs. METHODS: We herein developed and assessed a closed loop bioreactor system in which (i) a highly gas-permeable silicone material was used to fabricate cell culture bags and (ii) dynamic flow was introduced to allow for dissociation of activated T-cell aggregates. RESULTS: Using this system, we find superior T-cell proliferation compared with conventional bag materials and flasks, especially at later time points. Furthermore, intermittent dynamic flow could easily break apart T-cell clusters. CONCLUSIONS: Our novel closed loop bioreactor system is amenable to enhanced T-cell proliferation and has broader implications for being easily scaled for use in larger need settings.

Therapeutic Delivery Specifications Identified Through Compartmental Analysis of a Mesenchymal Stromal Cell-Immune Reaction.

Despite widespread preclinical success, mesenchymal stromal cell (MSC) therapy has not reached consistent pivotal clinical endpoints in primary indications of autoinflammatory diseases. Numerous studies aim to uncover specific mechanisms of action towards better control of therapy using in vitro immunomodulation assays. However, many of these immunomodulation assays are imperfectly designed to accurately recapitulate microenvironment conditions where MSCs act. To increase our understanding of MSC efficacy, we herein conduct a systems level microenvironment approach to define compartmental features that can influence the delivery of MSCs' immunomodulatory effect in vitro in a more quantitative manner than ever before. Using this approach, we notably uncover an improved MSC quantification method with predictive cross-study applicability and unveil the key importance of system volume, time exposure to MSCs, and cross-communication between MSC and T cell populations to realize full therapeutic effect. The application of these compartmental analysis can improve our understanding of MSC mechanism(s) of action and further lead to administration methods that deliver MSCs within a compartment for predictable potency.

Stromalized microreactor supports murine hematopoietic progenitor enrichment.

There is an emerging need to process, expand, and even genetically engineer hematopoietic stem and progenitor cells (HSPCs) prior to administration for blood reconstitution therapy. A closed-system and automated solution for ex vivo HSC processing can improve adoption and standardize processing techniques. Here, we report a recirculating flow bioreactor where HSCs are stabilized and enriched for short-term processing by indirect fibroblast feeder coculture. Mouse 3 T3 fibroblasts were seeded on the extraluminal membrane surface of a hollow fiber micro-bioreactor and were found to support HSPC cell number compared to unsupported BMCs. CFSE analysis indicates that 3 T3-support was essential for the enhanced intrinsic cell cycling of HSPCs. This enhanced support was specific to the HSPC population with little to no effect seen with the Lineagepositive and Lineagenegative cells. Together, these data suggest that stromal-seeded hollow fiber micro-reactors represent a platform to screening various conditions that support the expansion and bioprocessing of HSPCs ex vivo.

Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion.

Adult bone marrow mesenchymal stromal cells (MSCs) have cross-functional, intrinsic potency that is of therapeutic interest. Their ability to regenerate bone, fat, and cartilage, modulate the immune system, and nurture the growth and function of other bone marrow hematopoietic stem/progenitor cells have all been evaluated by transplant applications of MSCs. These applications require the isolation and expansion scaled cell production. To investigate biophysical properties of MSCs that can be feasibly utilized as predictors of bioactivity during biomanufacturing, we used a low-density seeding model to drive MSCs into proliferative stress and exhibit the hallmark characteristics of in vitro aging. A low-density seeding method was used to generate MSCs from passages 1-7 to simulate serial expansion of these cells to maximize yield from a single donor. MSCs were subjected to three bioactivity assays in parallel to ascertain whether patterns in MSC age, size, and shape were associated with the outcomes of the potency assays. MSC age was found to be a predictor of adipogenesis, while cell and nuclear shape was strongly associated to hematopoietic-supportive potency. Together, these data evaluate morphological changes associated with cell potency and highlight new strategies for purification or alternatives to assessing MSC quality.

An engineered biomarker system to monitor and modulate immune clearance of cell therapies.

BACKGROUND AIMS: Cell transplants offer a new opportunity to deliver therapies with novel and complex mechanisms of action. Understanding the pharmacology of cell transplants is important to deliver this new therapy effectively. Currently, however, there are limited techniques to easily track cells after intravenous administration due to the dispersion of the graft throughout the entire body. METHODS: We herein developed an engineered cell system that secretes a luciferase enzyme to sensitively detect cell transplants independent of their locale by a simple blood test. We specifically studied a unique feature of cell transplant pharmacology-namely, immune clearance-using mesenchymal stromal cells (MSCs) as a proof-of-concept cell therapy. MSCs are a clinically relevant cell therapy that has been explored in several disease indications due to their innate properties of altering an immune response. RESULTS: Using this engineered reporter, we observed specific sensitivity of cell therapy exposure to the preparation of cells, cytolysis of MSCs in an allogeneic setting and a NK cell-mediated destruction of MSCs in an autologous setting. CONCLUSIONS: Our cellular tracking method has broader implications at large for assessing in vivo kinetics of various other cell therapies.