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Visualizing Apoptosis in 3D Cell Cultures

Apoptosis, or programmed cell death, is an important cellular mechanism that is critical in development and tissue homeostasis.  Apoptosis also plays a role in cancer biology.  For example, visualizing apoptosis in response to treatments is one way to characterize potential therapies.

While 3D cell cultures are ideal for mimicking a tumor microenvironment, obtaining apoptosis data after treating the cultures can prove difficult.

To overcome this difficulty and to make apoptosis data accessible to researchers using Cell-Mate3D™ matrix, we optimized the commonly used Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent that enables quick and reliable imaging of apoptotic cells in culture.

Cell-Mate3D™ cultures were setup, treated, and analyzed as followed:

  • AU565 human breast cancer cells (HER2-positive) were embedded into the Cell-Mate3D™ matrix.
  • One sample was left untreated and an equivalent sample was treated with 100 μM Taxol for 12 days.
  • Samples were stained with 15 μM CellEvent™ Caspase-3/7 reagent (three times the recommended concentration).
  • Green-fluorescent apoptotic cells were clearly seen in the Taxol-treated sample by inverted confocal microscopy.
  • Caspase staining appears brighter in Taxol treated cultures compared to non-Taxol treated cultures.

Figure 1. Detection of apoptotic cells in the Cell-Mate3D™ matrix using CellEvent™ Caspase-3/7 Green reagent. AU565 breast cancer cells were embedded in the Cell-Mate3D™ matrix and cultured for 12 days. Cells were untreated (LEFT) or treated (RIGHT) with 100 μM Taxol for 12 days to induce apoptosis. Cells were then incubated with the Invitrogen™ CellEvent™ Caspase-3/7 Green Detection Reagent (15 μM) for 30 min. to label apoptotic cells with green fluorescence, counterstained with the Invitrogen™ NucBlue™ Live ReadyProbe™ Reagent, and imaged using an inverted confocal microscope at 20x magnification.

Reagents Used

Have a question?  Call us 855 849 BRTI (2784) or email info@brtilifesciences.com

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Creating a Tumor Microenvironment with a Hypoxic Gradient using Cell-Mate3D

Cancerous tumors are heterogeneous tissues with a dynamic microenvironment. They exhibit an oxygen gradient with outer regions of well-oxygenated (normoxic) tissue alongside poorly-oxygenated regions experiencing hypoxia (1).

Hypoxia is essential for tumor development and many studies have shown that tumor cells in hypoxic regions distant from blood vessels show resistance to chemotherapy or radiation therapy (1).

2D cultures and the lack of an in vitro model that can recapitulate a hypoxic, 3D tumor microenvironment, are limiting to researchers who want to target cells in this context.

We show that Cell-Mate3D™ exhibits features of a tumor microenvironment by demonstrating that by day 3 in culture:

  • Cells in the matrix express HIF-1α
  • A hypoxic gradient within the matrix is created without the use of hypoxic chambers

Cell-Mate3D Creates a Hypoxic Gradient

The ability to mimic a physiologically relevant tumor microenvironment that contains an oxygen gradient would serve as a useful tool for cancer researchers studying tumor biology and potential treatments and therapies (4).

HIF-1α is a transcription factor that is activated when cells experience a hypoxic environment (2). When HeLa cells are embedded into the Cell-Mate3D matrix, a hypoxic gradient is formed after 3 days (Figure 1). This is demonstrated by sectioning and staining a cross section of the matrix and performing HIF-1α staining. Imaging and analysis show that the HIF-1α staining intensity is low for in the outer 400-500μm of the matrix, and that expression is increased in the center of the matrix at Day 3.

This observation indicates that culturing cells in Cell-Mate3D creates a hypoxic gradient and more closely mimics a solid tumor microenvironment compared to 2D culture.

Figure 1. Cell-Mate3D cultures were created at a density of 4 million HeLa cells per 100μL of Cell-Mate3D matrix. After one day in culture, cryosectioning and staining showed little HIF-1α expression (a,b). After three days in culture, HIF-1α staining was more intense (d,e) which is indicative of hypoxia. Relative Fluorescence Intensity (RFI) profiles of (a) and (d) show that hypoxic conditions are time dependent. After three days in culture, HIF-1α RFI (red) is elevated up to four fold compared to one day in culture (c,f). Furthermore, the intensity profile of Day 3 cultures (f) indicates the presence of an oxygen gradient. The RFI of HIF-1α begins to increase approximately 400-500μm from the edge of the matrix.

Cell stains used:

  • Rabbit anti human HIF-1α (H-206) – Santa Cruz Biotech (SC-10790)
  • NucBlue® Live ReadyProbes® Reagent – ThermoFisher (R37605)
  • NucBlue and ReadyProbes are a registered trademark of ThermoFisher Scientific

Want to create your own hypoxic gradient without the use of expensive chambers? The Cell-Mate3D matrix is suitable for many cell types including cancer cells, fibroblasts, and stem cells.

Contact us today to schedule a free consultation on creating your own in vitro tumor microenvironment or developmental model.  Email info@brtilifesciences.com
Learn more about Cell-Mate3D

 

References

  1. Tannock IF.  Tumor physiology and drug resistance. Cancer Metastasis Rev 20: 123–132. (2001)
  2. LaGory EL, Giaccia AJ. The ever-expanding role of HIF in tumour and stromal biology. Nat Cell Biol. Apr;18(4):356-65. (2016)
  3. Vaupel  P, Mayer, A. Hypoxia in cancer: significance and impact on clinical outcomeCancer Metast. Rev. Jun;26(2):225-39. (2007)
  4. Saggar JK, Yu M, Tan Q, Tannock IF (2013) The tumor microenvironment and strategies to improve drug distribution. Jun 10;3:154. (2013)

 

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Tissue Engineering and Regenerative Medicine

Abstract:

Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10–14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expres- sion representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Click here to download full paper…

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BRTI Life Sciences to provide “tissue-like” microenvironment to support pancreatic islets for JDRF funded University of Minnesota study.

BRTI Life Sciences’ unique 3D matrix will be included in a study at the University of Minnesota of immunoisolated islets in a structured biomimetic environment to treat insulin-dependent diabetes. The study, which is fully funded by the Juvenile Diabetes Research Foundation (JDRF), employs a device, including islets embedded in BRTI’s unique microenvironment, that will be implanted under the skin of nonhuman primates. The device is designed to eliminate the need for immunosuppression drugs to protect the islets.  This novel approach may lower the cost and risk of islet transplantation for patients with insulin-dependent diabetes, freeing them from the need for routine insulin injections.
To learn more, see the JDRF funded abstract.
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Cell-Mate3D™ Featured at Regenerative Medicine Conference at Hilton Head

Podium Presentation:

Induction of Cerebral Organoids from Human Induced Pluripotent Stem Cells using a Chemically Defined Hydrogel and Defined Culture Medium. Timothy D. O’Brien
https://www.regenerativemedicineworkshop.com/users/timothy-d-obrien

Poster Presentation:

Culture and Trilineage Differentiation of hMSCs Within a Novel 3D Microenvironment. Applications for in vitro and in vivo Biomedical Research. Beth A. Lindborg, Yi Wen Chai, Connor B. Ulrich, Timothy D. O’Brien, John H. Brekke
https://www.regenerativemedicineworkshop.com/poster-abstracts?field_pro_last_name_value=Lindborg

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New publication using Cell-Mate3D

“A chitosan-hyaluronan based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells (MSCs)” was recently accepted in Tissue Engineering Part A.  This article describes how Cell-Mate3D can improve differentiation potential and induce more complex structures compared to 2D cultures.

Click here for the abstract.

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