FAQs on Cultured Cells
Stretch Chambers and Stretching Stimulation
- Does autoclaving affect cell adhesion on stretch chambers?
Autoclaving does not affect adhesion, however you should not use aluminum foil. Use special sterilized bags.
- Can we reuse the stretch chambers?
The stretch chambers are intended for one-time use. Cross-contamination between experiments cannot be completely removed, and reuse of stretch chambers may compromise the integrity of the experiment.
- What are the materials and characteristics of the stretch chambers?
The material of our stretch chambers is a thin silicone elastomer whose main part is made of Polydimethylsiloxane (PDMS). The surface has strong hydrophobic and weak cell adhesiveness. In cell culture, the chambers must be first coated with an extracellular matrix (e.g. fibronectin, collagen, laminin, gelatin) to strengthen the cell adhesiveness of culture surface (see coating protocols). PDMS chambers bounce back from stretching and compression with their original properties intact. Thus, the chambers demonstrate good reproducibility in applications requiring continuous mechanical stretching over prolonged periods. An optically transparent, ultra-thin (100 – 200 μm) membrane at the well bottom not only makes stretch chambers compatible with optical microscopy techniques, but with fluorescence detection and microscopy as well.
- Is a uniform stress load applied on every cell in the stretch chamber?
Our unique stretching system enables uniform stress loading due to the materials and methods employed and the shapes of stretch chambers. The STREX Cell Stretching System is designed to achieve stretching in a single, parallel direction, with only a very weak secondary load. Research has demonstrated that the STREX system enables highly reproducible cyclic stretching over prolonged periods at ratios of 1 – 20%. (Ref) Naruse. K., et al. (1998), Oncogene,17:455-463.
- What is the difference in cell response between sustained stretching and cyclic stretching?
It is reported that the signal transduction systems and biochemical responses stimulated in the cells under sustained cellular stretching and those under periodic cellular stretching differs. (Ref) Sasamoto et al, 2005, 288, C1012-22.
- Are the stretch chambers sterilized?
Stretch Chambers as sold are non-sterile. The chamber must be autoclaved for 20 minutes at 180°C. Aluminum foil should not be used in the autoclaving process. Rather, autoclave bags (sterilization pouches) are recommended.
Note: Chambers are disposable, and heat-resistant from 0° to 180°C. Product quality and cell adhesion performance are not guaranteed when the chambers are reused, or used outside the range of heat resistance.
- Are you able to manufacture chambers larger than shown in your catalog?
We manufacture stretch chambers for mass cultures by special order.
- Can we preserve coated chambers for future use?
We recommend coating the chambers just before use.
- Are there any solutes that may emerge from the stretching system?
PDMS or polymerization initiators with low molecular weight may emerge, however we have never received a report saying these substances do harm on the experiments.
- The culture medium is not of uniform thickness. Why is that?
Due to the thinness of the elastic silicone membrane, the chamber may slack in the center. The ultra-thin film (100 – 200 μm) facilitates observation of samples. Our experience shows that this variation in chamber thickness does not affect uniform stress load in a considerable way.
- Why does the culture medium evaporate after a few days while using an STB-1400 stretching system?
The STB-1400 is not suitable for long-term culture, as it requires daily replenishment of culture fluid. We recommend using our stretching systems suitable for long-term culture. Additionally, check that the lid of the system is put on during the experiment. Placing a 35 mm or 60 mm dish with water at any location within stretching system will slow the evaporation of culture fluid. The velocity of evaporation of culture fluid heavily depends on the condition of the incubator. Check the condition of evaporation dishes, and confirm that sufficient coolant is in the plastic container attached with the stretching system and it circulates properly. If you do not have any problems with the above and still have the evaporation problem, try to put cotton wool with purified water nearby the chamber of the stretching system.
- We use reagents with phenols on RNA extraction. How much will the silicone materials of the chambers degrade when we add the reagents directly?
We recommend that you dispose of the chambers after single use as they are not intended for multiple use.
- Do you have any recommendation of gels to use for 3D cyclic stretching?
We recommend collagen gels for use in 3D cyclic stretching at this time. We have successfully used the following gel in house: KOKEN’s DME-02 Neutral collagen solution DMEM 2 mg/mL Atelocollagen, DMEM.
- Can we coat chambers under UV rays?
It is not recommended to coat chambers under UV rays. UV exposure after coating may denature proteins in the extracellular matrix on the chamber and may affect adhesiveness.
- Cells adhesion appears to be different from chamber to chamber. What could be the Cause?
