Filling the gap: the workforce of tomorrow for CGT manufacturing as the sector advances.

Workforce education and development are key cornerstones in advancing and maturing the Cell & Gene Therapy sector. A skilled worker shortage can significantly impact and delay progress as well as the quality of output for any developer, thereby negatively impacting a patient's access to life-saving treatments. Several roundtable discussions were held at the International Society for Cell & Gene Therapy (ISCT) 2023 Annual Meeting to dive deeper into the current state of workforce development and solutions to address this bottleneck. One roundtable discussion was co-hosted by the Alliance for Regenerative Medicine (ARM) and ISCT, which focused on the gap analysis provided for the United States Cell & Gene Therapy (CGT) sector, highlighting the lack of skilled workers in manufacturing and quality control. In this manuscript, the roundtable participants continue this conversation, review the roles and staffing requirements in both academic and industry as well as small and large company settings. The adoption of increased manufacturing automation is one promising solution to propel the sector forward. However, automation alone won't replace on-site staff, but will lower the bar to entry for a larger pool of people and require different training. This paper also addresses the workforce development and training paradigm shift as advanced manufacturing techniques are implemented, which will differ considerably based on the type of manufacturing efforts, thus emphasizing the need for a well-thought-out strategy to up-skill and re-skill the technical workforce to adapt to these advancements. Organizations such as ISCT and ARM have a role to play in propelling the field forward, providing awareness and education to stakeholders at all levels, as well as acting as a convener and participating as a key stakeholder in discussions and partnerships between academia and industry towards solutions for training the best personnel for CGT manufacturing. This scope includes novel digital tools and technologies to simplify training to increase access to new talent pools interested in careers in a rapidly advancing sector.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags.

BACKGROUND: A major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags. METHODS: Mo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized. RESULTS: Monocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels. CONCLUSIONS: Our findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell-based immunotherapies.

In recent years, cell-based therapies targeting the immune system have emerged as promising strategies for cancer treatment. This review summarizes manufacturing challenges related to production of antigen presenting cells as a patient-tailored cancer therapy. Understanding cell-material interactions is essential because in vitro cell culture manipulations to obtain mature antigen-producing cells can significantly alter their in vivo performance. Traditional antigen-producing cell culture protocols often rely on cell adhesion to surface-treated hydrophilic polystyrene flasks. More recent commercial and investigational cancer immunotherapy products were manufactured using suspension cell culture in closed hydrophobic fluoropolymer bags. The shift to closed cell culture systems can decrease risks of contamination by individual operators, as well as facilitate scale-up and automation. Selecting closed cell culture bags over traditional open culture systems entails different handling procedures and processing controls, which can affect product quality. Changes in culture vessels also entail changes in vessel materials and geometry, which may alter the cell microenvironment and resulting cell fate decisions. Strategically designed culture systems will pave the way for the generation of more sophisticated and highly potent cell-based cancer vaccines. As an increasing number of cell-based therapies enter the clinic, the selection of appropriate cell culture vessels and materials becomes a critical consideration that can impact the therapeutic efficacy of the product, and hence clinical outcomes and patient quality of life.

Hutchinson-Gilford progeria syndrome as a model for vascular aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a de novo genetic mutation that leads to the accumulation of a splicing isoform of lamin A termed progerin. Progerin expression alters the organization of the nuclear lamina and chromatin. The life expectancy of HGPS patients is severely reduced due to critical cardiovascular defects. Progerin also accumulates in an age-dependent manner in the vascular cells of adults that do not carry genetic mutations associated with HGPS. The molecular mechanisms that lead to vascular dysfunction in HGPS may therefore also play a role in vascular aging. The vascular phenotypic and molecular changes observed in HGPS are strikingly similar to those seen with age, including increased senescence, altered mechanotransduction and stem cell exhaustion. This article discusses the similarities and differences between age-dependent and HGPS-related vascular aging to highlight the relevance of HGPS as a model for vascular aging. Induced pluripotent stem cells derived from HGPS patients are suggested as an attractive model to study vascular aging in order to develop novel approaches to treat cardiovascular disease.

Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens.

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. We investigated the role of cytotoxic T-lymphocytes (CTL) counteracting and recognizing LSC. Leukemia-associated antigens (LAA) represent immunogenic structures to target LSC. We enriched the LSC-containing fraction of 20 AML patients and hematopoietic stem cells (HSC) of healthy volunteers. Using microarray analysis and qRT-PCR we detected high expression of several LAA in AML cells but also in LSC. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC compared with HSC. PRAME, RHAMM and WT1 are furthermore also lower expressed on leukemic bulk. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk than on LSC. In colony forming unit (CFU) immunoassays, T cells stimulated against various LAA indicated a significant inhibition of CFUs in AML patient samples. The LAA PRAME, RHAMM and WT1 showed highest immunogenic responses with a range up to 58-83%. In a proof of principle xenotransplant mouse model, PRAME-stimulated CTL targeted AML stem cells, reflected by a delayed engraftment of leukemia (p = 0.0159). Taken together, we demonstrated the expression of several LAA in LSC. LAA-specific T cells are able to hamper LSC in immunoassays and in a mouse model, which suggests that immunotherapeutic approaches have the potential to target malignant stem cells.

