Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts.

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described.

Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing.

Background Aims: Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing.

Methods: To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR).

DeepIFC: Virtual fluorescent labeling of blood cells in imaging flow cytometry data with deep learning.

Imaging flow cytometry (IFC) combines flow cytometry with microscopy, allowing rapid characterization of cellular and molecular properties via high-throughput single-cell fluorescent imaging. However, fluorescent labeling is costly and time-consuming. We present a computational method called DeepIFC based on the Inception U-Net neural network architecture, able to generate fluorescent marker images and learn morphological features from IFC brightfield and darkfield images.

Immunomodulatory Functions of Adipose Mesenchymal Stromal/Stem Cell Derived From Donors With Type 2 Diabetes and Obesity on CD4 T Cells.

For adipose stromal/stem cell (ASCs)-based immunomodulatory therapies, it is important to study how donor characteristics, such as obesity and type 2 diabetes (T2D), influence ASCs efficacy. Here, ASCs were obtained from 2 groups, donors with T2D and obesity (dASCs) or nondiabetic donors with normal-weight (ndASCs), and then cultured with anti-CD3/CD28-stimulated allogeneic CD4 T cells. ASCs were studied for the expression of the immunomodulators CD54, CD274, and indoleamine 2, 3 dioxygenase 1 (IDO) in inflammatory conditions.

ATMP-classified, scalable, autologous cell spray for the treatment of skin wounds and assessment of its effects on wound healing clinically and on a molecular level.

Background: Autologous split-thickness skin grafts (STSGs) are the standard of care for closure of deep and large burns. However, perforation and extensive fishnet-like expansion of the grafts to achieve greater area wound coverage can lead to treatment failures or esthetically poor healing outcomes and scarring. The purpose of this study was to validate an autologous advanced therapy medicinal product (ATMP)-compliant skin cell suspension and evaluate its efficacy to promote epithelialization.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment.

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture.

The Utilization of Freezing Steps in Mesenchymal Stromal Cell (MSC) Manufacturing: Potential Impact on Quality and Cell Functionality Attributes.

Some recent reports suggest that cryopreserved and thawed mesenchymal stromal cells (MSCs) may have impaired functional properties as compared to freshly harvested MSCs from continuous cultures. A cryopreservation step in the manufacturing process brings important benefits, since it enables immediate off-the-shelf access to the products and a completion of all quality testing before batch release and administration to the patient. Cryopreservation is also inevitable in MSC banking strategies.

HLA-DR expression in clinical-grade bone marrow-derived multipotent mesenchymal stromal cells: a two-site study.

Background: Contrary to the minimal criteria proposed by the International Society for Cell and Gene Therapy for defining multipotent mesenchymal stromal cells (MSC), human leukocyte antigen (HLA)-DR expression is largely unpredictable in ex vivo-expanded clinical-grade cultures. Although activation of MSC in culture does not appear to affect their functionality, a large study investigating the impact of HLA-DR expression on cell identity and potency is still missing in the literature.

Methods: A retrospective analysis of HLA-DR expression in 130 clinical batches of bone marrow (BM)-MSC from two independent Good Manufacturing Practice-compliant production facilities was performed in order to identify the consequences on critical quality attributes as well as potential activation cues and dynamics of MSC activation in culture.

The use of unlicensed bone marrow-derived platelet lysate-expanded mesenchymal stromal cells in colitis: a pre-clinical study.

Background: Mesenchymal stromal cells (MSCs) are a promising candidate for treatment of inflammatory disorders, but their efficacy in human inflammatory bowel diseases (IBDs) has been inconsistent. Comparing the results from various pre-clinical and clinical IBD studies is also challenging due to a large variation in study designs.

Methods: In this comparative pre-clinical study, we compared two administration routes and investigated the safety and feasibility of both fresh and cryopreserved platelet-lysate-expanded human bone marrow-derived MSCs without additional licensing in a dextran sodium sulfate (DSS) colitis mouse model both in the acute and regenerative phases of colitis.

A microfluidic oxygen sink to create a targeted cellular hypoxic microenvironment under ambient atmospheric conditions.

Unlabelled: Physiological oxygen levels within the tissue microenvironment are usually lower than 14%, in stem cell niches these levels can be as low as 0-1%. In cell cultures, such low oxygen levels are usually mimicked by altering the global culture environment either by O removal (vacuum or oxygen absorption) or by N supplementation for O replacement. To generate a targeted cellular hypoxic microenvironment under ambient atmospheric conditions, we characterised the ability of the dissolved oxygen-depleting sodium sulfite to generate an in-liquid oxygen sink.

Automated image analysis detects aging in clinical-grade mesenchymal stromal cell cultures.

Background: Senescent cells are undesirable in cell therapy products due to reduced therapeutic activity and risk of aberrant cellular effects, and methods for assessing senescence are needed. Early-passage mesenchymal stromal cells (MSCs) are known to be small and spindle-shaped but become enlarged upon cell aging. Indeed, cell morphology is routinely evaluated during MSC production using subjective methods.

Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

Background: Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory.

In vitro Treg expansion favors the full-length splicing isoform of CTLA4.

Aim: We compared fresh and in vitro expanded human Tregs for their CTLA4 splicing isoform expression.

Methods: The CD4(+)CD25(+)CD127(low/-)phenotype was used for sorting Tregs and mRNA levels were measured with relative qRT-PCR.

Results: In fresh Tregs the level of soluble CTLA4 (sCTLA4) was half of that of full-length CTLA4, whereas in expanded cells sCTLA4 level was tenfold lower.

Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study.

Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.