Journal articles on the topic 'Ex Vivo Vein Culture System'
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Cialdai, Francesca, Stefano Bacci, Virginia Zizi, Aleandro Norfini, Michele Balsamo, Valerio Ciccone, Lucia Morbidelli, et al. "Optimization of an Ex-Vivo Human Skin/Vein Model for Long-Term Wound Healing Studies. Ground Preparatory Activities for the ‘Suture in Space’ Experiment Onboard the International Space Station." International Journal of Molecular Sciences 23, no. 22 (November 16, 2022): 14123. http://dx.doi.org/10.3390/ijms232214123.
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This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.
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Prandi, Francesca, Marco Piola, Monica Soncini, Claudia Colussi, Yuri D’Alessandra, Eleonora Penza, Marco Agrifoglio, et al. "Adventitial Vessel Growth and Progenitor Cells Activation in an Ex Vivo Culture System Mimicking Human Saphenous Vein Wall Strain after Coronary Artery Bypass Grafting." PLOS ONE 10, no. 2 (February 17, 2015): e0117409. http://dx.doi.org/10.1371/journal.pone.0117409.
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Maffei, S., G. Galeati, G. Pennarossa, T. A. L. Brevini, and G. Gandolfi. "188 DEVELOPMENT OF AN EFFECTIVE WHOLE-OVARY PERFUSION SYSTEM." Reproduction, Fertility and Development 27, no. 1 (2015): 185. http://dx.doi.org/10.1071/rdv27n1ab188.
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The different structures of a mammalian ovary require complex 3-dimensional interactions to function properly. It is difficult to access the ovary in vivo and to study its physiology in vitro, it is necessary to dissect its different parts and culture them individually. Although informative, this approach prevents the understanding of the role played by their interactions. Perfusion systems are available for ovaries of laboratory animals while organs of larger species have been maintained in culture only for a few hours. This has prompted us to develop a system that can preserve the function of a whole sheep ovary for a few days ex vivo so that it is available for analysis in controlled conditions. Twenty-four sheep ovaries were collected at the local abattoir; 18 were assigned randomly to 3 experimental groups (media A, B, and C) and 6 were immediately fixed in 10% formaldehyde and used as fresh controls. Whole ovaries were cultured for up to 4 days using a semi-open perfusion system. Organs were perfused through the ovarian artery, at a flow rate of 1.5 mL min–1 with basal medium (M199, 25 mM HEPES, 2 mM l-glutamine and 100 µg mL–1 antibiotic-antimycotic solution) supplemented with 0.4% fatty acid free BSA (medium A); or 0.4% BSA heat shock fraction (medium B); or 10% FBS, 50 ng mL–1 IGF-1, and 50 mg bovine insulin (medium C). Ovaries were stimulated with FSH (Folltropin®-V, Bioniche Animal Health Inc., Belleville, Ontario, Canada) changing medium in a pulsatile manner (1 mg mL–1 for 2 h; 0.5 mg mL–1 for 2 h; 0 mg mL–1 for 20 h), with the same cycle repeated each day of culture. At every change, aliquots were collected for oestradiol (E2) and progesterone (P4) quantification. After culture, ovaries were examined for follicular morphology, cell proliferation, and apoptotic rate. Statistical analysis was performed using one-way ANOVA (SPSS 20, IBM, Armonk, NY, USA). In media A and B, all morphological parameters showed a small but significant decrease compared to fresh control, only after 3 days of culture. The different BSA in medium B did not affect follicle morphology but significantly increased cell proliferation (medium A, 28.59 ± 3.26%; medium B, 32.04 ± 2.67%) and decreased apoptosis (medium A, 32.51 ± 5.92%; medium B, 24.55 ± 2.55%). In both media, steroid concentration increased after FSH pulses (E2 range 1.95–10.50 pg mL–1; P4 range 0.34–3.08 ng mL–1), reaching levels similar to those measurable in peripheral plasma. The presence of FBS, IGF-1, and insulin in medium C allowed extension of the culture period to 4 days with a percentage of intact follicles comparable to that observed after 3 days in media A and B. Moreover, proliferation rates were comparable to fresh controls. Steroid pattern changed with P4 values dropping close to zero (range 0.03–1.18 ng mL–1) and E2 level (range 23.59–94.98 pg mL–1) increasing 10-fold, achieving a concentration similar to that measured in the ovarian vein around oestrous. Our data indicate that it is possible to support viability of large animal whole ovaries for up to 4 days, providing a physiologically relevant model for studying ovarian functions in vitro. Research was supported by AIRC IG 10376 and by the Carraresi Foundation.
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Chan, Tim, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, et al. "Abstract 2821: Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2821. http://dx.doi.org/10.1158/1538-7445.am2022-2821.
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Abstract Current chimeric antigen receptor (CAR)-T manufacturing utilizes viral vectors and extensive ex vivo expansion at large central facilities leading to an exhausted CAR-T phenotype, high costs and long vein-to-vein times. While allogeneic CAR-T can reduce delays in patient treatment, they require extensive manipulation of donor cells, severe lymphodepletion and demonstrate short persistence limiting their therapeutic window. The UltraCAR-T® platform is designed to overcome these limitations by utilizing non-viral multigene delivery and a rapid, decentralized manufacturing process without ex vivo activation or expansion of T cells. Patient’s own T cells are collected and manufactured at the medical center and re-infused one day after gene transfer. Here we describe the next generation UltraCAR-T platform that addresses the inhibitory tumor microenvironment by incorporating a novel mechanism for intrinsic downregulation of one or more checkpoint inhibitor (CPI) genes. Our design achieves intrinsic CPI blockade without gene editing and is aimed at avoiding systemic toxicity and the high cost of combining CPI antibodies. Next generation UltraCAR-T cells simultaneously express CAR, membrane bound IL-15 (mbIL15), kill switch, and incorporate intrinsic CPI blockade. To illustrate the ability of this platform, we designed exemplary non-viral transposons to generate UltraCAR-T cells against multiple tumor targets incorporating intrinsic blockade of either one (PD-1) or two (PD-1 and TIGIT) CPI genes. Healthy donor T cells were transfected using the UltraPorator™ electroporation system to manufacture UltraCAR-T cells without ex vivo activation or expansion. The co-expression of CAR, mbIL15 and kill switch was confirmed by flow cytometry and western blot. Activated UltraCAR-T showed significant reduction in CPI gene expression compared to control CAR-T cells lacking the CPI blockade and did not show unintended off-target activity. Downregulation of CPI gene(s) on UltraCAR-T enhanced cytotoxicity and inflammatory cytokine production, especially at low effector to target (E:T) cell ratios, when co-cultured with PD-L1+/CD155+ tumor cells. Single-cell cytokine proteomics showed significant increase in polyfunctionality of UltraCAR-T with intrinsic downregulation of CPI gene(s). In vivo, a single infusion of receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific UltraCAR-T with intrinsic PD-1 blockade resulted in rapid expansion, an increase in preferred T cell memory (TSCM/TCM) populations, and significantly improved overall survival of ROR1+ PD-L1+ tumor bearing mice. These preclinical data highlight the improved efficacy of incorporating intrinsic CPI blockade in UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Tim Chan, Cheryl Bolinger, Sean Scott, Mengyan Du, Carol Poortman, Byron Koenitzer, Taranjit Athwal, Lindsey Shepard, R. Daniel Slone, Shourik Dutta, Steven Zilko, James M. Dunleavey, Giorgio Zenere, Jacques Plummer, Bernward Klocke, Christian Zinser, Shamim Ahmad, Douglas E. Brough, Rutul R. Shah, Helen Sabzevari. Incorporation of intrinsic checkpoint blockade enhances functionality of multigenic autologous UltraCAR-T® cells manufactured using non-viral gene delivery and rapid manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2821.
