TheraKan services

Approximately 123 million people in the US are diagnosed with a chronic disease such as diabetes, heart disease, respiratory disease or cancer every year,  requiring long-term drug treatment (1, 2, 3,4).  While more and more treatment strategies are being tailored to individual patients, many drugs still exert significant adverse side effects on the immune system. Immuno-suppression increases risk of infections (5, 6). Drugs that hyper-activate the immune system cause tissue damage and organ failure (7).  Conversely, many drugs are now known to provide therapeutic benefits on the immune system. This knowledge is uncovered in patients, only after decades of use (8).   Current pre-clinical drug tests and assays have many limitations that contribute to immuno-toxic drugs being advanced to clinical trials.  Mouse models are expensive, labor intensive to use and have significant genetic differences in the immune system that can demonstrate drug effectiveness while masking toxic side effects  (7,9, 10). Commonly used in vitro assays, which tend to examine immune cells alone plated in tissue culture dishes, do not provide a realistic picture immune response in tissues.

To overcome existing limitations in pre-clinical drug testing, we offer the TheraKan system, which will facilitate measurement of immune response to 3D cell cultures (Figure 1). 

 

 

 

 

 

The TheraKan system can be used to measure multiple cellular immune responses (cell proliferation, recruitment, activation) in the same samples within a single experiment, thereby increasing efficiency, decreasing variability and lowering cost in data collection (Table 2)

 

 

 

 

 

We offer several different services using the TheraKan system: 1. monocyte/macrophage infiltration assay, 2.  peripheral mononuclear blood cell (PBMC) infiltration assay 3. Cytokine assay. Sample data from these  assays are shown below.

Monocyte/Macrophage infiltration assay

Monocytes are derived from the bone marrow that play important roles in wound healing, infection and cancer.  Monocytes circulate in the blood until they are attracted to tissues through chemokine gradients. Once in the tissues,  they differentiate into macrophages in response to cytokines. Macrophages scavenge debris, engulf  foreign particles, secrete growth factors, and interact with a variety of immune cells including T cells to regulate their activities.

This assay can be used to detect and quantify the number of  macrophages recruited to 3D cultures over time. Macrophages may be detected by flow cytometry or microscopy  approaches.
You can use this assay to address issues such as:
  • How do potential drug targets/molecular factors regulate the overall recruitment of macrophages over time
  • How do potential drug targets/molecular factors regulate the rate and direction of macrophage migration over time?
  • How do drug compounds affect overall macrophage recruitment over time?
  • How do drug compounds affect the rate and direction of macrophage migration over time?
Macrophage infilration assay with mouse mammary carcinoma cells

mcherry+ Raw264.7 macrophages recruited to PyVmT 3D cell cultures established in the TheraKan after 24 hours. Image shows mcherry (red) overlayed with PyVmT cells (unlabeled) at 10x magnification.

24 hours
48 hours
Macrophage infilration assay with human breast cancer cells

mcherry+ Raw264.7 macrophages recruited to MDA-MB-231 3D cell cultures established in the TheraKan after 24 and 48 hours. Image shows mcherry (red) cells at 10x magnification.

device entrance

Movie:Macrophage infilration assay with mouse mammary carcinoma cells.

mcherry+ Raw264.7 macrophages recruited to PyVmT 3D cell cultures established in the TheraKan after 24 hours.  Movie shows mcherry+ cells migrating within the TheraKan device, near the entrance.

PBMC infiltration assay

Peripheral mononuclear blood cells (PBMCs) are blood cells that have a round nucleus. They include lymphocytes (T cells, B cells and NK cells) and monocytes/macrophages.  They are typically extracted from whole blood and are a reliable means to examine the functions of certain immune cell types.
This assay can be used to detect and quantify the number of  primary peripheral mononuclear cells (PBMCSs) recruited to 3D cultures over time. Macrophages may be detected by flow cytometry or microscopy  approaches.
You can use this assay to address issues such as:
  • How do potential drug targets/molecular factors regulate the overall recruitment of PBMCs over time
  • How do potential drug targets/molecular factors regulate the rate and direction of PBMC migration over time?
  • How do drug compounds affect overall PBMC recruitment over time?
  • How do drug compounds affect the rate and direction of PBMC migration over time?

CD8+

Detection of PBMCs in 3D cultures of breast cancer cells using the TheraKan system.

MDA-MB-231 breast cancer cells were grown in 3D cultures in TheraKan system containing PBMCs. After 24 hours, 3D cultures were analyzed by flow cytometry for PBMCs expressing CD8+ and CD68+.

Detection of PBMCs in 3D cultures of pancreatic cancer cells using the TheraKan system.

Panc-1 pancreatic cancer cells were grown in 3D cultures in TheraKan system containing PBMCs. After 24 hours, 3D cultures were analyzed by flow cytometry for PBMCs expressing CD8+ and CD68+.

CD68+

Cytokine assay

Cytokines are a class of soluble molecules that regulate the migration and activity of immune cells. Cytokines are expressed by multiple tissue types and  by immune cells in response to injury, infection or chronic diseases such as cancer.  There many different types of cytokines including: chemokines and interleukins.
This assay can be used to detect and quantify the different types of cytokines associated with immun response over time. Cytokine expression may be quantified by ELISA  approaches.
You can use this assay to address issues such as:
  • How do potential drug targets/molecular factors regulate cytokine expression over time?
  • How do drug compounds affect overall cytokine expression  over time?
  • How are changes in cytokine expresion associated with changes in immune cell recruitment and activity over time ?
  • What kinds of cytokines function as hallmarks of drug activity?

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Detection of Interleukin 6 in 3D cultures of pancreatic cancer cells using the TheraKan system.

Panc-1 pancreatic cancer cells were grown in 3D cultures in TheraKan system with or without PBMCs. After 24 hours, medium from 3D cultures were analyzed by ELISA for IL6 expression

Detection of Interleukin 2 in 3D cultures of breast epithelial cells using the TheraKan system.

MCF10A breast epithelial cells were grown in 3D cultures in the TheraKan systemand treated with or without heat killed S. aureus bacteria.  PBMCs were added to the TheraKan system.  After 24 hours, 3D cultures were analyzed by ELISA for IL2 expression.

Detection of CCL2 in 3D cultures of breast epithelial cells using the TheraKan system.

MDA-MB-231 breast cancer cells or MCF10A benign breast epithelial cells were grown in 3D cultures in TheraKan system with or without PBMCs. After 24 hours, 3D cultures were analyzed by ELISA for CCL2 expression.

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