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Potential reasons
1. Not enough media.
2. No serum in media or incompatible media.
3. Matrix not compatiable with cells.
4. Cells are contaminated.
5. Cells are senescent or overpassaged.
6. When adherent cells were being lifted off plate for seeding of 3D culture, they became over trypsinized.
Solutions
1. We recommend submerging 3D cell cultures in media. The volume will depend on the size well that is being used.
2. We recommend incubating 3D cell cultures for 24 hours in serum containing media to allow cells to recover, before starting experiment.
3.Make sure the matrix you are using is biocompatible.
4. Check for mycoplasm, bacteria, fungal or yeast contamination. Signs of contamination include: visible signs of particles under the microscopy, cloudy media and abrupt changes in pH in phenol red containing media. Repeat experiment. Add antibiotics to medium to reduce chances of contamination. Check reagents and equipment.
5. Check passage number. Use earlier passage cells.
6. Reduce concentration of trypsin. Reduce duration of tryspinization. Use a different method for cell dettachment. There are other enzymatic approaches that are commercially available that too many to list here, but search "alternatives or replacement of trypsin."
Potential reasons
1.Devices were not closed- inner and outer chambers were not out of frame.
2. Inner chamber not pushed all of the way down, leaving a gap at the bottom in between the inner and outer chambers.
Solutions
1.Swivel the inner and outer chambers so that chamber openings are not aligned to each other.
2.Make sure that the inner chamber is flush to the bottom of the outer chamber.
The chamber devices are designed to ensure a tight fit to prevent leakage, so that 3D cultures can be established consistently, while allowing the chambers to swivel back and forth. The lubricant makes it easier to assemble the devices and swivel the chambers to open and close the system. The lubricant is inert and non-toxic, and has not been shown to affect cell systems established in the device.
If you choose not to use the lubricant, you can still assemble the devices but requires much more effort, without guarentee that you can disassemble them. Pre-incubating devices at 37 degrees celcius may help in this regard.
Potential reasons
1. Not enough media in the TheraKan devices.
2. No serum in media or incompatible media.
3. Matrix not compatiable with cells.
4. Cells are contaminated.
5. Cells are senescent or overpassaged.
6. When adherent cells were being lifted off plate for seeding of 3D culture, they became over trypsinized.
Solutions
1.We recommend 500 ul or more media in devices.
2. After establishing 3D cell cultures in devices, we recommend incubating 3D cell cultures in serum containing media for 24 hours to allow cells to recover before starting experiments. Make sure the media you are using is compatible with your cell line.
3. Make sure the matrix you are using is biocompatible.
4. Check for mycoplasm, bacteria, fungal or yeast contamination. Signs of contamination include: visible signs of particles under the microscopy, cloudy media and abrupt changes in pH in phenol red containing media. Repeat experiment. Add antibiotics to medium to reduce chances of contamination. Check reagents and equipment.
5. Check passage number. Use earlier passage cells.
6.Reduce concentration of trypsin. Reduce duration of tryspinization. Use a different method for cell dettachment. There are other enzymatic approaches that are commercially available that too many to list here, but search "alternatives or replacement of trypsin."
You can potentially culture any cell type with these devices. Devices have been tested with rodent and human cell types. 3D cultures in devices depend on factors including matrix and media. You may need to determine the appropriate matrix and media conditions for your cell type.
For guidance, you may access:
protocols https://www.fenniklifesciences.com/protocols
publications https://www.fenniklifesciences.com/copy-of-publications
Potential reasons
1. Matrix is responsible.
2. Cell type/cell line is responsible.
Solutions
1. Some matrices such as Matrigel and collagen may affect the cell structures formed in 3D. It may be necessary to test different matrices, or different combinations of matrices to determine which one(s) are optimal for your experiment.
2.Some cell lines may form symmetrical or assymetrical spheroids depending on their transformative state. For example, normal or benign epithelial cell lines form symmetric spheroids. Malignant cell lines tend form assymmetric cell clusters. These are not technical problems but may be inherent characteristics of your cell type/cell line.
You can measure the number of immune cells infiltrated to devices from the test chamber, using live cell imaging approaches, or immunostaining and detection of cells by microscopy or flow cytometry.
You can examine biomarker expression in 3D cultures established in the TheraKan device using microscopy or flow cytometry techniques.
You can detect expression of soluble factors including cytokine levels by ELISA.
For further guidance, please go to protocols section at https://www.fenniklifesciences.com/protocols or publications at https://www.fenniklifesciences.com/protocols
Other potential data may include detection of gene expression by qPCR or protein expression by immunoblotting of 3D cultures. As we develop new protocols and methods for the TheraKan device, our protocols and publication sections will be updated. So please check back from time to time.
Potential reasons
1. Immune cells were not labeled sufficiently.
2. Cell extraction from 3D cultures was not efficient for flow cytometry.
3. Not enough mmune cells were plated.
4. Incubation time not long enough.
5. Not enough cells in the 3D cultures.
6. 3D cultures did not contain enough viable cells.
Solutions
1. Make sure the antibody labeling was sufficient for flow cytometry or microscopy.
2. Count your cells after extraction from matrix. Processing samples for flow cytometry tends to result in cell loss. Increase the number of cells in the test chamber and 3D cultures if necessary.
3. Increase the number of cells in the test chamber.We recommend starting with 500,000 cells/4 ml volume.
4. Increase incubation time to allow more immune cells to migrate from test chambers into devices. We recommend a time course experiment, incorporating a 24 hour timepoint.
5. Increase the numbers of cells in 3D cultures. We recommend starting with 100,000/250 ul matrix. You may want to test varying numbers to cells to opimize your experiment.
6. Make sure your cells were viable before they were embedded in 3D cultures by count cells with trypan blue. Make sure there was enough medium in devices. We recommend at least 500 ul or more.
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