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Jackman, Rachael P., Marcus O. Muench, John W. Heitman, Susanne Marschner, Raymond P. Goodrich, and Philip J. Norris. "Prevention of Alloimmunization and Induction of Partial Tolerance Following Transfusion with Pathogen Reduced Platelets in Mice." Blood 118, no. 21 (November 18, 2011): 718. http://dx.doi.org/10.1182/blood.v118.21.718.718.
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Abstract Abstract 718 Introduction: The presence of donor white blood cells (WBC) in transfused blood products can induce alloimmunization, and reducing or eliminating this response may prove to be of clinical benefit. The use of a pathogen reduction method based on UV light illumination in the presence of riboflavin has been shown to induce changes in WBCs that result in a failure to bind to, or induce proliferation of allogeneic PBMCs in vitro. In addition, a study in rats has shown a reduction in alloimmunization in vivo using this treatment. Transfusion of cells illuminated with UV light at other doses without riboflavin has been shown to induce some degree of tolerance with a reduced antibody response to subsequent allogeneic transfusions. We sought to assess both the degree of alloimmunization in mice given pathogen reduced versus untreated allogeneic platelets, as well as determine if cells from mice given pathogen reduced platelets exhibited signs of tolerance ex vivo. Methods: Peripheral blood was collected from C57Bl/6 and Balb/cJ mice into CPDA-1, and platelet rich plasma (PRP) was prepared by gentle centrifugation. WBCs were isolated from the remainder of the blood and were added back to a portion of the PRP to generate either WBC-enriched or WBC-poor PRP. These products were either left untreated or pathogen reduced using the Mirasol pathogen reduction technology system, which uses a combination of riboflavin and UV illumination. These products were transfused via tail vein injection into Balb/cJ mice. Two weeks after transfusion the treated mice were sacrificed, and peripheral blood and spleens were collected. Serum levels of circulating alloantibodies were measured by flow cytometry. Splenocytes were cultured for 48 hours in the presence or absence of C57Bl/6 splenocytes, and levels of secreted cytokines were measured in culture supernatants using multiplexing techniques. Groups were compared using one-way ANOVA with Tukey's multiple comparison post-test, α=0.05. Results: Mice given allogeneic PRP transfusions had significantly elevated levels of alloantibodies compared with non-transfused control mice, whereas mice given syngeneic PRP or pathogen reduced PRP did not. Mice given either the WBC-enriched PRP or WBC-poor PRP generated alloantibodies, though higher levels of antibodies were observed with WBC-enriched PRP. Levels of IFN-γ, TNF-α, IL-10 and GM-CSF were significantly higher following secondary allogeneic challenge of cells from mice given untreated allogeneic PRP compared with those given no transfusion or syngeneic PRP, but not with those given pathogen reduced PRP. Levels of IL-1β, IL-4, IL-5, IL-6, IL-12(p70), and IL-13 were significantly reduced following secondary allogeneic challenge of cells from mice given pathogen reduced allogeneic PRP compared with those given no transfusion or syngeneic PRP. Conclusions: Treatment of allogeneic PRP with riboflavin and UV light prior to transfusion blocks alloimmunization in mice. Furthermore, secondary cytokine responses to allogeneic cells ex vivo are reduced, in some cases bellow the levels observed in cells from mice without prior exposure, suggesting induction of tolerance. Disclosures: Marschner: CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment. Norris:CaridianBCT Biotechnologies: Consultancy, Research Funding.
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Gardner, Rebecca, Corinne Summers, Dan Weber, Michael C. Jensen, and Colleen Delaney. "Lentiviral Transduction Of Notch Ligand Expanded Cord Blood HSC’s To Express a CD19-Specific CAR Generates NK and Myeloid Progeny With Antitumor Activity." Blood 122, no. 21 (November 15, 2013): 900. http://dx.doi.org/10.1182/blood.v122.21.900.900.
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Abstract Over the past decade, significant progress has been made using chimeric antigen receptors (CAR) to redirect the specificity of T cells against an array of cell surface tumor antigens. Using this same technology, we hypothesized that CD34+ hematopoietic stem and progenitor cells (HSPC) derived from cord blood (CB) could be transduced with lentivirus to efficiently express a CAR specific for CD19 that would enable targeted lysis through its continued expression in differentiated progeny effector cells. As previously reported, the Notch-mediated ex vivo expansion of CB HSPC is a clinically validated cell therapy product that is well tolerated, can be given off the shelf without HLA matching, and provides transient myeloid engraftment in both the HCT and intensive chemotherapy setting. CB-derived CD34+ cells are selected using the Miltenyi CliniMACS or AutoMACS platform and plated in StemSpan serum free expansion media supplemented with IL-6, IL-3, TPO, Flt3L, and SCF and in the presence of immobilized Notch ligand and retronectin. The cells are cultured for 14-16 days resulting in >150 fold expansion of CD34+ cells on average and a final heterogeneous cell product consisting of 14.5% (range 6.2-26) CD34+, 20.5% (6-36) CD15+, 11.3 (1.8-23) CD14+, 5.4%(2.2-13.6) CD56+CD16+, and 0.1(0-0.2)CD20+ which is cryopreserved for future clinical use. Importantly, no T cells are derived from this culture system. Off the shelf expanded units have been infused into 45 patients and no serious adverse events have been noted except for one allergic reaction attributed to DMSO. Additionally, there has been no persistent engraftment beyond day 180 in the HCT setting and 14 days post infusion in the chemotherapy setting. To engineer anti-CD19 activity into this product, cells are transduced at an MOI of 3 on day 3 of culture using a self inactivating lentivirus that encodes for a second generation CD19 CAR with 41BB costimulation as well as an EGFR tag which lacks an intracellular signaling domain and serves as a marker of transduction and a selection marker. Transduction efficiency ranges from 20-40% and equally transduces the CD34+ and CD34- populations. Copy number analysis demonstrates between 1-4 copies/cell. Expression of the transgene does not affect the final culture phenotype at 14 days and transgene expression is seen in all cell subsets and is stable over the culture period. Due to concerns of expression of the CAR on HSC with potential signaling capacity, irradiated LCL was added on day 7 of culture at a 1:1 ratio to provide antigen stimulation and did not negatively affect the growth of the cultures, nor the expression of the transgene. Functional NK cells were derived in vitro from the end culture population by an additional week of culture with RPMI supplemented with L-glutamine, human serum, IL-2 and IL-15. The CD19-CAR expressing NK cells had enhanced cytotoxic activity in a CRA against LCL compared with non-transduced NK cells (50 v 30%) whereas both killed K562 targets equally (75 v 80%). Ongoing experiments are being conducted to further evaluate the expression, signaling capacity and function of the CAR on each of the cell compartments. The NOG mouse model was used for in vivo functional assays to look at effects of CD19 CAR expression on human repopulating ability and to assess anti-CD19 activity. NOG mice underwent sub-lethal irradiation followed by tail vein injection of the expanded progeny of 20,000 selected CD34+ CB cells. There was no effect on early engraftment of transduced cells, however there was no detectable CD19 engraftment in the mice transplanted with CD19 CAR expressing cells compared with untransduced cells, in which the CD19 population was >20% of the engrafted cells, indicating anti-CD19 activity. NK cell populations were increased using NS0-IL15 secreting cells, irradiated and injected subcutaneously three times per week starting at week 3 to provide enhanced effector function. Current studies are ongoing to evaluate the anti-CD19 activity using the Raji CD19+ tumor model in the NOG mice. These initial results are promising as a way to engineer a graft versus leukemia effect into cord blood transplant. Furthermore, transduction of a CD19 CAR into our existing universal donor expanded CB cell therapy product allows for infusion of an anti-CD19 cell product to be given immediately following identification of a patient with clinical need for therapy, for example one in relapse or with persistent MRD. Disclosures: Jensen: ZetaRx: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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Nishimoto, Mitsutaka, Ke Zeng, Meixian Huang, Mi-Ae Lyu, Nina D. Shah, Swaminathan P. Iyer, Robert Z. Orlowski, and Simrit Parmar. "Cord Blood Regulatory T Cells Prevent Mutiple Myeloma Progression By Suppressing Inflammation." Blood 134, Supplement_1 (November 13, 2019): 3095. http://dx.doi.org/10.1182/blood-2019-128418.
