Perfusion Bioreactor Research Articles

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Scaffold geometry and computational fluid dynamics simulation supporting osteogenic differentiation in dynamic culture

Geometry of porous scaffolds is critical to the success of cell adhesion, proliferation, and differentiation in bone tissue engineering. In this study, the effect of scaffold geometry on osteogenic differentiation of MC3T3-E1 pre-osteoblasts in a perfusion bioreactor was investigated. Three geometries of oligolactide-HA scaffolds, named Woodpile, LC-1000, and LC-1400, were fabricated with uniform pore size distribution and interconnectivity using stereolithography (SL) technique, and tested to evaluate for the most suitable scaffold geometry. Compressive tests revealed sufficiently high strength of all scaffolds to support new bone formation. The LC-1400 scaffold showed the highest cell proliferation in accordance with the highest level of osteoblast-specific gene expression after 21 days of dynamic culture in a perfusion bioreactor; however, it deposited less amount of calcium than the LC-1000 scaffold. Computational fluid dynamics (CFD) simulation was employed to predict and explain the effect of flow behavior on cell response under dynamic culture. The findings concluded that appropriate flow shear stress enhanced cell differentiation and mineralization in the scaffold, with the LC-1000 scaffold performing best due to its optimal balance between permeability and flow-induced shear stress.

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Efficient calculation of fluid-induced wall shear stress within tissue engineering scaffolds by an empirical model

Mechanical stimulation, such as fluid-induced wall shear stress (WSS), is known that can influence the cellular behaviours. Therefore, in some tissue engineering experiments in vitro, mechanical stimulation is applied via bioreactors to the cells in cell culturing to study cell physiology and pathology. In 3D cell culturing, porous scaffolds are used for housing the cells. It is known that the scaffold porous geometries can influence the scaffold permeability and internal WSS in a bioreactor (such as perfusion bioreactor). To calculate the WSS generated on cells within scaffolds, usually computational fluid dynamics (CFD) simulation is needed. However, the limitations of the computational method for WSS calculation are: (i) the high time cost of the CFD simulation (in particular for the highly irregular geometries); (ii) accessibility to the CFD model for some cell culturing experimentalists due to the knowledge gap. To address these limitations, this study aims to develop an empirical model for calculating the WSS based on scaffold permeability. This model can allow the tissue engineers to efficiently calculate the WSS generated within the scaffold and/or determine the bioreactor loading without performing the computational simulations.

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Generation of colon cancer–derived tumor-infiltrating T cells (TILs) for adoptive cell therapy

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7−). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay)

CFD supported scale up of perfusion bioreactors in biopharma

The robust scale up of perfusion systems requires comparable conditions over all scales to ensure equivalent cell culture performance. As cells in continuous processes circulate outside the bioreactor, performance losses may arise if jet flow and stirring cause a direct connection between perfusion feed and return. Computational fluid dynamics can be used to identify such short circuit flows, assess mixing efficiencies, and eventually adapt the perfusion setup. This study investigates the scale up from a 2 L glass bioreactor to 100 L and 500 L disposable pilot scale systems. Highly resolved Lattice Boltzmann Large Eddy simulations were performed in single phase and mixing efficiencies (Emix) furthermore experimentally validated in the 2 L system. This evaluation gives insight into the flow pattern, the mixing behavior and information on cell residence time inside the bioreactors. No geometric adaptations in the pilot scale systems were necessary as Emix was greater than 90% for all conditions tested. Two different setups were evaluated in 2 L scale where the direction of flow was changed, yielding a difference in mixing efficiency of 10%. Nevertheless, since Emix was confirmed to be >90% also for both 2 L setups and the determined mixing times were in a similar range for all scales, the 2 L system was deemed to be a suitable scale down model. The results demonstrate how computational fluid dynamic models can be used for rational process design of intensified production processes in the biopharmaceutical industry.

Experimental measurements and CFD modelling of hydroxyapatite scaffolds in perfusion bioreactors for bone regeneration.

In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors.

A New Model of Esophageal Cancers by Using a Detergent-Free Decellularized Matrix in a Perfusion Bioreactor.

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Peripheral blood mononuclear cells contribute to myogenesis in a 3D bioengineered system of bone marrow mesenchymal stem cells and myoblasts.

