Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

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In vitro Models of the Small Intestine for Studying Intestinal Diseases

Frontiers in Microbiology ◽

10.3389/fmicb.2021.767038 ◽

2022 ◽

Vol 12 ◽

Author(s):

Sang-Myung Jung ◽

Seonghun Kim

Keyword(s):

Small Intestine ◽

State Of The Art ◽

Ex Vivo ◽

3D Culture ◽

In Vitro Models ◽

Cellular Functions ◽

Current State ◽

Culture Models ◽

Composition Structure

The small intestine is a digestive organ that has a complex and dynamic ecosystem, which is vulnerable to the risk of pathogen infections and disorders or imbalances. Many studies have focused attention on intestinal mechanisms, such as host–microbiome interactions and pathways, which are associated with its healthy and diseased conditions. This review highlights the intestine models currently used for simulating such normal and diseased states. We introduce the typical models used to simulate the intestine along with its cell composition, structure, cellular functions, and external environment and review the current state of the art for in vitro cell-based models of the small intestine system to replace animal models, including ex vivo, 2D culture, organoid, lab-on-a-chip, and 3D culture models. These models are described in terms of their structure, composition, and co-culture availability with microbiomes. Furthermore, we discuss the potential application for the aforementioned techniques to these in vitro models. The review concludes with a summary of intestine models from the viewpoint of current techniques as well as their main features, highlighting potential future developments and applications.

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3D culture technologies of cancer stem cells: promising ex vivo tumor models

Journal of Tissue Engineering ◽

10.1177/2041731420933407 ◽

2020 ◽

Vol 11 ◽

pp. 204173142093340 ◽

Cited By ~ 2

Author(s):

Chengye Zhang ◽

Zhaoting Yang ◽

Da-Long Dong ◽

Tae-Su Jang ◽

Jonathan C. Knowles ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Ex Vivo ◽

Three Dimensional ◽

3D Culture ◽

Cell Matrix ◽

Culture Models ◽

Three Dimensional Culture ◽

Three Dimensional Models

Cancer stem cells have been shown to be important in tumorigenesis processes, such as tumor growth, metastasis, and recurrence. As such, many three-dimensional models have been developed to establish an ex vivo microenvironment that cancer stem cells experience under in vivo conditions. Cancer stem cells propagating in three-dimensional culture systems show physiologically related signaling pathway profiles, gene expression, cell–matrix and cell–cell interactions, and drug resistance that reflect at least some of the tumor properties seen in vivo. Herein, we discussed the presently available Cancer stem cell three-dimensional culture models that use biomaterials and engineering tools and the biological implications of these models compared to the conventional ones.

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Optophoresis Accurately Predicts Response to Chemotherapy in CLL.

Blood ◽

10.1182/blood.v104.11.4834.4834 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4834-4834

Author(s):

Patricia McNeeley ◽

Alan Saven ◽

Ilona Kariv ◽

Jorge Nieva ◽

Philippe Marchand ◽

...

Keyword(s):

Near Infrared ◽

Ex Vivo ◽

Chemotherapeutic Agents ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Functional Responses ◽

Chemotherapy Administration ◽

Response To Chemotherapy ◽

Fluorescent Tags

Abstract In vitro drug resistance assays have shown efficacy in predicting response to chemotherapeutic drugs in a number of solid tumors. In chronic lymphocytic leukemia (CLL) many assays are limited by the difficulty involved in culturing these cells ex vivo. We have developed a novel, optical-based methodology that is sensitive to broad cellular physical characteristics, such as morphology, size, refractive index, density and surface properties. This measurement, known as Optophoresis, quantifies cell motion induced by exposure to a moving optical gradient, generated from a near-infrared laser beam (Forster AH, et al. Anal Biochem 2004, 327(1):14–22 and Wang MM, et al. Appl Opt 2003, 42(28):5765–73). In Optophoresis small numbers of cells are analyzed intact, in their native state. No labels are required for quantification of functional responses, but cell subpopulations may be identified using fluorescent tags. We have used this assay to predict response to chemotherapeutic agents in patients with CLL. Methods: We performed Optophoresis with six drugs (fludarabine, chlorambucil, vincristine, cyclophosphamide, cladribine and prednisolone) on 74 CLL patient samples. 68 of these were classic B-CLL; there was one hairy cell variant, two were T-CLL, two evolved to PLL and one evolved to lymphoma. 21 of the samples were from patients for whom clinical data on response to chemotherapy was available for 33 drug treatments. Response to therapy was defined as a decrease in RAI stage. Results: 21 patients aged 58 to 93 years; 71% Rai stage III or IV were included in this analysis. Six patients were evaluated prospectively, with the assay performed before chemotherapy administration, and 15 patients were evaluated retrospectively with the assay performed after a course of treatment. Optophoresis accurately predicted response to chemotherapy in 88% of the 33 treatments evaluated. In the four instances for which the Optophoresis results did not match the retrospective clinical outcome; the patient was historically sensitive to treatment and Optophoresis results indicated current resistance to the drug. Subsequent resistance to drug therapy is known to occur in a significant percentage of treated CLL patients. Conclusion: Optophoresis of CLL cells accurately predicts response to chemotherapy in CLL. Further studies using results of Optophoresis to guide CLL treatment are warranted.