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Introduction: Regulatory T cells (Tregs) are potent immunosuppressors and are being developed as adoptive immunotherapy for inflammatory disorders and autoimmune diseases. Specifically, cord blood (CB) Tregs are emerging as a promising treatment for graft vs. host disease (GVHD) as well as immune mediated bone marrow failure disorders including aplastic anemia, hypoplastic myelodysplasia and primary myelofibrosis. We and others have shown that when compared to peripheral blood (PB), CB Tregs have a higher expression of Helios, a marker of thymic Tregs; exert superior suppression on proliferating conventional T cells and do not secrete inflammatory IL-17 or express RORүt under stressful conditions. Such a stable suppressor profile coupled with their ready availability as an off-the-shelf product positions CB Tregs to be a very attractive therapeutic agent. Since multiple myeloma, a plasma cell clonal malignancy, exists in an inflammatory microenvironment of hematopoietic and non-hematopoietic cells which is likely related to disease progression, we hypothesized that adoptive therapy with CB Tregs may block myeloma progression by exerting their anti-inflammatory function. Methods: We used the NOD-SCID IL2rγnull (NSG) mice to first establish the myeloma tumor model, where mice were injected intravenously via tail vein with 3 x 106 Firefly luciferase labeled MM1S cells. For generating CB Tregs for adoptive therapy, Treg cell enrichment was performed from the whole CB unit using CD25+ magnetic beads and LS column (MIltenyl) followed by continuous culture in the presence of CD3/28 T-cell activator beads, and human recombinant interleukin-2 for 14 days. Expanded CB Treg phenotype was confirmed using flow cytometry analysis and the in-vitro suppressive capacity was assessed by CellTrace Violet Cell Proliferation assay. The mice were injected intravenously via tail vein with Firefly luciferase labeled MM1S cells on day 0 with or without 10 x 106 ex-vivo expanded CB Treg cells on day -1. We monitored the progression of MM1S cells by in vivo bioluminescence imaging (BLI) using the IVIS imaging system (Xenogen) 10 minutes after intraperitoneal injection of D-luciferin. Imaging data were analyzed and quantified with Living Image Software (Xenogen). We also assessed the weight and survival as well as the plasma cytokines levels. At the time of euthanasia, organs were harvested and analyzed for myeloma burden by using immunohistochemistry as well as flow analysis. Results: The expanded CB Tregs showed a consistent phenotype of CD4+25+127-FoxP3high and more than 80% suppression of the proliferating conventional T cells (Tcons). The progression of MM1S cells in the mice injected with CB Treg (n=9) was significantly delayed as shown in figure 1A, where minimal evidence of myeloma cells is visualized on day 31 compared to widespread tumor in the control arm (n=9). CB Treg recipients preserved their weight for a longer time (Figure 1B) and had improved overall survival (p=0.039) (Figure 1C). The prevention of development of myeloma by CB Tregs correlated with the lower levels of the inflammatory cytokine, interleukin-6 (IL-6) (murine) especially on day 35 post myeloma inoculation (Figure 1D). The circulating myeloma cells in the CB Treg recipients were significantly lower as compared to control mice. Upon euthanasia on day 25 post inoculation, myeloma cells were barely detectable in bone marrow and spleen in the CB Treg recipients as compared to large tumor burden in the control arm (Figure 1E). Conclusions: Our results demonstrate that ex-vivo expanded CB Treg cells prevent the growth of myeloma cells in a xenogenic model via suppression of inflammatory cytokines such as IL-6. These data provide additional insight to the underpinnings of myeloma progression and may contribute to the development of the novel immunotherapies in the future. Figure 1 Disclosures Shah: University of California, San Francisco: Employment; Genentech, Seattle Genetics, Oncopeptides, Karoypharm, Surface Oncology, Precision biosciences GSK, Nektar, Amgen, Indapta Therapeutics, Sanofi: Membership on an entity's Board of Directors or advisory committees; Indapta Therapeutics: Equity Ownership; Celgene, Janssen, Bluebird Bio, Sutro Biopharma: Research Funding; Poseida: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Nkarta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Teneobio: Consultancy, Membership on an entity's Board of Directors or advisory committees. Orlowski:Poseida Therapeutics, Inc.: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Choi, Yoonsu, Zheng Li, Xukui Wang, Hui Li, Feng Li, Daniel Y. Lee, Parijat Bhatnagar, et al. "Combination of Gene Therapy and Nanoparticle Imaging for Improving T-Cell Therapy." Blood 116, no. 21 (November 19, 2010): 1479. http://dx.doi.org/10.1182/blood.v116.21.1479.1479.
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Abstract Abstract 1479 Despite a functioning immune system, malignant tumors develop strategies to avoid immune recognition and elimination by T cells. Gene therapy can be used to render T cells capable of targeting tumors in an antigen-specific manner. The ability to genetically manipulate a T cell ex vivo provides new opportunities to enhance their biological activity in vivo. To explore new treatment approaches we propose a novel paradigm in which T cells are developed as biological delivery vehicles for desired genes and nanoparticles. We have combined gene therapy with nanotechnology to generate T cells that express a chimeric antigen receptor (CAR) selectively expressed on T cells operating within the tumor microenvironment and to introduce radiolabeled gold nanoparticles for tracking T cells in vivo. T cells can home to tumor microenvironments to exert their immunoreceptor-dependent cytolytic activity. However, despite the ability of T-cell receptors (TCRs) and CAR to recognize tumor cells, the anti-tumor effect can be incomplete. The ability to transfer a wide range of materials into T cells via electroporation can be exploited to adapt T cells as biological delivery agents capable of targeting specific tissues. This method may also circumvent the limited biodistribution of “raw” nanomaterials due to rapid clearance by the liver and other reticuloendothelial system (RES) organs. Gold is an attractive biocompatible nanomaterial which can be synthesized in compliance with current good manufacturing practice (cGMP) guidelines required for clinical-grade production and can be chemically functionalized for specific imaging or therapeutic functions. We first tested whether commercially available nanoparticles can be electro-transferred into cultured and primary human T cells. 43 nm diameter latex nanoparticles and 7 nm gold nanoparticles (GNPs) were electro-transferred into T cells and visualized by TEM and confocal imaging (Figure 1). We have modified GNPs on the surface with the chelator, diethylenetriaminepentaacetic acid (DTPA), for stable coordination with 111In followed by GNP PEGylation. We have shown that these radiolabeled GNPs can be readily electro-transferred into T cells suitable for combined single-photon emission computed tomography (SPECT) and computed tomography (CT) (Figure 2). We used a clinical SPECT/CT scanner to detect 111In-GNP in T cells (∼2.1 × 104 nanoparticles/cell) at a sensitivity of ∼760 cells/mL. After developing electroporation protocols of nanoparticles and in vivo imaging of 111In-GNPs, additional sensitivity was achieved by modifying the chelating chemistry using the macrocyclic chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), to bind 64Cu to GNPs, increasing the number of gold particles/cell, and using 64Cu-labeled GNPs for imaging by positron emission tomography (PET). Before tail vein injection to a mouse, 11.4 mCi was detected from 10 million T cells (suspended in 300 μL PBS) electroporated using a BTX ECM830 device with the following settings: 1 kV/cm, 4 ms duration, single square pulse (Figure 3). The estimated concentration of nanoparticles transferred into T cells was ∼2.3 × 105 nanoparticles/cell as determined by a gamma counter (2470 Wizard, PerkinElmer) and nanoparticle titration. While 20 nm GNPs were used for 111In labeling, 7 nm GNPs were chosen for 64Cu labeling because of the improved (10-fold) electroporation efficiency. We are currently investigating whether multi-functional GNPs encapsulating chemotherapy drugs can add both in vivo T-cell imaging capability and enhanced cytotoxicity to CAR-redirected T cells. In aggregate, this may improve the potency of clinical-grade genetically modified T cells as a vehicle with the imaging capability for targeted delivery of drug-loaded GNPs to the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.
