Studying the Effects of Confinement on Cell Division

On Microfluidic Future I like reviewing advancements in therapeutic or diagnostic devices because I’m really drawn to those areas of research. Every once in a while, however, I take interest in research for the sake for knowledge, like the Root Chip. I recently came across an article from Dino Di Carlo of UCLA that describes a microfluidic device used to study cancer cells. The article, “Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments” appeared in PLoS ONE, which is an open access journal (very cool!).

Specifically, Di Carlo’s device is used to study the effects of the mechanical environment on cancer cells during division. It’s commonly known that the course of the cell cycle is affected by soluble factors, but the cell’s mechanical interaction with the environment also affects its morphology, differentiation and cell cycle. Changes in confinement and substrate elasticity were tested using the HeLa cervical cancer cell line in this study. The authors looked for several deviations from standard cell division including delayed mitosis, multi-daughter mitosis events, unevenly sized daughter cells and induction of cell death.

Device Design

A. The device features posts to confine cells with continuous perfusion. B. The device is deformable, bringing the two layers into contact. C. The device has variable elasticity and confinement height.

A. The device features posts to confine cells with continuous perfusion. B. The device is deformable, bringing the two layers into contact. C. The device has variable elasticity and confinement height.

Di Carlo’s device has a bottom layer and an elevated PDMS layer supported by posts with varying height for control over cell confinement. In a relaxed state, there is a 15 µm clearance between the posts and bottom layer. When pressure is applied to the device, the two layers meet which confines the cells between posts and reduces the clearance to 3 µm or 7 µm. Additionally, the top layer has an elasticity of 130 KPa or 1 MPa. The device is designed to allow media to flow throughout all the confining chambers, eliminating the possibility of cell death due to a toxic environment.

Results

Induced multi-daughter division resulted in 3, 4 and even 5 daughters

Induced multi-daughter division resulted in 3, 4 and even 5 daughters

In an unconfined environment, a HeLa cell would normally ball up into a sphere during mitosis, which would take no longer than 140 minutes. But with increased confinement and stiffness, the authors witnessed multi-daughter mitosis (one cell dividing into three or four daughter cells), unbalanced daughter sizes, prolonged mitosis and cell death. Resulting control cells from division would often be spheres with a diameter of 20 µm, while the confined cells would be highly asymmetric with diameters 40-80 µm. Increases in stiffness and confinement generally increased the odds of abnormal cell division, with some clear observed patterns. Under low compression of 7µm and stiffness of 130 KPa, 90% of multi-daughter divisions resulted in three cells. When a stiffness of 1 MPa was applied to the same low compression, 85% of multi-daughter divisions resulted in four cells. The authors believe that the cells aren’t able to effectively deform the stiffer substrate and are limited in how spherical they can be before mitosis. The confined shape may also affect chromosomes lining up at the metaphase plane(s), resulting in a bias toward multi-daughter divisions. The multi-daughter cell divisions can produce viable cells, which subsequently can undergo their own multi-daughter division, and also generate daughters that re-fuse after division.

The authors also hypothesized that when the cells are forced to divide in a discoid shape, signaling and regulation may be affected. Diffusion or active transport of signals would take much longer to traverse the large cross-section of the cell, and the force of cytoskeletal elements might be diminished across the same large distance.

Discussion

This work has produced some findings that may not be totally surprising, but are definitely peculiar. A follow-up to the findings generated here would surely add to the increasing knowledge base of cancer cell behavior. In its current form, this information wouldn’t lead to any new treatments, although under high confinement 70% of cell cycles resulted in cell death, which holds potential in therapeutic applications. Studying diseases help us learn more about healthy cells because we can see what goes wrong when specific elements fail, but I’m also interested in seeing how healthy cells react under the same mechanical conditions. The microfluidic device itself also has potential beyond the study of cellular life cycles: One area in particular includes investigating the effects of mechanical strain in osteocyte and chondrocyte differentiation.

Reference:

ResearchBlogging.org

Henry Tat Kwong Tse, Westbrook McConnell Weaver, & Dino Di Carlo (2012). Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments PLoS ONE, 7 (6)

Detecting Ovarian Cancer with a Cell Phone and a Microfluidic Chip

This post was chosen as an Editor's Selection for ResearchBlogging.org

Author's note: This post was chosen as an Editor's Selection at ResearchBlogging.org. Thanks for the support!

Ovarian Cancer

Ovarian cancer is the fifth leading cause of cancer related mortality among women. Like many diseases, there is a stark difference in survival rates depending on detection times. When ovarian cancer is detected at stage I, there is a 90% 5 year survival rate. Compare that with the 33% 5 year survival rate when the ovarian cancer is detected in stage III and IV. This disease is unfortunately asymptomatic at early stages, drastically eliminating the odds of discovery with enough time to make a difference.

While using traditional diagnostics like imaging, biopsy, and genetic analysis is impractical for regular screening, there are alternative methods used for women who are high-risk for ovarian cancer or who have family history. Transvaginal sonography can be used annually although it has been shown to have limited efficacy. Blood serum can also be tested to indicate ovarian cancer, but this method only has a sensitivity of 72% at specificity of 95%. Sensitivity and specificity are used to measure how well a system can detect something. To calculate specificity in our case, imagine 100 women without ovarian cancer are tested, and only 5 women are incorrectly told that they have ovarian cancer. This would undoubtedly be corrected in a follow up test. But to calculate sensitivity, imagine 100 women with ovarian cancer and 28 women are incorrectly told that they do not have it.