In this work, a 3D environment obtained using fibrin scaffold and two cell populations, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), and primary skeletal muscle cells (SkMs), was assembled. Peripheral blood mononuclear cells (PBMCs) fraction obtained after blood filtration with HemaTrate® filter was then added to the 3D culture system to explore their influence on myogenesis. The best cell ratio into a 3D fibrin hydrogel was 1:1 (BM-MSCs plus SkMs:PBMCs) when cultured in a perfusion bioreactor; indeed, excellent viability and myogenic event induction were observed. Myogenic genes were significantly overexpressed when cultured with PBMCs, such as MyoD1 of 118-fold at day 14 and Desmin 6-fold at day 21. Desmin and Myosin Heavy Chain were also detected at protein level by immunostaining along the culture. Moreover, the presence of PBMCs in 3D culture induced a significant downregulation of pro-inflammatory cytokine gene expression, such as IL6. This smart biomimetic environment can be an excellent tool for investigation of cellular crosstalk and PBMC influence on myogenic processes.

Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments.

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2weeks on collagen scaffolds. Microenvironments of various size/volume (6-12mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

The Influence of Fluid Shear Stress on Bone and Cancer Cells Proliferation and Distribution.

We investigated the potential correlation between the fluid shear stress and the proliferation of bone prostate cancer cells on the surface of nanoclay-based scaffolds in a perfusion bioreactor. Human mesenchymal stem cells (hMSCs) were seeded on the scaffolds to initiate bone growth. After 23 days, prostate cancer cells (MDAPCa2b) were cultured on top of the osteogenically differentiated hMSCs. The scaffolds were separated into two groups subjected to two distinct conditions: (i) static (no flow); and (ii) dynamic (with flow) conditions to recapitulate bone metastasis of prostate cancer. Based on measured data, Computational Fluid Dynamics(CFD) models were constructed to determine the velocity and shear stress distributions on the scaffold surface. Our experimental results show distinct differences in the growth pattern of hMSCs and MDAPCa2b cells between the static and dynamic conditions. Our computational results further suggest that the dynamic flow leads to drastic change in cell morphology and tumorous distribution. Our work points to a strong correlation between tumor growth and local interstitial flows in bones.

Computational fluid dynamics analysis of the fluid environment of 3D printed tissue scaffolds within a perfusion bioreactor: the effect of pore shape

Computational fluid dynamics analysis of the fluid environment of 3D printed tissue scaffolds within a perfusion bioreactor: the effect of pore shape

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells’ maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

Computational modeling and experimental characterization of fluid dynamics in micro-CT scanned scaffolds within a multiple-sample airlift perfusion bioreactor

The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructure of scaffolds on the extracellular matrix deposition also play a crucial role during perfusion cultures. In the present study, a numerical simulation was implemented at the pore level of the scaffold for fluid flow through porous media during perfused culture. Micro-computed tomography was used to obtain the digital 3D image of the complex geometry of a PLLA scaffold, offering a detailed analysis from a volume-based methodology without simplifications of the results as for pore or Darcy’s law-models. Predictions about the uniformity of the flow field through the scaffolds-bioreactor system have been assessed by quantifying the cell viability of a perfusion culture while using pre-osteoblastic cells seeded on 24 PLLA scaffolds for up to 6 days.

Cancer Exosomes: An Overview and the Applications of Flow

Cancer is one of the most prevalent and disruptive diseases affecting the population, and as such, is the subject of major research efforts. Recently, these efforts have been put towards understanding the role that exosomes can play in the progression of cancer. Exosomes are small extracellular vesicles ranging from 40–150 nm in size that carry bioactive molecules like proteins, DNA, RNA, miRNA, and surface receptors. One of the most important features of exosomes is their ability to easily travel throughout the body, extending the reach of parent cell’s signaling capabilities. Cancer derived exosomes (CDEs) carry dangerous cargo that can aid in the metastasis, and disease progression through angiogenesis, promoting epithelial to mesenchymal transition, and immune suppression. Exosomes can transport these molecules to cells in the tumor environment as well as distant premetastatic locations making them an extremely versatile tool in the toolbelt of cancer. This review aims to compile the present knowledge and understanding of the involvement of exosomes in the progression of cancer as well as current production, isolation, and purification methods, with particular interest on flow perfusion bioreactor and microfluidics systems, which allow for accurate modeling and production of exosomes.