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Hartman, Robert A., Kevin M. Bell, Richard E. Debski, James D. Kang, and Gwendolyn A. Sowa. "Novel ex-vivo mechanobiological intervertebral disc culture system." Journal of Biomechanics 45, no. 2 (January 2012): 382–85. http://dx.doi.org/10.1016/j.jbiomech.2011.10.036.
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Davies, CM, DB Jones, MJ Stoddart, K. Koller, E. Smith, CW Archer, and RG Richards. "Mechanically loaded ex vivo bone culture system 'Zetos': Systems and culture preparation." European Cells and Materials 11 (April 12, 2006): 57–75. http://dx.doi.org/10.22203/ecm.v011a07.
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Moro, Carlos Fernández, Sougat Misra, Soledad Pouso, Marita Wallenberg, Rainer Heuchel, Matthias Löhr, Mikael Björnstedt, Marco Del Chiaro, and Caroline Verbeke. "Establishing an ex vivo culture system for normal pancreatic tissue." Pancreatology 14, no. 3 (June 2014): S120—S121. http://dx.doi.org/10.1016/j.pan.2014.05.789.
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Tsiridis, Eleftherios, Neelam Gurav, Guy Bailey, Rod Sambrook, and Lucy Di Silvio. "A novel ex vivo culture system for studying bone repair." Injury 37, no. 3 (September 2006): S10—S17. http://dx.doi.org/10.1016/j.injury.2006.08.019.
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Vorobyova, D. A. Vorobyova, A. M. Lebedevа Lebedevа, M. S. Vagida Vagida, O. I. Ivanova Ivanova, E. I. Felker Felker, V. N. Gontarenko Gontarenko, A. V. Shpektor Shpektor, L. B. Margolis Margolis, and E. Yu Vasilieva Vasilieva. "Immunological Analysis of Human Atherosclerotic Plaques in ex vivo Culture System." Kardiologiia 11_2016 (December 2, 2016): 78–85. http://dx.doi.org/10.18565/cardio.2016.11.78-85.
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Companjen, Arjen R., L. I. van der Wel, Liu Wei, J. D. Laman, and E. P. Prens. "A modified ex vivo skin organ culture system for functional studies." Archives of Dermatological Research 293, no. 4 (April 24, 2001): 184–90. http://dx.doi.org/10.1007/s004030100219.
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Sun, Xiaofei, Xing Fu, Min Du, and Mei-Jun Zhu. "Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells." Open Biology 8, no. 4 (April 2018): 170256. http://dx.doi.org/10.1098/rsob.170256.
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Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPK α 1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α 1 in intestinal health.
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Won, Natalie, Jorge Castillo-Prado, Xinzhu Tan, John Ford, David Heath, Laura Ioana Mazilescu, Markus Selzner, and Ian M. Rogers. "Ex Vivo Perfusion Using a Mathematical Modeled, Controlled Gas Exchange Self-Contained Bioreactor Can Maintain a Mouse Kidney for Seven Days." Cells 11, no. 11 (June 2, 2022): 1822. http://dx.doi.org/10.3390/cells11111822.
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Regenerative medicine requires better pre-clinical tools in order to increase the efficiency of novel therapies transitioning to the clinic. Current monolayer cell culture methods are suboptimal for effectively testing new therapies and live mouse models are expensive, time consuming and require invasive procedures. Fetal organ culture, organoids, microfluidics and culture of thick sections of adult organs all aim to fill the knowledge gap between monolayer culture and live mouse studies. Here we report on an ex vivo organ perfusion system that can support whole adult mouse organs. Ex vivo perfusion of healthy and diseased mouse organs allows for real-time analysis that provides immediate feedback and accurate data collection throughout the experiment. Having a suitable normothermic ex vivo perfusion system for mouse organs provides a tool that will help contribute to our understanding of kidney physiology and disease and can take advantage of the many mouse models of human disease that already exist. Furthermore, an ex vivo kidney perfusion system can be used for testing novel cell therapies, drug screening, drug validation and for the detection of nephrotoxic substances. Critical to the success of mouse ex vivo organ perfusion is having a suitable bioreactor to maintain the organ. Here we have focused on the mouse kidney and mathematically modeled, built and validated a bioreactor that can maintain a kidney for 7 days. The long duration of the ex vivo perfusion will help to advance studies on kidney disease and can rapidly test for new regenerative medicine therapies compared to whole animal studies.
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Severn, Charlotte E., and Ashley M. Toye. "Exploration of a 3D Scaffold Culture System for Ex Vivo Blood Production." Blood 124, no. 21 (December 6, 2014): 4371. http://dx.doi.org/10.1182/blood.v124.21.4371.4371.
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Abstract The human body produces 2.5 million red blood cells (RBC) every second in the bone marrow, where differentiation of immature erythroblasts to reticulocytes occurs largely within erythroblastic islands. Erythropoiesis can be routinely replicated in 2D liquid culture using hematopoietic stem cells (HSC) isolated from adult peripheral blood. However current 2D culture methodologies eventually exhaust the input of HSC. The use of 3D scaffolds to better mimic erythropoiesis in the bone marrow would increase the RBC yield and longevity of ex vivo cultures, whilst reducing use of exogenous cytokines and minimizing handling requirements. As a starting point we are utilizing a porous collagen coated synthetic polyurethane (PU) scaffold (0.5cm by 0.5cm) for static 3D cultures provided by the Mantalaris group at Imperial College London (Mortera-Blanco et al., 2011). The PU scaffolds were seeded with lineage depleted or CD34+ population isolated from adult peripheral blood and maintained in serum-free erythroid expansion media with SCF, IL-3 and Dexamethasone, alone, or with erythropoietin (EPO) or thrombopoietin (TPO). Advantages of using the lineage depleted population is that it provides a larger diversity of stem cells for establishment of the niche, potentially facilitating the use of rare patient blood samples which may only be available in small sample volumes with low numbers of CD34+ cells. Scaffolds are productive using both cellular inputs, with significant cellular egress for up to 5 weeks regardless of whether exogenous EPO or TPO were included. As anticipated, the highest increase in cell production from the scaffolds was observed using CD34+ in the presence of EPO, which also provided a significant reduction in cell death. Histology and immunofluorescence were used to explore the cell populations within the scaffold. No mature macrophages were detected but GPA+ cells within the scaffold was observed until the end of culture, suggesting that cellular expansion is occurring without establishment of the classical macrophage niche. We also characterized the cells that continually egress from the CD34+ scaffold cultures using flow cytometry. Typically the cellular output exhibited approximately 20-60% CD34 positivity dropping to <15% post day 24 of culture and 10-30% GPA positivity, the remainder of the population was largely CD61 positive, with a contingent of CD14 positive monocytes. Mature cell surface markers for erythroid and megakaryocytic lineages were detected in up to half of the population, when the harvested 3D egress cells were further cultured in erythroid or megakaryocyte 2D culture systems. This suggests a high proportion of the cells that egress from the scaffold are megakaryocyte erythroid progenitors (MEPs) that are consistently expanding within the scaffold environment for the entire culture period. Since CD34+ expansion here has been achieved in the absence of macrophages, we wanted to explore their effects in 2D with the intention of introducing macrophages or macrophage derived functionality into our next generation scaffolds. Importantly, macrophage inclusion significantly increased proliferation of expanding erythroblasts compared to erythroblasts alone. Expansion of CD34+ cells in co-culture gave a statistically significant average fold increase of 528 compared to 301 for the control at day 7 (p = 0.0126 and 0.0162 for days 5 and 7 respectively (n=5)). Flow cytometry at the endpoint of the experiment showed a larger CD34+ population and a reduced GPA+ population when cells are in co-culture, suggesting the CD34+ cells are maintained in a more primitive state for longer. Therefore, co-culture has the additional benefit of improving early erythroblast expansion, alongside the recently reported enhanced expansion of erythroblasts during terminal differentiation (Ramos et al., 2013). In summary, we have demonstrated that a basic static PU scaffold can be utilized to increase hematopoietic stem cell culture longevity and facilitate generation of megakaryocyte or erythroid progenitors with expansion potential. This occurs in the absence of any detectable macrophage niche generation. We have also shown that co-culture with macrophages enhances erythroblast expansion in 2D. Further work is needed to determine whether inclusion of macrophages or macrophage derived proteins in our scaffolds will effectively boost progenitor production. Disclosures No relevant conflicts of interest to declare.