Human mesenchymal stromal cell adhesion and expansion on fluoropolymer surfaces modified with oxygen and nitrogen-rich plasma polymers.

Fluorinated ethylene propylene (FEP) vessels are of significant interest for therapeutic cell biomanufacturing applications due to their chemical inertness, hydrophobic surface, and high oxygen permeability. However, these properties also limit the adhesion and survival of anchorage-dependent cells. Here, we develop novel plasma polymer coatings to modify FEP surfaces, enhancing the adhesion and expansion of human mesenchymal stromal cells (hMSCs). Similar to commercially available tissue culture polystyrene vessels, oxygen-rich or nitrogen-rich surface chemistries can be achieved using this approach. While steam sterilization increased the roughness of the coatings and altered the surface chemistry, the overall wettability and oxygen or nitrogen-rich nature of the coatings were maintained. In the absence of proteins during initial cell attachment, cells adhered to surfaces even in the presence of chelators, whereas adhesion was abrogated with chelator in a protein-containing medium, suggesting that integrin-mediated adhesion predominates over physicochemical tethering in normal protein-containing cell seeding conditions. Albumin adsorption was more elevated on nitrogen-rich coatings compared to the oxygen-rich coatings, which was correlated with a higher extent of hMSC expansion after 3 days. Both the oxygen and nitrogen-rich coatings significantly improved hMSC adhesion and expansion compared to untreated FEP. FEP surfaces with nitrogen-rich coatings were practically equivalent to commercially available standard tissue culture-treated polystyrene surfaces in terms of hMSC yields. Plasma polymer coatings show significant promise in expanding the potential usage of FEP-based culture vessels for cell therapy applications.

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components.

BACKGROUND AIMS: The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). METHODS: Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. RESULTS: PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-ß1 (TGF-ß1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-ß1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-ß1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. CONCLUSIONS: PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

Labeling of mesenchymal stromal cells with iron oxide-poly(L-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the non-interference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. METHODS: We studied the effects of iron oxide-poly(l-lactide) nanoparticles in MSC with flow cytometry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immunologic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. RESULTS: It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. CONCLUSIONS: These particles qualify for studying MSC homing and trafficking via MRI.

Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy.

Conventional cell culture surfaces typically consist of polystyrene, with or without surface modifications created through plasma treatment or protein/peptide coating strategies. Other polymers such as fluorinated ethylene propylene are increasingly being implemented in the design of closed cell culture vessels, for example to facilitate the production of cells for cancer immunotherapy. Cultured cells are sensitive to culture vessel material changes through different mechanisms including cell-surface interactions, which are in turn dependent on the amount, type, and conformation of proteins adsorbed on the surface. Here, we investigate the protein deposition from cell culture medium onto untreated polystyrene and fluoropolymer surfaces using quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both of these non-polar surfaces showed comparable protein deposition kinetics and resulted in similar mechanical and topographical film properties. At protein concentrations found in typical serum-free media used to culture dendritic cells, two deposition phases can be observed. The protein layers form within the first few minutes of contact with the cell culture medium and likely consist almost exclusively of albumin. It is indicated that initial protein film formation will be completed prior to cell settling and initial cell contact will be established with the secondary protein layer. The structural properties of the protein film surface will strongly depend on the albumin concentration in the medium and presumably be less affected by the chemical composition of the cell culture surface.

Adhesion of human monocytes to oxygen- and nitrogen- containing plasma polymers: Effect of surface chemistry and protein adsorption.

The interactions between monocytes and biomaterials can potentially be modulated by controlling the chemical and structural surface properties of biomaterials. The objective of this study was to determine the effect of plasma-deposited functional organic coatings on monocyte adhesion and differentiation into macrophages. Organic coatings with varying oxygen and nitrogen concentration were prepared by low-pressure plasma co-polymerization of binary gas mixtures combining a hydrocarbon (butadiene/ethylene) and a heteroatom-containing gas (carbon dioxide/ammonia) to deposit either oxygen or nitrogen-containing coatings. The deposition parameters controlled the composition of the coatings and, consequently, the surface charge (between 26 mV and -28 mV) and wettability. The adhesion of myeloid leukemia cell lines U937 and NB4 as well as human monocytes to plasma polymerized coatings, was tested using cell culture medium with and without fetal bovine serum. The results showed that the concentration of [-NH2] and [-COOH] on the surface of the plasma polymers, controls the adhesion of U937 and NB4 cell lines to the coatings. Thus, above a certain composition threshold, i.e. [-NH2]=2.6-3.0% and [-COOH]=1.2-1.57 nmol/cm2, the surface facilitates adhesion of both cell lines, irrespective of the culture medium used. Based on qualitative observations the number of monocytes adhering to the coatings was proportional to the concentration of functional groups at the surface of the coatings. The surface plasmon resonance results, in line with cell culture experiments, indicated that the presence of albumin on the surfaces with [-NH2] and [-COOH] above the determined critical concentration may be an indicator of monocyte adhesion to these plasma polymers.

