Design of a Microscopy System for Quantitative Spatial and Temporal Analysis of Multicellular Interactions

The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture.Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.

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Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-020-00537-3 ◽

2021 ◽

Author(s):

Terry Riss ◽

O. Joseph Trask

Keyword(s):

Cell Culture ◽

Three Dimensional ◽

3D Culture ◽

Fluorescent Imaging ◽

Culture Models ◽

Assay Methods ◽

Diffusion Rates ◽

Cell Culture Models ◽

Dimensional Cell ◽

Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

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What Have In Vitro Co-Culture Models Taught Us about the Contribution of Epithelial-Mesenchymal Interactions to Airway Inflammation and Remodeling in Asthma?

Cells ◽

10.3390/cells9071694 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1694

Author(s):

Emmanuel Twumasi Osei ◽

Steven Booth ◽

Tillie-Louise Hackett

Keyword(s):

Extracellular Matrix ◽

Cell Proliferation ◽

Airway Inflammation ◽

Airway Epithelial ◽

Disease Pathogenesis ◽

3 Dimensional ◽

Lung Epithelial ◽

Culture Models

As the lung develops, epithelial-mesenchymal crosstalk is essential for the developmental processes that drive cell proliferation, differentiation, and extracellular matrix (ECM) production within the lung epithelial-mesenchymal trophic unit (EMTU). In asthma, a number of the lung EMTU developmental signals have been associated with airway inflammation and remodeling, which has led to the hypothesis that aberrant activation of the asthmatic EMTU may lead to disease pathogenesis. Monoculture studies have aided in the understanding of the altered phenotype of airway epithelial and mesenchymal cells and their contribution to the pathogenesis of asthma. However, 3-dimensional (3D) co-culture models are needed to enable the study of epithelial-mesenchymal crosstalk in the setting of the in vivo environment. In this review, we summarize studies using 3D co-culture models to assess how defective epithelial-mesenchymal communication contributes to chronic airway inflammation and remodeling within the asthmatic EMTU.

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Combining galiximab with the chemotherapeutic agents fludarabine or doxorubicin improves efficacy in animal models of lymphoma

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.3040 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 3040-3040 ◽

Cited By ~ 5

Author(s):

H. K. Hariharan ◽

T. Murphy ◽

D. Clanton ◽

L. Berquist ◽

P. Chu ◽

...

Keyword(s):

Clinical Trials ◽

Animal Models ◽

Clinical Benefit ◽

Therapeutic Potential ◽

Xenograft Model ◽

Chemotherapeutic Agents ◽

Model Systems ◽

Anti Cd20

3040 Background: Galiximab, a primatized monoclonal antibody that binds with high affinity to CD80 and mediates antibody- dependent, cell-mediated cytotoxicity in vitro, is currently under investigation for the treatment of follicular non-Hodgkin’s lymphoma (NHL). In a phase I/II monotherapy study, galiximab produced an overall response rate of 11%, and tumor reductions were observed in 46% of patients. Initial clinical trials also demonstrate that galiximab is well tolerated and suggest that combining galiximab with rituximab (anti-CD20) provides clinical benefit. These results are consistent with preclinical studies in murine lymphoma xenograft model systems, which demonstrate the superiority of combination therapy. Methods: To further define the therapeutic potential of galiximab, the Raji subcutaneous and the SKW disseminated lymphoma murine xenograft models were used to define the in vivo efficacy of galiximab alone or in combination with fludarabine or doxorubicin. Similar studies were performed with rituximab. Results: In the Raji model, both galiximab and rituximab exhibited maximal inhibition of the growth of preestablished (150-mg) tumors at a dose of 3 mg/kg/wk. Interestingly, higher doses of galiximab (but not rituximab) showed reduced inhibition. Galiximab (3 mg/kg/wk) inhibited tumor growth alone (P<0.0001 vs. control) and showed significantly enhanced activity when combined with fludarabine (50 or 100 mg/kg daily for 5 days; P<0.0002 vs. galiximab alone and P<0.003 vs. fludarabine alone). Similar results were observed with rituximab. In the SKW model, treatment with galiximab (5 mg/kg/wk for 6 doses) significantly enhanced survival compared with a control (P<0.0001) or doxorubicin (2.5 mg/kg/day for 3 doses; P<0.0001). Studies combining fludarabine or doxorubicin with both galiximab and rituximab are ongoing. Conclusions: Studies in animal models of lymphoma indicate that galiximab may provide clinical benefit when used in combination with chemotherapeutic agents such as fludarabine and doxorubicin, and provide a rationale for the investigation of these novel chemoimmunotherapy combinations in clinical trials. No significant financial relationships to disclose.