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Easterbrook, Jennifer, Sabrina Gordon-Keylock, Stanislav Rybtsov, Andrejs Ivanovs, Richard Anderson, and Alexander Medvinsky. "Investigating haematopoiesis in the human embryo using an ex vivo culture system." Experimental Hematology 44, no. 9 (September 2016): S68. http://dx.doi.org/10.1016/j.exphem.2016.06.122.
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Hughes, Daniel Ll, Aron Hughes, Zahir Soonawalla, Somnath Mukherjee, and Eric O’Neill. "Dynamic Physiological Culture of Ex Vivo Human Tissue: A Systematic Review." Cancers 13, no. 12 (June 8, 2021): 2870. http://dx.doi.org/10.3390/cancers13122870.
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Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h–60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.
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Crowe, Suzanne M., Spyridon Kintzios, Grigoris Kaltsas, and Clovis S. Palmer. "A Bioelectronic System to Measure the Glycolytic Metabolism of Activated CD4+ T Cells." Biosensors 9, no. 1 (January 9, 2019): 10. http://dx.doi.org/10.3390/bios9010010.
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The evaluation of glucose metabolic activity in immune cells is becoming an increasingly standard task in immunological research. In this study, we described a sensitive, inexpensive, and non-radioactive assay for the direct and rapid measurement of the metabolic activity of CD4+ T cells in culture. A portable, custom-built Cell Culture Metabolite Biosensor device was designed to measure the levels of acidification (a proxy for glycolysis) in cell-free CD4+ T cell culture media. In this assay, ex vivo activated CD4+ T cells were incubated in culture medium and mini electrodes were placed inside the cell free culture filtrates in 96-well plates. Using this technique, the inhibitors of glycolysis were shown to suppress acidification of the cell culture media, a response similar to that observed using a gold standard lactate assay kit. Our findings show that this innovative biosensor technology has potential for applications in metabolic research, where acquisition of sufficient cellular material for ex vivo analyses presents a substantial challenge.
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Parrish, Elizabeth M., Anaar Siletz, Min Xu, Teresa K. Woodruff, and Lonnie D. Shea. "Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system." REPRODUCTION 142, no. 2 (August 2011): 309–18. http://dx.doi.org/10.1530/rep-10-0481.
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Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell–cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus–oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.
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Chen, Shih-Heng, Hui-Wen Yao, Wen-Yen Huang, Kuei-Sen Hsu, Huan-Yao Lei, Ai-Li Shiau, and Shun-Hua Chen. "Efficient Reactivation of Latent Herpes Simplex Virus from Mouse Central Nervous System Tissues." Journal of Virology 80, no. 24 (September 27, 2006): 12387–92. http://dx.doi.org/10.1128/jvi.01232-06.
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ABSTRACT For decades, numerous ex vivo studies have documented that latent herpes simplex virus (HSV) reactivates efficiently from ganglia, but rarely from the central nervous systems (CNS), of mice when assayed by mincing tissues before explant culture, despite the presence of viral genomes in both sites. Here we show that 88% of mouse brain stems reactivated latent virus when they were dissociated into cell suspensions before ex vivo explant culture. The efficient reactivation of HSV from the mouse CNS was demonstrated with more than one viral strain, viral serotype, and mouse strain, further indicating that the CNS can be an authentic latency site for HSV with the potential to cause recurrent disease.
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Shih, Chu-Chih, Mickey C. T. Hu, Jun Hu, Yehua Weng, Paul J. Yazaki, Jeffrey Medeiros, and Stephen J. Forman. "A secreted and LIF-mediated stromal cell–derived activity that promotes ex vivo expansion of human hematopoietic stem cells." Blood 95, no. 6 (March 15, 2000): 1957–66. http://dx.doi.org/10.1182/blood.v95.6.1957.
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Abstract The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion.
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Pan, Xiuwei, Qiong Sun, Haibo Cai, Yun Gao, Wensong Tan, and Weian Zhang. "Encapsulated feeder cells within alginate beads for ex vivo expansion of cord blood-derived CD34+ cells." Biomaterials Science 4, no. 10 (2016): 1441–53. http://dx.doi.org/10.1039/c6bm00191b.
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A co-culture system based on encapsulated feeder cells within alginate beads was developed through optimizing the detailed aspects of the cell culture system to expand CD34-positive (CD34+) cells ex vivo.
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Baydoun, Treizeibré, Follet, Vanneste, Creusy, Dercourt, Delaire, et al. "An Interphase Microfluidic Culture System for the Study of Ex Vivo Intestinal Tissue." Micromachines 11, no. 2 (January 30, 2020): 150. http://dx.doi.org/10.3390/mi11020150.
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Ex vivo explant culture models offer unique properties to study complex mechanisms underlying tissue growth, renewal, and disease. A major weakness is the short viability depending on the biopsy origin and preparation protocol. We describe an interphase microfluidic culture system to cultivate full thickness murine colon explants which keeps morphological structures of the tissue up to 192 h. The system was composed of a central well on top of a porous membrane supported by a microchannel structure. The microfluidic perfusion allowed bathing the serosal side while preventing immersion of the villi. After eight days, up to 33% of the samples displayed no histological abnormalities. Numerical simulation of the transport of oxygen and glucose provided technical solutions to improve the functionality of the microdevice.
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Schukur, Lina, Barbara Geering, and Martin Fussenegger. "Human whole-blood culture system for ex vivo characterization of designer-cell function." Biotechnology and Bioengineering 113, no. 3 (September 30, 2015): 588–97. http://dx.doi.org/10.1002/bit.25828.
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Jakubikova, Jana, Danka Cholujova, Richard W. Groen, Jungnam Joo, Sun-Young Kong, Teru Hideshima, Rikio Suzuki, et al. "Mimicking Myeloma Niche Ex Vivo." Blood 124, no. 21 (December 6, 2014): 2076. http://dx.doi.org/10.1182/blood.v124.21.2076.2076.
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Abstract Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p<0.001) in 3-D vs. 2-D MM-MSCs. Similarly, activation of integrins including VLA-2, VLA-4 and VLA-5 on the MSCs surface was also increased in 3-D MM-MSCs, as determined by confocal microscopy and flow cytometry analysis. Importantly, MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche. Gene expression analyses of 3-D MM-MSCs revealed upregulation of BMP-2, MGP, PTGIS, COL14A1 and other genes and down-regulation of DKK1, ADAM9, OPCML genes and others compared to 2-D MM-MSCs. We also measured significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p<0.001) in 3-D vs. 2-D MM-MSCs, by multiplex luminex analysis. Next, we co-cultured tumor cells from MM patients (12 MM patients) with either autologous or allogeneic MM-MSCs in 3-D vs. 2-D model. Plasma (CD38/CD138+) cells in 3-D co-culture were increased in 8/12 MM patients and equivalent to 2-D in 4/12 patients. By co-culturing MM cell lines (OPM1, RPMI-S, OCIMY5 and KMS11) labeled with CFSE fluorescent dye with various MSCs, we evaluated expression of side population (SP) cells, identified by Hoechst staining, and gating on CFSE positive MM cells, as low Hoechst stained cells. Our results showed that the SP fraction was significantly lower in 3-D compared to 2-D in co-cultures of various MM-MSCs with all 4 MM cell lines. Finally, we validated drug resistance to melphalan, bortezomib, lenalidomide, and carfilzomib in 3-D co-cultures of CFSE labeled primary tumor cells with various MM-MSCs. Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.