In producing the stretch chambers, STREX works to assure that there are no creases or other imperfections on any of the surfaces. However, given the exceptionally thin membranes involved, creases can easily develop. Therefore it is important to use great care with the bottom surface when seeding the cells. To avoid creases, first treat a culture dish by dripping a bit of ethanol on it. Then set the chamber on the dish and tilt the dish and chamber, taking care not to introduce any air between the two. Allow some time for the ethanol to evaporate. The chamber will then be in optimal condition for seeding and culturing the cells with high adhesion.
- Do we have to hydrophilicate the silicone film before applying the coating agents?
Stretch chambers are hydrophilicated with plasma processing before shipping.
- How do you confirm the completion of coating?
Some cells adhere without coating. For example, some cells secrete collagens, such as smooth muscle cells and fibroblasts. You can check the degree of coatings on the chambers using antibodies, however this method sacrifices a chamber.
- Do we have to air-dry after four-hour standing?
We coat with fibronectin just before using and have never done air-drying. However, some users have reported air-drying after coating with collagen.
- Do you coat with fibronectins without pretreatment on the methyl groups on the surface?
We do not recommend conducting any treatments on the surface of the chambers. Some chamber treatment during the manufacturing process such as plasma processing may cause nonspecific absorption with cells. If you want to observe specific bonding between fibronectins, collagens and integrins, you may special order the chambers without plasma processing, giving them strong hydrophobic properties.
- Which extracellular matrix works best for cell adhesion? Fibronectin, gelatin, or collagen?
Cell adhesion to the stretch chamber membrane is entirely dependent on the cells bonding to the extracellular matrix coating. The cells themselves vary in adhesive properties from one cell type to another, and even vary between different cell lines of the same cell type. Therefore, before conducting any stretching experimentation or research, it is important to be aware of the adhesiveness and the required conditions for whatever coating is to be employed.
See coating protocols as a reference.
- Adhesion to the stretch chamber was confirmed under the microscope before the stretch stimulus was initiated, yet after the stretching, the cells were no longer adhered. What could have caused the cells to detach from the chamber surface?
There are three possible causes:
1. Cell density
The cell concentration of the culture may be too dense. Generally speaking, over-confluence will cause the adhesive force between cells to increase beyond the adhesive force between the cells and the extracellular matrix. This relative decrease in extracellular adhesion can lead to cells detaching from the stretch chamber.
2. Enzyme treatment
Trypsin and other enzyme treatment can damage cells. However, this may not be obvious in experiments conducted with standard dishes, because the binding to the dishes’ plastic wells is nonspecific. By contrast, when stretch chambers are employed, adhesion is attained solely by the extracellular matrix coating. Thus, relatively severe enzyme damage will cause stretch chamber adhesion to fail. Please see the tip on trypsin treatment (below) that addresses this issue.
Cells will not adhere to the stretch chamber if the coating is insufficient. This insufficiency is indicated when the chamber surface easily repels liquid after the applied coating has been absorbed. Extend the coating application and setting time should this occur.
- Cells seeded onto the stretch chamber sometimes aggregate at the center of the chamber. Is there any technique to avoid this aggregation?
Cells may be migrating toward the center of the stretch chamber due to the vibration of the incubator. If that is
the issue, seed the cells normally, then after 15 minutes, gently tilt the chamber from side to side.
- Are rounded cells difficult to be stretched even though they adhere to the chamber?
Rounded cells are generally more difficult to be stretched in comparison with flat-shaped cells.
- In a long-term experiment with the cells in culture under continuing stress, what is the longest period that the stretching stimulus can be applied?
Generally speaking, cells in the incubator can be stretched for a period of two weeks. However, in these long-term experiments, the culture fluid has to be replaced at 2-3 day intervals. In addition, there must be enough motor coolant to ensure the motor is running at a safe temperature throughout the long-term culture. Insufficient coolant may cause the temperature in and around the motor to rise, in turn destroying the cells and/or damaging the equipment.
- What is the optimal cell count to be placed on the chamber?
In general, 200,000 cells / chamber (4 mL of culture medium) is proper from our experience.
- Is it preferred to do the coating at room temperature?
The manufacturer recommends the coating at room temperature.
- We are planning an experiment using neurons. Is It okay to coat them with laminin?
We do not foresee any problems with laminin coating, but unfortunately we do not have any experience using laminin coating.
- What density of cells is adequate when we put them on chambers?
It depends on the experiment you are planning. As a general guide, we say that the cells of 5 × 104 cm2 are adequate.
Cell to Use
- Do you have any movies of cells recorded in time-lapse capturing?
Unfortunately no movie is available. Please refer to the following paper for photos of fluorescent images of GFP-actin transferred cells in the stretched state on stretch chambers.