Mining for osteogenic surface topographies: In silico design to in vivo osseo-integration.

Stem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships - the relationships between substrate parameters and the phenotypes they induce - have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated.

Effect of high-dose irradiation on human bone-marrow-derived mesenchymal stromal cells.

Cell therapy using multipotent mesenchymal stromal cells (MSCs) is of high interest in various indications. As the pleiotropic effects mediated by MSCs rely mostly on their unique secretory profile, long-term persistence of ex-vivo-expanded cells in the recipient may not always be desirable. Irradiation is a routine procedure in transfusion medicine to prevent long-term persistence of nucleated cells and could therefore also be applied to MSCs. We have exposed human bone-marrow-derived MSCs to 30 or 60 Gy of γ-irradiation and assessed cell proliferation, clonogenicity, differentiation, cytokine levels in media supernatants, surface receptor profile, as well as expression of proto-oncogenes/cell cycle markers, self-renewal/stemness markers, and DNA damage/irradiation markers. Irradiated MSCs show a significant decrease in proliferation and colony-forming unit-fibroblasts. However, a subpopulation of surviving cells is able to differentiate, but is unable to form colonies after irradiation. Irradiated MSCs showed stable expression of CD73 and CD90 and absence of CD3, CD34, and CD45 during a 16-week follow-up period. We found increased vascular endothelial growth factor (VEGF) levels and a decrease of platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB in culture media of nonirradiated cells. Irradiated MSCs showed an inverse pattern, that is, no increase of VEGF, and less consumption of PDGF-AA and PDGF-AB/BB. Interestingly, interleukin-6 (IL-6) levels increased during culture regardless of irradiation. Cells with lower sensitivity toward γ-irradiation showed positive ß-galactosidase activity 10 days after irradiation. Gene expression of both irradiated and nonirradiated MSCs 13-16 weeks after irradiation with 60 Gy predominantly followed the same pattern; cell cycle regulators CDKN1A (p21) and CDKN2A (p16) were upregulated, indicating cell cycle arrest, whereas classical proto-oncogenes, respectively, and self-renewal/stemness markers MYC, TP53 (p53), and KLF4 were downregulated. In addition, DNA damage/irradiation markers ATM, ATR, BRCA1, CHEK1, CHEK2, MDC1, and TP53BP1 also mostly showed the same pattern of gene expression as high-dose γ-irradiation. In conclusion, we demonstrated the existence of an MSC subpopulation with remarkable resistance to high-dose γ-irradiation. Cells surviving irradiation retained their trilineage differentiation capacity and surface marker profile but changed their cytokine secretion profile and became prematurely senescent.

Essential components for ex vivo proliferation of mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are highly interesting candidates for clinical applications in regenerative medicine. Due to their low occurrence in human tissues, extensive in vitro expansion is necessary to obtain sufficient cell numbers applicable as a clinical dose in the context of cellular therapy. Current cell culture media formulations for the isolation and expansion of MSCs include fetal calf serum (FCS), human AB serum (ABS), or human platelet lysate (PL) as a supplement. However, these established supplements are inherently ill-defined formulations that contain a variety of bioactive molecules in varying batch-to-batch compositions and the risk of transmitting pathogens that escape routine screening procedures. In this study, we have comparatively characterized the capacity of commonly used basal media, such as the Minimum Essential Medium alpha (αMEM), Dulbecco's modified Eagle's medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), and RPMI 1640 as well as human- and animal-derived supplements, that is, PL, ABS, and FCS to stimulate cell proliferation. MSC proliferation was observed to be optimal in the PL-supplemented αMEM. Using a combinatorial approach, we then assessed a library of soluble factors, including mitogens (TGF-ß1, Activin A, bFGF, EGF, IGF-I, PDGF-BB, and VEGF), chemokines (CCL21, CCL25, CXCL12, and RANTES), proteins (human serum albumin), lipids (e.g., oleic acid, linoleic acid, and arachidonic acid), and hormones (dexamethasone, insulin, and TSH), to create a defined medium as well as coating of cell culture surfaces to promote robust MSC proliferation in vitro. A combination of recombinant human factors partially met the nutritional requirements of bone marrow-derived MSCs, and was able to promote cell proliferation comparable to about 5% PL if supplemented with auxiliary 0.6%-1.2% PL. Maximal MSC proliferation was achieved by combining 5% PL with a cocktail of recombinant factors and did not depend on coating of cell culture surfaces.

GMP-compliant isolation and expansion of bone marrow-derived MSCs in the closed, automated device quantum cell expansion system.

The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-ß1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100×10(6) of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.

GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC.

BACKGROUND: Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. METHODS AND FINDINGS: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. CONCLUSIONS: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.