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Reiter, Chad E. N., Lakshman Sandirasegarane, Ellen B. Wolpert, Marianne Klinger, Ian A. Simpson, Alistair J. Barber, David A. Antonetti, Mark Kester, and Thomas W. Gardner. "Characterization of insulin signaling in rat retina in vivo and ex vivo." American Journal of Physiology-Endocrinology and Metabolism 285, no. 4 (October 2003): E763—E774. http://dx.doi.org/10.1152/ajpendo.00507.2002.
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Insulin receptor (IR) signaling cascades have been studied in many tissues, but retinal insulin action has received little attention. Retinal IR signaling and activity were investigated in vivo in rats that were freely fed, fasted, or injected with insulin by phosphotyrosine immunoblotting and by measuring kinase activity. A retina explant system was utilized to investigate the IR signaling cascade, and immunohistochemistry was used to determine which retinal cell layers respond to insulin. Basal IR activity in the retina was equivalent to that in brain and significantly greater than that of liver, and it remained constant between freely fed and fasted rats. Furthermore, IR signaling increased in the retina after portal vein administration of supraphysiological doses of insulin. Ex vivo retinas responded to 10 nM insulin with IR β-subunit (IRβ) and IR substrate-2 (IRS-2) tyrosine phosphorylation and AktSer473 phosphorylation. The retina expresses mRNA for all three Akt isoforms as determined by in situ hybridization, and insulin specifically increases Akt-1 kinase activity. Phospho-AktSer473 immunoreactivity increases in retinal nuclear cell layers with insulin treatment. These results demonstrate that the retinal IR signaling cascade to Akt-1 possesses constitutive activity, and that exogenous insulin further stimulates this prosurvival pathway. These findings may have implications in understanding normal and dysfunctional retinal physiology.
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Böhlen, Sebastian, Sebastian Konzok, Jennifer Labisch, Susann Dehmel, Dirk Schaudien, Stephan Behrens, Florian Schmieder, Armin Braun, Frank Sonntag, and Katherina Sewald. "Using a micro-physiological system to prolong the preservation of ex vivo lung tissue." Current Directions in Biomedical Engineering 7, no. 2 (October 1, 2021): 207–10. http://dx.doi.org/10.1515/cdbme-2021-2053.
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Abstract Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.
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Watanabe, Ryota, Aira Matsugaki, Takuya Ishimoto, Ryosuke Ozasa, Takuya Matsumoto, and Takayoshi Nakano. "A Novel Ex Vivo Bone Culture Model for Regulation of Collagen/Apatite Preferential Orientation by Mechanical Loading." International Journal of Molecular Sciences 23, no. 13 (July 4, 2022): 7423. http://dx.doi.org/10.3390/ijms23137423.
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The anisotropic microstructure of bone, composed of collagen fibers and biological apatite crystallites, is an important determinant of its mechanical properties. Recent studies have revealed that the preferential orientation of collagen/apatite composites is closely related to the direction and magnitude of in vivo principal stress. However, the mechanism of alteration in the collagen/apatite microstructure to adapt to the mechanical environment remains unclear. In this study, we established a novel ex vivo bone culture system using embryonic mouse femurs, which enabled artificial control of the mechanical environment. The mineralized femur length significantly increased following cultivation; uniaxial mechanical loading promoted chondrocyte hypertrophy in the growth plates of embryonic mouse femurs. Compressive mechanical loading using the ex vivo bone culture system induced a higher anisotropic microstructure than that observed in the unloaded femur. Osteocytes in the anisotropic bone microstructure were elongated and aligned along the long axis of the femur, which corresponded to the principal loading direction. The ex vivo uniaxial mechanical loading successfully induced the formation of an oriented collagen/apatite microstructure via osteocyte mechano-sensation in a manner quite similar to the in vivo environment.
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Cramer, E. E. A., K. Ito, and S. Hofmann. "Ex vivo Bone Models and Their Potential in Preclinical Evaluation." Current Osteoporosis Reports 19, no. 1 (January 11, 2021): 75–87. http://dx.doi.org/10.1007/s11914-020-00649-5.
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Abstract Purpose of Review Novel therapies for damaged and diseased bone are being developed in a preclinical testing process consisting of in vitro cell experiments followed by in vivo animal studies. The in vitro results are often not representative of the results observed in vivo. This could be caused by the complexity of the natural bone environment that is missing in vitro. Ex vivo bone explant cultures provide a model in which cells are preserved in their native three-dimensional environment. Herein, it is aimed to review the current status of bone explant culture models in relation to their potential in complementing the preclinical evaluation process with specific attention paid to the incorporation of mechanical loading within ex vivo culture systems. Recent Findings Bone explant cultures are often performed with physiologically less relevant bone, immature bone, and explants derived from rodents, which complicates translatability into clinical practice. Mature bone explants encounter difficulties with maintaining viability, especially in static culture. The integration of mechanical stimuli was able to extend the lifespan of explants and to induce new bone formation. Summary Bone explant cultures provide unique platforms for bone research and mechanical loading was demonstrated to be an important component in achieving osteogenesis ex vivo. However, more research is needed to establish a representative, reliable, and reproducible bone explant culture system that includes both components of bone remodeling, i.e., formation and resorption, in order to bridge the gap between in vitro and in vivo research in preclinical testing.
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Gao, Chunxu, Shannon Hitchcock, Heather Deutsch, Tadimeti Rao, Chidozie Amuzie, and Ravi Malaviya. "Development of a murine intestinal explant model to examine immune mechanisms of graft versus host disease." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 55.46. http://dx.doi.org/10.4049/jimmunol.200.supp.55.46.
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Abstract Graft-versus host disease (GvHD) is a major course of mortality and morbidity in transplant recipients. Cytokine signaling plays a key role in the development of GvHD. To study the immune mechanisms relevant to intestinal pathology associated with GvHD, we developed a murine ex vivo model on intestinal explants derived from Xeno-GvHD to characterize mouse and human cytokine secretion and signaling. Intestinal tissues from Xeno-GvHD induced by transfer of human PBMC to irradiated NSG mice and control naïve mice were dissected and cultured. Histological analysis of the explants and measurement of lactate dehydrogenase (LDH) in the ex vivo culture system indicate that intestinal explants remain viable in culture for up to 72 hours, with an increased cytotoxicity developing over this time. Release of mouse proinflammatory cytokines including RANTES, IL-6, KC, and GM-CSF were significantly higher in GvHD explants compared to naïve controls. Among the human cytokines, RANTES was a key cytokine detected only from Xeno-GvHD explants. The patterns of cytokine/chemokine release were shown to be consistent in the ex vivo culture over time and on explants from different sites of intestine including ileum and colon. Furthermore, histochemical analysis of GvHD explants revealed much stronger immunoreactivity for RANTES compared to naïve explants. Overall, our results indicate inflammatory milieu in the intestines of GvHD mice leading to sustained production of proinflammatory cytokines in the ex vivo culture. These features indicate that the explant system is a relevant model for studying the mechanism(s) of inflammatory responses in intestinal GvHD.
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Santamaria-Martínez, A., J. Epiney, D. Srivastava, D. Tavernari, D. Milowich, I. Letovanec, G. Ciriello, A. Cairoli, and E. Oricchio. "P1290: A TISSUE-BASED EX VIVO CULTURE SYSTEM TO ANTICIPATE TEHRAPY RESPONSE IN LYMPHOMA." HemaSphere 6 (June 2022): 1175–76. http://dx.doi.org/10.1097/01.hs9.0000848024.58707.55.