- Can we stretch myocardial cells?
Yes. Please see this reference.
- Can we load shear stress on cells?
The system is intended to work with adhesive cells, therefore it is difficult to load shear stress with this system.
- Can we stretch cells undergoing gene transfer?
You can conduct gene transfer directly on stretch chambers. However, the transfer work damages cells so that it becomes difficult to snap images of high quality. We recommend that you conduct the transfer on cells in dishes prior to be placed on the stretch chamber.
Experiments after Stretching
- How can cell proteins or mRNA be obtained from the stretch chamber after culturing?
1. Western blotting: Wash with PBS, add electrophoresis sample buffer directly to the stretch chamber, then collect the cell lysate with a cell scraper.
2. Immunoprecipitation: Wash with PBS, add cell solubilizer directly to the stretch chamber, then collect the cell lysate with a cell scraper.
3. mRNA:Wash with PBS suitable for RNA, add cell solubilizer directly to the stretch chamber, then collect the cell lysate with a cell scraper.
- Is there an assay to measure tension load for fibroblasts?
We recommend phosphorylation of ERK with Western Blotting.
- Could you show any report saying the amount of mRNA in gene of mechanosensitive channels changes drastically under stretching stimulation?
Some genes in mechanosensitive channels are well known, however many of them have not been identified yet. So we cannot show the references on this topic at this time.
- Is there any report saying the amount of β-actin in cells is changed under stresses?
Amount of β-actin in cells is not considered to change so drastically under stresses, except for long-term experiments.
Fixation, Staining, and Observation of Cells
- Can we conduct fluorescent staining and observations with the membrane stretched?
Yes. Follow the protocol in page 20 of the manual or use our trial kit with a stretching stand.
- We are planning to stretch the cells on chambers to observe them after fixation and staining. Do your chambers degrade on the fixation and staining?
If you use acetone or chloroform, the silicone film of chambers may swell a little. You have no problems with methanol.
Tip: Splicing the silicone chamber film into approx. 5 mm x 5 mm makes your work easier. You can observe good images putting the cell plane side onto the cover glass.
- Can we use immersion lens in the observation of fixed and fluorescent-stained cells after stretching? Is there any limitations in the observation?
You can use immersion lens. However, the choice of chamber is limited in accordance with the size of the object lens to use.
- The microscope observations appear out of focus.
Do you use oil-immersion lens? The existing PDMS films swell when they are exposed on oils and make the observation difficult. We recommend using water-immersion lens if available.
Do you use fixed samples for fluorescence observations? We recommend splicing samples with a razor after fixation and staining to enclose them on cover glass in the up-side-down state, then observe them. In this case there is no problem with the observations with oil-immersion lens. We provide order-made chambers with the thickness of 100 μm for real-time imaging.
- Can we conduct stretching with biomembrane attached on stretch chambers (to conserve the orientation of cells)?
Yes. Usually we stretch the cell sheets with cell sheet cramp. Otherwise we can exchange inner chamber sheet with biomembrane in the stretching with double-structured transwell.
- Is there a method for standard observing and photographing stretch-stressed cells in their stretched position, using a standard optical microscope?
With the STB-1400 Cell Stretching System this is accomplished by employing a static stretch chamber stand, which holds the samples in the stretched state. Alternatively, cells can be observed and photographed using one of the microscope-mountable stretching systems established directly on the microscope stage.
- STB-150 has the limitation of 20 Minutes in continuous stretching duration. Can we conduct continuous observation of 60 minutes (approx.) with long intervals between each stretching?
Yes. You can conduct continuous observation of one hour if you limit the frequency of stretching up to 10 times/min. In this case, you need 1) thermal insulator and 2) thermostatic reflux system.
- Could you show some approximate figure of this stretching force, depicted with mechanical units?
The stretching force cannot be measured with our equipment. An example of a system which measures loading force on cells under stretching is shown in this paper.
- Do you have any recommendations for antiseptics to add to the coolant container?
We have no recommendation for any particular brand, but you may use the antiseptics used in your laboratory, or the antiseptics for incubator water jackets. Alternatively, you can get along with no antiseptics during an experiment if you change water when you start another experiment.
- Is It okay to wash inside silicone tubes for coolant for circulation with neutral or alkaline detergent?
No problem. You are welcome to request replacement of silicone tubes.
- On sterilization: Is it safe to reflux ethanol in plastic container? Is it safe to use hypochlorous acid?
Reflux with ethanol is okay. Highly concentrated hypochlorous acid may harm pump valves. Please be cautious on the density in use of hypochlorous acid (density of 50-100 ppm is okay).