Improved endothelial cell proliferation on laminin-derived peptide conjugated nanofibrous microtubes using custom made bioreactor

Cardiovascular diseases (CVD) are currently considered as one of the major reasons for death worldwide. The blockage of minor vessels such as the coronary arteries may be linked to more severe occurrences that might be fatal. The gold standard approach involves the transplantation of secondary vessels or the use of synthetic vascular grafts. Electrospun nanofiber (NF) based grafts produced with synthetic polymers might be simply modified to resemble the original structure of vessels providing desirable physical features and potentially improving cellular behavior including cell attachment, growth, and differentiation. Although poly lactic-co-glycolic acid (PLGA), is well-known, commercially available, degradable synthetic, has good mechanical and biocompatibility properties, PLGA is inadequate in terms of cell recognition signals. To overcome the bioactivity problem of PLGA, bioactive peptides are the most extensively utilized approach for surface modification. On the other hand, seeding and cultivation of tube-like conduits are challenging due to their shapes, and dynamic seeding and culture are considered beneficial for these grafts. Herein, we attempted to enhance the Endothelial Cells (ECs) attachment and proliferation on PLGA electrospun NF-based vascular grafts by both the conjugation of laminin-derived peptide IKVAV and perfusion culture with the custom-made bioreactor system. The bioreactor and its flow and pressure were simulated and decided using COMSOL Multiphysics 5.4. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation were increased by both functionalization of PLGA graft with IKVAV and using a custom-made perfusion bioreactor for cell seeding and cultivation within 7 days (d). This tubular vascular graft could be a potential tissue-engineered scaffold for the restoration of the venous system.

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

Role of Co-Culture with Fibroblasts and Dynamic Culture Systems in 3-Dimensional MCF-7 Tumor Model Maturation

In vitro tumor models that are 3-dimensional (3D) have emerged as a significant area in the field of cancer research over the past several years. In order for breast cancer cell lines to grow and develop, it is important to select a scaffold, determine a strategy for seeding cells into the scaffold, then cultivate the cells under a variety of conditions. Therefore, we cultivated MCF-7 cells and fibroblasts on a gelatin-alginate scaffold alone or in co-culture under static or dynamic culture conditions in order to produce a 3D tumor model. MCF-7 and fibroblast were seeded into Gellatin-Alginate by Centrifugation and Incubation. After that, the cell proliferation was examined by MTT assay and the cell number determination in the scaffold. The morphology of MCF-7 was observed by H&E staining and SEM. The results showed that the co-culture of MCF-7 cells and fibroblasts in the scaffold exhibited an increase in cell mass size. Their mass morphologies feature a significant number of MCF-7 cells with a round structure that persists for an extended period of time. Perfusion bioreactors also demonstrate an increase in the size of cell mass (3 times higher than static culture). As a result, the long-term stability of the structure offers the possibility of cancer biology research and drug testing, especially the sustained release or actions experiements. HIGHLIGHTS This research focuses on aspects of the tissue engineering concept that support the proliferation and development of specific structures within the mass. By experimenting, we were able; to develop co-culture with MCF-7 breast cancer cells and fibroblasts in a bioreactor system to form the superior cell mass up to 500 mm to preserve the cell mass structure for a long duration (28 days) to have a particularly stable mass structure, with a large number of live cells GRAPHICAL ABSTRACT

The monoculture of cord-blood-derived CD34+ cells by an automated, membrane-based dynamic perfusion system with a novel cytokine cocktail.

Human leukocyte antigen (HLA)-matched cord blood (CB) transplantation is a procedure for the treatment of certain hematological malignancies, hemoglobinopathies, and autoimmune disorders. However, one of the challenges is to provide a sufficient number of Tcell-depleted hematopoietic stem and progenitor cells. Currently, only 4%-5% of the CB units stored in CB banks contain enoughCD34+ cells for engrafting 70-kg patients. To support this clinical need, we have developed an automated expansion protocol for CB-derived CD34+ cells in the Quantum system's dynamic perfusion bioreactor using a novel cytokine cocktail comprised of stemcell factor (SCF), thrombopoietin (TPO), fms-like tyrosine kinase 3 ligand (Flt-3L), interleukin-3 (IL-3), IL-6, glial cell line-derived neurotrophic factor (GDNF), StemRegenin 1 (SR-1), and a fibronectin-stromal-cell-derived factor-1 (SDF-1)-coated membrane. In an 8-day expansion of a 2×106 positively selected CD34+ cell inoculum from 3 donor lineages, the mean cell harvest and cell viability were1.02×108 cells and 95.5%, respectively, and the mean frequency of the CD45+CD34+ immunophenotype was 54.3%. Themean differentiated cell frequencies were 0.5% for lymphocytes, 15.8% for neutrophils, and 15.4% for platelets. These resultsdemonstrate that the automated monoculture protocol can support the expansion of CD34+ cells with minimal lymphocyte residual.