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Tunc-Ozcan, Elif, Adriana B. Ferreira, and Eva E. Redei. "Modeling Fetal Alcohol Spectrum Disorder: Validating an Ex Vivo Primary Hippocampal Cell Culture System." Alcoholism: Clinical and Experimental Research 40, no. 6 (May 10, 2016): 1273–82. http://dx.doi.org/10.1111/acer.13090.
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Kim, Se Eun, Kyung Mi Shim, and Seong Soo Kang. "Evaluation of short-term ex vivo culture with air/liquid system of porcine cornea." Journal of Biomedical Research 16, no. 3 (September 2015): 93–97. http://dx.doi.org/10.12729/jbr.2015.16.3.093.
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Fischer, Stephen E., Yiwei Ma, Caitlin Smith, Anirudhasingh Sodha, and Yukang Zhao. "Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells." Blood 120, no. 21 (November 16, 2012): 4411. http://dx.doi.org/10.1182/blood.v120.21.4411.4411.
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Abstract Abstract 4411 Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo. In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation. Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures: No relevant conflicts of interest to declare.
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Maffei, Sara, Giovanna Galeati, Georgia Pennarossa, Tiziana A. L. Brevini, and Fulvio Gandolfi. "Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries." Reproduction, Fertility and Development 28, no. 12 (2016): 1893. http://dx.doi.org/10.1071/rd15101.
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We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n = 6), not perfused and fixed; Groups SM72 and SM72-FSH (n = 6 each), perfused with a simple medium for 72 h with or without FSH; Groups CM96 and CM96-FSH (n = 6 each), perfused with a complex medium for 96 h with or without FSH; Group CM96-FSH-cryo, (n = 3) cryopreserved and perfused for 96 h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2 mm and then for these follicles to reach the ovulatory size of 4 mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.
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Wick, Nikolaus, Pipsa Saharinen, Juha Saharinen, Elisabeth Gurnhofer, Carl W. Steiner, Ingrid Raab, Dejan Stokic, et al. "Transcriptomal comparison of human dermal lymphatic endothelial cells ex vivo and in vitro." Physiological Genomics 28, no. 2 (January 2007): 179–92. http://dx.doi.org/10.1152/physiolgenomics.00037.2006.
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The in vivo functions of lymphatic endothelial cells depend on their microenvironment, which cannot be fully reproduced in vitro. Because of technical limitations, gene expression in uncultured, “ex vivo” lymphatic endothelial cells has not been characterized at the molecular level. We combined tissue micropreparation and direct cell isolation with DNA chip experiments to identify 159 genes differentiating human lymphatic endothelial cells from blood vascular endothelial cells ex vivo. The same analysis performed with cultured primary cells revealed that only 19 genes characteristic for lymphatic endothelium ex vivo retained this property upon culture, while 27 marker genes were newly induced. In addition, a set of panendothelial genes could be recognized. The propagation of lymphatic endothelial cells in culture stimulated transcription of genes associated with cell turnover, basic metabolism, and the cytoskeleton. On the other hand, there was downregulation of genes encoding extracellular matrix components, signaling via transmembrane tyrosine kinase pathways and the chemokine (C-C) ligand 21. Direct ex vivo analysis of the lymphatic endothelial cell transcriptome is helpful for the understanding of the physiology of the lymphatic vascular system and of the pathogenesis of its diseases.
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Waldschmidt, Johannes M., Stefan J. Fruttiger, Dagmar Wider, Johannes Jung, Andreas R. Thomsen, Tanja N. Hartmann, Justus Duyster, et al. "Ex vivo propagation in a novel 3D high-throughput co-culture system for multiple myeloma." Journal of Cancer Research and Clinical Oncology 148, no. 5 (January 24, 2022): 1045–55. http://dx.doi.org/10.1007/s00432-021-03854-6.
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Abstract Purpose Multiple myeloma (MM) remains an incurable hematologic malignancy which ultimately develops drug resistance and evades treatment. Despite substantial therapeutic advances over the past years, the clinical failure rate of preclinically promising anti-MM drugs remains substantial. More realistic in vitro models are thus required to better predict clinical efficacy of a preclinically active compound. Methods Here, we report on the establishment of a conical agarose 3D co-culture platform for the preclinical propagation of primary MM cells ex vivo. Cell growth was compared to yet established 2D and liquid overlay systems. MM cell lines (MMCL: RPMI-8226, U266, OPM-2) and primary patient specimens were tested. Drug sensitivity was examined by exploring the cytotoxic effect of bortezomib and the deubiquitinase inhibitor auranofin under various conditions. Results In contrast to 2D and liquid overlay, cell proliferation in the 3D array followed a sigmoidal curve characterized by an initial growth delay but more durable proliferation of MMCL over 12 days of culture. Primary MM specimens did not expand in ex vivo monoculture, but required co-culture support by a human stromal cell line (HS-5, MSP-1). HS-5 induced a > fivefold increase in cluster volume and maintained long-term viability of primary MM cells for up to 21 days. Bortezomib and auranofin induced less cytotoxicity under 3D vs. 2D condition and in co- vs. monoculture, respectively. Conclusions This study introduces a novel model that is capable of long-term propagation and drug testing of primary MM specimens ex vivo overcoming some of the pitfalls of currently available in vitro models.
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Nakamura, Yoshihiko, Kiyoshi Ando, Jamel Chargui, Hiroshi Kawada, Tadayuki Sato, Takashi Tsuji, Tomomitsu Hotta, and Shunichi Kato. "Ex Vivo Generation of CD34+ Cells From CD34− Hematopoietic Cells." Blood 94, no. 12 (December 15, 1999): 4053–59. http://dx.doi.org/10.1182/blood.v94.12.4053.
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Abstract The human Lin−CD34− cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin−CD34− cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin−CD34− cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34+ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34+ cells in the human CD34− cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin−CD34− hematopoietic stem cells.
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Nakamura, Yoshihiko, Kiyoshi Ando, Jamel Chargui, Hiroshi Kawada, Tadayuki Sato, Takashi Tsuji, Tomomitsu Hotta, and Shunichi Kato. "Ex Vivo Generation of CD34+ Cells From CD34− Hematopoietic Cells." Blood 94, no. 12 (December 15, 1999): 4053–59. http://dx.doi.org/10.1182/blood.v94.12.4053.424k41_4053_4059.
Full textAbstract:
The human Lin−CD34− cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin−CD34− cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin−CD34− cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34+ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34+ cells in the human CD34− cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin−CD34− hematopoietic stem cells.
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Liu, Yan Qiu, and Nan Huang. "Stability Assessment of Minor-Caliber Vessel Perfusion System for Ex Vivo." Applied Mechanics and Materials 631-632 (September 2014): 594–97. http://dx.doi.org/10.4028/www.scientific.net/amm.631-632.594.
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Biggish animal models were applied in perfusion system for ex vivo research. But the disadvantages is the needs for more research funding and better surgical skills to perform which many researchers lack. A minor-caliber vessel perfusion system for ex vivo was designed and constructed to simulate pulsatile situation in vivo. The parameters mimicked the physiological measures of New Zealand rabbit. Pulse is 250 beats per minute. Diastolic blood pressure is 12.666KPa, and systolic blood pressure is 14.665KPa. The vessel perfusion system can regulate pulse pressure, the state of laminar flow, and shear stress. To culture several vessels simultaneously for control, the pressures data of 4 baths was analyzed with one way analysis of variance. The results show that the perfusion system is stable, and pressure difference of 4 baths is not significant.
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Ong, Li Ming, Xiubo Fan, Pak Yan Chu, Florence Gay, Justina Ang, Zhihong Li, Jianzhu Chen, SaiKiang Lim, Ralph Bunte, and William Hwang. "Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice." Blood 116, no. 21 (November 19, 2010): 3722. http://dx.doi.org/10.1182/blood.v116.21.3722.3722.
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Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.