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Technical Note: Design of a Flow Perfusion Bioreactor System for Bone Tissue-Engineering Applications

Tissue Engineering ◽

10.1089/107632703322066723 ◽

2003 ◽

Vol 9 (3) ◽

pp. 549-554 ◽

Cited By ~ 263

Author(s):

Gregory N. Bancroft ◽

Vassilios I. Sikavitsas ◽

Antonios G. Mikos

Keyword(s):

Tissue Engineering ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Technical Note ◽

Perfusion Bioreactor ◽

Engineering Applications ◽

Flow Perfusion ◽

Bioreactor System ◽

Flow Perfusion Bioreactor

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Flow-perfusion bioreactor system for engineered breast cancer surrogates to be used in preclinical testing

Journal of Tissue Engineering and Regenerative Medicine ◽

10.1002/term.2026 ◽

2015 ◽

Vol 11 (4) ◽

pp. 1242-1250 ◽

Cited By ~ 7

Author(s):

Lauren E. Marshall ◽

Kayla F. Goliwas ◽

Lindsay M. Miller ◽

Andrew D. Penman ◽

Andra R. Frost ◽

...

Keyword(s):

Breast Cancer ◽

Perfusion Bioreactor ◽

Preclinical Testing ◽

Flow Perfusion ◽

Bioreactor System ◽

Flow Perfusion Bioreactor

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Targeted Treatments: Pre-Clinical Evaluation Of Efficacy By Tracking Clonal Diversity In CLL Xenograft Models

Blood ◽

10.1182/blood.v122.21.875.875 ◽

2013 ◽

Vol 122 (21) ◽

pp. 875-875

Author(s):

Nicholas J Davies ◽

Ceri E Oldreive ◽

Angelo Agathanggelou ◽

Clive Gould ◽

Guy Pratt ◽

...

Keyword(s):

Conflicts Of Interest ◽

Ex Vivo ◽

Clonal Evolution ◽

Xenograft Model ◽

Response To Therapy ◽

Response To Treatment ◽

Proliferating Cells ◽

Targeted Treatments ◽

Xenograft Models

Abstract Progressive CLL with DNA damage response defects caused by ATM or TP53 inactivation is still a therapeutic problem. There is currently a growing appreciation of the subclonal complexity in this type of leukaemia, mostly supported by whole genome analyses of CLL samples such as SNP arrays and next generation sequencing, both highlighting the role of subclonal variations in progression and therapeutic response. Although revealing, these analyses do not inform about the clonal make-up of individual proliferating cells and such information might be necessary to determine effectiveness of novel targeted treatments. In order to determine and follow the clonal evolutionary millieu in individual CLL cases we developed multi-colour fluorescence in situ hybridization (MCF) to analyse CLL samples at the single cell level. We screened 134 samples for the presence of 11q and 17p deletions of which 25 were identified with one of these cytogenetic defects and these were screened alongside 28 samples with normal 17p and 11q loci for 13q and 6q deletion as well as trisomy 12. We found that all but four of the samples with 11q or 17p deletions had at least one other genomic abnormality, whilst only two of the 28 samples with normal 17p and 11q loci harboured two genomic abnormalities. We subsequently performed MCF on the 27 samples with multiple genetic abnormalities and generated evolutionary trees for each of the samples. Two types of clonal evolution were identified: linear and branched, with the latter being the more common. We were able to analyse eleven of these samples post-treatment and found that whilst the clonality of some samples was largely unaffected by treatment, others showed treatment-induced differences in the subclonal make-up. Furthermore, some samples also exhibited signs of evolution with the generation of novel subclones upon treatment. Pre-clinical testing of novel therapeutic agents in xenograft models requires that the subclonal architecture of engrafted samples is representative of the donor sample. To this end, modifications were made to two primary CLL xenograft models. Firstly, samples from three different CLLs with a complex karyotype and multiple subclones were engrafted with autologous ex-vivo stimulated T-cells into NOG mice. All CLL subclones displayed a capacity to engraft and proliferate in this xenograft model. Furthermore, the MCF protocol was implemented to assess subclonality in vivo. Mice were randomised to Rituximab or control saline treatment three times over a period of five days. Analysis of engrafted cells in the spleen a week later displayed a good response to Rituximab with a significantly lower number of hCD45+ CD19+ CD5+cells. However, upon isolation of CLL cells by FACS and assessment of clonal architecture using MCF it was shown that Rituximab treatment affected all subclones with a differential subclonal response. This resulted in a greater proportion of the CLL subclones with the greatest genetic complexity harbouring both 11q and 6q deletions still remaining. Finally, to recapitulate patient response to therapy, T-cell depleted, pre-treatment PBMC from a patient poorly responsive to bendamustine + Rituximab were engrafted in humanised mice. As in the patient, bi-weekly therapy for 3 weeks resulted in a limited, poor response to therapy with a corresponding small reduction of CFSE-labelled CLL PBMC and hCD45+ CD19+ cells. We conclude that the majority of CLLs display a branched pattern of evolution and that the subclone dynamic is an important determinant of CLL proliferation and response to treatment. We suggest that novel targeted therapies should be tested in the context of their ability to eradicate CLL subclones with the highest proliferative capacity, as these subclones are most likely to evolve. The development of an MCF protocol, in combination with the xenograft model, provides a powerful tool to help predict overall and subclonal responses to therapy in the patient. Disclosures: No relevant conflicts of interest to declare.