Not only are these tests inconclusive, they are extremely invasive. In the case of transvaginal sonography, an instrument is inserted in the vagina to check the ovaries. With blood serum testing, blood obviously must be drawn. Biochips currently exist to detect ovarian cancer based on protein biomarkers or DNA sequences, but these rely on fluorescence or chemiluminescence and are designed to be used in laboratory settings. None of the previous methods lend themselves to be used in point-of-care (POC) settings. An ideal POC device would not require expensive parts, be usable by limited trained personnel or be too complex. This would allow it to be used in resource-rich and resource-limited settings, especially if it does not need a continuous power source.

Detecting Ovarian Cancer with Urine

Researchers from Harvard Medical School have developed a cell phone system to detect ovarian cancer that should address the lacking areas of diagnosis so far. “Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care” was featured in the 2011 issue 11 of Lab on a Chip. Utkan Demirci et al. demonstrate a method to non-invasively detect ovarian cancer efficiently with urine and a cell phone. At the heart of this system is an enzyme-linked immunosorbent assay (ELISA). ELISA is a very common technique used in protein detection. In this case, a sandwich ELISA is used to detect the ovarian cancer biomarker Human epididymis protein 4 (HE4). Antibodies targeted to HE4 are conjugated to horseradish peroxidase which catalyzes a substrate and causes blue color to develop. We should then be able to ascertain the amount of HE4 originally in solution by quantifying the resulting color. This process takes place in three different microfluidic channels on a microchip the size of a stamp. These three channels allow a sample to be treated in triplicate or for many samples to be tested at once.

After the urine is loaded in the microfluidic chanel, ELISA is performed resulting in a colorimetric change

After the urine is loaded in the microfluidic chanel, ELISA is performed resulting in a colorimetric change

Cell Phone and CCD Imaging

Two methods were used to detect the change in color. The first method utilized a cell phone (more specifically Sony-Ericsson i790). This took advantage of the built in camera and processing power, allowing all processing steps to be carried out on the single device. The second method uses a lensless charge-coupled device (CCD). CCDs are found in digital cameras and have completely changed the way we capture images. In fact, the cell phone used has its own CCD inside. The CCD is used directly with a computer which analyzes the image with MATLAB. Both methods take a picture of the three microfluidic channels on the chip and compare the colors of the channels to previously measured standards.

Cell phone takes an image of ELISA results and compares the color to calibrated curves

Cell phone takes an image of ELISA results and compares the color to calibrated curves

Calibration and Testing

Before this system can be tested on actual samples, it has to be calibrated with known samples. HE4 was evaluated from 1,250 to 19.5 ng/mL, which was its detection limit. I’m unsure how much urine is actually needed. Each sample was diluted twenty times, and each channel can only handle 96.75 µL including the ELISA solutions. In order to make sure that ELISA was occurring correctly on the microchip, the colored solution was transferred to a 96-well microplate and the optical density was measured with a spectrophotometer. This was validated and a strong correlation between HE4 concentration and color was found for the CCD and cell phone with high R2 values above 0.90. After this calibration, the system was used to differentiate between the urine samples of 19 women with ovarian cancer and 20 women without ovarian cancer. The standard microplate technique and the cell phone and CCD methods were able to distinguish between the normal and cancer samples with statistical significance. When operating at a specificity of 90%, the cell phone and CCD tests achieved 89.5% and 84.2% sensitivity respectively. These results indicate that the new methods can efficiently and effectively detect ovarian cancer in urine.

Strengths

  • Both the CCD and cell phone methods demonstrated their ability to distinguish the difference between healthy and ovarian cancer urine.
  • These methods are extremely portable and can be used in a POC setting.
  • No complex machines or techniques are needed, which makes it cost-effective and allows operation by minimally trained personnel.
  • The low price of these tests makes them more accessible to be used to annually screen high-risk women or to check the efficacy of treatment.
  • Urine is an attractive diagnostic fluid because it is non-invasive and not intimidating.
  • It is unclear how well this could work in early detection at stage I of ovarian cancer because the samples used had later stage cancer. It is possible that the current configuration may not be able to differentiate between normal and early stage if HE4 levels vary between stages.
  • This test could be applied to other diseases with established biomarkers and sandwich ELISAs.

Further Development

  • It is unclear how well this could work in early detection at stage I of ovarian cancer because the samples used had later stage cancer. It is possible that the current configuration may not be able to differentiate between normal and early stage if HE4 levels vary between stages.
  • This test could be applied to other diseases with established biomarkers and sandwich ELISAs.

Reference:

ResearchBlogging.org

Wang, S., Zhao, X., Khimji, I., Akbas, R., Qiu, W., Edwards, D., Cramer, D., Ye, B., & Demirci, U. (2011). Integration of cell phone imaging with microchip ELISA to detect ovarian cancer HE4 biomarker in urine at the point-of-care Lab on a Chip, 11 (20) DOI: 10.1039/C1LC20479C