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Labadie, R. F., J. F. Antaki, J. L. Williams, S. Katyal, J. Ligush, S. C. Watkins, S. M. Pham, and H. S. Borovetz. "Pulsatile perfusion system for ex vivo investigation of biochemical pathways in intact vascular tissue." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 2 (February 1, 1996): H760—H768. http://dx.doi.org/10.1152/ajpheart.1996.270.2.h760.
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We have constructed and performed initial validation of an innovative perfusion system that allows exposure of intact segments of vascular tissue to realistic physiological and hemodynamic environments ex vivo. Computer-controlled opening and closing of an in-line gate valve allows generation of arterial pressure waveforms. The control algorithm predicted resultant pressure waveforms with a high degree of accuracy (Pearson correlation coefficient > 0.97). To document vascular homeostasis ex vivo, vasomotor bioassays and morphological studies were performed. The bioassays consisted of injecting epinephrine (2 x 10(-3) mg/ml) into the perfusion system followed by acetylcholine (100 microM) while concurrently measuring vessel diameter with a laser micrometer, significant vasomotion was measured for canine carotid arteries (n = 4) bioassayed after 1, 24, and 48 h of perfusion (P < 0.03). Additionally, human saphenous vein segments were perfused for 24 h (n = 4) and viewed with laser confocal scanning microscopy and transmission electron microscopy; photomicrographs show typical vascular morphology. We conclude that the vascular perfusion system described herein is well suited for investigating the response of intact vascular tissue to hemodynamic variables.
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Jaroscak, Jennifer, Kristin Goltry, Alan Smith, Barbara Waters-Pick, Paul L. Martin, Timothy A. Driscoll, Richard Howrey, et al. "Augmentation of umbilical cord blood (UCB) transplantation with ex vivo–expanded UCB cells: results of a phase 1 trial using the AastromReplicell System." Blood 101, no. 12 (June 15, 2003): 5061–67. http://dx.doi.org/10.1182/blood-2001-12-0290.
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AbstractAllogeneic stem cell transplantation with umbilical cord blood (UCB) cells is limited by the cell dose a single unit provides recipients. Ex vivo expansion is one strategy to increase the number of cells available for transplantation. Aastrom Biosciences developed an automated continuous perfusion culture device for expansion of hematopoietic stem cells (HSCs). Cells are expanded in media supplemented with fetal bovine serum, horse serum, PIXY321, flt-3 ligand, and erythropoietin. We performed a phase 1 trial augmenting conventional UCB transplants with ex vivo–expanded cells. The 28 patients were enrolled on the trial between October 8, 1997 and September 30, 1998. UCB cells were expanded in the device, then administered as a boost to the conventional graft on posttransplantation day 12. While expansion of total cells and colony-forming units (CFUs) occurred in all cases, the magnitude of expansion varied considerably. The median fold increase was 2.4 (range, 1.0-8.5) in nucleated cells, 82 (range, 4.6-266.4) in CFU granulocyte-macrophages, and 0.5 (range, 0.09-2.45) in CD34+ lineage negative (lin–) cells. CD3+ cells did not expand under these conditions. Clinical-scale ex vivo expansion of UCB is feasible, and the administration of ex vivo–expanded cells is well tolerated. Augmentation of UCB transplants with ex vivo–expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients. Recipients of ex vivo–expanded cells continue to have durable engraftment with a median follow-up of 47 months (range, 41-51 months). A randomized phase 2 study will determine whether augmenting UCB transplants with ex vivo–expanded UCB cells is beneficial.
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Siminski, J. T., T. J. Kavanagh, E. Chi, and G. Raghu. "Long-term maintenance of mature pulmonary parenchyma cultured in serum-free conditions." American Journal of Physiology-Lung Cellular and Molecular Physiology 262, no. 1 (January 1, 1992): L105—L110. http://dx.doi.org/10.1152/ajplung.1992.262.1.l105.
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Current concepts of the pathogenesis of lung injury and repair are derived from in vitro cellular and in vivo investigations. Studies with viable ex vivo models may offer additional insights into disease processes, since essential cellular interactions would be maintained. However, a major limiting factor has been the availability of a model that maintains normal parenchymal structure, viability, and homeostasis beyond 4 wk in serum-free conditions. We have succeeded in establishing an ex vivo lung culture system which reproducibly maintains parenchymal architecture for up to 9 wk. Our method is a simple, modified version of previously utilized techniques. Thin slices of mature murine lung were inflated with agar-defined medium and cultured on Gelfoam saturated with serum-free medium. Normal pulmonary parenchyma, with the exception of endothelial cells, was maintained for up to 60 days as assessed chronologically by light and electron microscopy. The integrity of the microvasculature and endothelial cells was lost beyond 7 days. The adult lung ex vivo culture system maintained necessary epithelial and interstitial cellular interactions in the alveolar wall without systemic circulatory influences. Future studies with this model may provide important insights in assessing the pathogenesis of many acute and chronic lung diseases and clarify existing controversies raised from in vitro and in vivo studies.
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Takebe, Naoko, Thomas MacVittie, Xiangfei Cheng, Ann M. Farese, Emily Welty, and Barry Meisenberg. "Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System." Blood 106, no. 11 (November 16, 2005): 1068. http://dx.doi.org/10.1182/blood.v106.11.1068.1068.
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Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.
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Han, Jin-Yeong, Rhee-Young Koh, Su-Yeong Seo, Joo-In Park, Hyuk-Chan Kwon, Jae-Seok Kim, Young-Ho Lee, and Hyo-Jin Kim. "Co-Transplantation of Hematopoietic Stem Cells and GM-CSF/G-CSF/CSF-Transfected Mesenchymal Stem Cells in SCID Mice." Blood 104, no. 11 (November 16, 2004): 4950. http://dx.doi.org/10.1182/blood.v104.11.4950.4950.
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Abstract Mesenchymal stem cells (MSC) are multipotent and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and recently even in human trials. In this study, we transfected culture-expanded MSC with GM-CSF, G-CSF, and CSF cytokine genes and then co-transplanted with HSC to further promote HSC engraftment. Mononuclear cells were harvested from the various sources and seeded in long-term culture for ex vivo MSC expansion. The phenotype and purity of MSC were assessed by flow cytometry. We transferred the above three cytokine genes into ex vivo expanded MSC, confirmed transfection by fluorescent microscope of GFP, and thereafter did co-transplantation with HSC. A total of 1x107 HSC plus MSC/uL were introduced to tail vein of SCID mice. After 3–7 weeks later, with venous blood from the eyeballs, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization (FISH) studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX/XY-positive human cells significantly increased in co-transplanted mice and even higher with the cytokine gene-transfected MSC in the order of GM-CSF, SCF, and G-CSF transfections (P<0.05). These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Now we are planning to deliver genes involved in homing and cell adhesions, e.g., CXCR4, VLA, or TPO into ex vivo expanded MSC and do co-transplantation with HSC to further increase the efficiency of stem cell transplantation.
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Frayne, Jan, Deborah E. Daniels, Jan Frayne, Timothy J. Satchwell, Joseph Hawksworth, Belinda K. Singleton, Tatyana N. Andrienko, et al. "Ex vivo Engineering of Red Blood Cells." Blood 134, Supplement_1 (November 13, 2019): SCI—5—SCI—5. http://dx.doi.org/10.1182/blood-2019-121117.
Full textAbstract:
Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with >105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of >12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway. Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin. We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations. As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring: Diagnostic and 'Universal' transfusion products: Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3. Cytokine independent lines Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines. Model disease systems In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806 Disclosures No relevant conflicts of interest to declare.
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Matsunaga, Takuya, Ikuta Tanaka, Masayoshi Kobune, Yutaka Kawano, Maki Tanaka, Takehide Akiyama, Kageaki Kuribayashi, Takafumi Ninomiya, Hirofumi Hamada, and Yoshiro Niitsu. "Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells." Blood 106, no. 11 (November 16, 2005): 1892. http://dx.doi.org/10.1182/blood.v106.11.1892.1892.
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Abstract To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 106 CD34+ cells) was calculated to be 10.5 units (2 x 1011 PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.