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Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

10.1101/2021.11.30.470581 ◽

2021 ◽

Author(s):

Abderhman Abuhashem ◽

Anna-Katerina Hadjantonakis

Keyword(s):

Protein Degradation ◽

Protein Function ◽

Mammalian Cell Culture ◽

Elongation Factor ◽

Model Systems ◽

Mammalian Development ◽

Endogenous Protein ◽

Culture Models ◽

Genome Activation

Targeted protein degradation methods offer a unique avenue to assess a protein's function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBPF36V tag fused to Negative elongation factor b (Nelfb) locus, a ubiquitously expressed protein regulator of transcription. In the first validation of targeted endogenous protein degradation across mammalian development, we demonstrate that irrespective of the route of administration the dTAG system is safe, rapid, and efficient in embryos from the zygote to midgestation stages. Additionally, acute early depletion of NELFB revealed a specific role in zygote-to-2-cell development and Zygotic Genome Activation (ZGA).

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Tissue Engineering of Autologous Cartilage Transplants for Rhinology

American Journal of Rhinology ◽

10.2500/105065898782102972 ◽

1998 ◽

Vol 12 (1) ◽

pp. 59-64 ◽

Cited By ~ 41

Author(s):

A. Naumann ◽

N. Rotter ◽

J. Bujía ◽

J. Aigner

Keyword(s):

Tissue Engineering ◽

Reconstructive Surgery ◽

3 Dimensional ◽

Combined Use ◽

Extracellular Matrix Molecules ◽

Immunohistochemical Methods ◽

Increasing Demand ◽

Cell Carriers ◽

Dimensional Cell

In reconstructive surgery there is increasing demand for cartilage transplants to fill defects, especially nose and/or outer ear defects. Tissue engineering is one of the most modern pathways to generate autologous cartilage transplants. Isolated chondrocytes obtained from a tiny patient's biopsy were seeded on bioresorbable preshaped cell carriers to provide a 3-dimensional cell arrangement as in vivo. The combined use of these cell carriers in form of a non-woven mesh and a constant medium perfusion was performed to generate a cartilage-like cell-polymer-construct, which was finally subcutanously implanted in nude mice for full maturation. After explantation of 6 months, expression of cartilage specific extracellular matrix molecules was obvious by using histochemical and immunohistochemical methods. These data show that tissue engineering with isolated multiplied human chondrocytes from a tiny biopsy seeded on bioresorbable polymer is a promising system to generate autologous cartilage transplants for replacements in reconstructive surgery.

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Advances in Modeling the Immune Microenvironment of Colorectal Cancer

Frontiers in Immunology ◽

10.3389/fimmu.2020.614300 ◽

2021 ◽

Vol 11 ◽

Author(s):

Paul Sukwoo Yoon ◽

Nuala Del Piccolo ◽

Venktesh S. Shirure ◽

Yushuan Peng ◽

Amanda Kirane ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Microenvironment ◽

Immune Cells ◽

3D Culture ◽

Model Systems ◽

Preclinical Model ◽

Advantages And Disadvantages ◽

Culture Models

Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death in the US. CRC frequently metastasizes to the liver and these patients have a particularly poor prognosis. The infiltration of immune cells into CRC tumors and liver metastases accurately predicts disease progression and patient survival. Despite the evident influence of immune cells in the CRC tumor microenvironment (TME), efforts to identify immunotherapies for CRC patients have been limited. Here, we argue that preclinical model systems that recapitulate key features of the tumor microenvironment—including tumor, stromal, and immune cells; the extracellular matrix; and the vasculature—are crucial for studies of immunity in the CRC TME and the utility of immunotherapies for CRC patients. We briefly review the discoveries, advantages, and disadvantages of current in vitro and in vivo model systems, including 2D cell culture models, 3D culture systems, murine models, and organ-on-a-chip technologies.

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Cytokine Production and Cytotoxicity of Calcium Silicate–based Sealers in 2- and 3-dimensional Cell Culture Models

Journal of Endodontics ◽

10.1016/j.joen.2020.03.011 ◽

2020 ◽

Vol 46 (6) ◽

pp. 818-826

Author(s):

Alexis Gaudin ◽

Mirek Tolar ◽

Ove Andreas Peters

Keyword(s):

Cell Culture ◽

Calcium Silicate ◽

Cytokine Production ◽

3 Dimensional ◽

Culture Models ◽

Cell Culture Models ◽

Dimensional Cell

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Three-Dimensional Cell Culture Systems for Studying Hepatitis C Virus

Viruses ◽

10.3390/v13020211 ◽

2021 ◽

Vol 13 (2) ◽

pp. 211

Author(s):

Chui-Wa So ◽

Glenn Randall

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Three Dimensional ◽

Hcv Infection ◽

Culture Systems ◽

Cell Culture Systems ◽

Culture Models ◽

Dimensional Cell

Hepatocytes, the major target of hepatitis C virus (HCV), are highly polarized. HCV infection requires extensive trafficking to distinct subcellular domains in the polarized hepatocyte. Polarized cells and three-dimensional organoids are commonly used to study liver functions and differentiation. Researchers have begun adapting these cell culture models that morphologically and physiologically resemble hepatocytes in vivo to study HCV infection. This review summarizes the use of three-dimensional cell culture systems in studies of HCV infection.

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