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Traditional and Advanced Cell Cultures in Hematopoietic Stem Cell Studies

Cells ◽

10.3390/cells8121628 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1628 ◽

Cited By ~ 2

Author(s):

Antonio Carlos Ribeiro-Filho ◽

Débora Levy ◽

Jorge Luis Maria Ruiz ◽

Marluce da Cunha Mantovani ◽

Sérgio Paulo Bydlowski

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Ex Vivo ◽

3D Culture ◽

Bone Marrow Microenvironment ◽

Main Function ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Culture Models

Hematopoiesis is the main function of bone marrow. Human hematopoietic stem and progenitor cells reside in the bone marrow microenvironment, making it a hotspot for the development of hematopoietic diseases. Numerous alterations that correspond to disease progression have been identified in the bone marrow stem cell niche. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells determine the balance between the proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation. However, our understanding of human hematopoiesis and the associated niche biology remains limited due to accessibility to human material and the limits of in vitro culture models. Traditional culture systems for human hematopoietic studies lack microenvironment niches, spatial marrow gradients, and dense cellularity, rendering them incapable of effectively translating marrow physiology ex vivo. This review will discuss the importance of 2D and 3D culture as a physiologically relevant system for understanding normal and abnormal hematopoiesis.

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Tracking Photodynamic- and Chemotherapy-Induced Redox-State Perturbations in 3D Culture Models of Pancreatic Cancer: A Tool for Identifying Therapy-Induced Metabolic Changes

Journal of Clinical Medicine ◽

10.3390/jcm8091399 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1399 ◽

Cited By ~ 4

Author(s):

Mans Broekgaarden ◽

Anne-Laure Bulin ◽

Jane Frederick ◽

Zhiming Mai ◽

Tayyaba Hasan

Keyword(s):

Pancreatic Cancer ◽

Photodynamic Therapy ◽

High Throughput ◽

Redox State ◽

Cancer Metabolism ◽

Ex Vivo ◽

3D Culture ◽

High Throughput Analysis ◽

Orthotopic Mouse Model ◽

Culture Models

The metabolic plasticity of cancer cells is considered a highly advantageous phenotype that is crucial for disease progression and acquisition of treatment resistance. A better understanding of cancer metabolism and its adaptability after treatments is vital to develop more effective therapies. To screen novel therapies and combination regimens, three-dimensional (3D) culture models of cancers are attractive platforms as they recapitulate key features of cancer. By applying non-perturbative intensity-based redox imaging combined with high-throughput image analysis, we demonstrated metabolic heterogeneity in various 3D culture models of pancreatic cancer. Photodynamic therapy and oxaliplatin chemotherapy, two cancer treatments with relevance to pancreatic cancer, induced perturbations in redox state in 3D microtumor cultures of pancreatic cancer. In an orthotopic mouse model of pancreatic cancer, a similar disruption in redox homeostasis was observed on ex vivo slices following photodynamic therapy in vivo. Taken together, redox imaging on cancer tissues combined with high-throughput analysis can elucidate dynamic spatiotemporal changes in metabolism following treatment, which will benefit the design of new metabolism-targeted therapeutic approaches.

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