Success of our customer is our primary goal – if you need help with using of InoCure products or need specific products please do not hesitate to contact our specialists. We are prepared to help to satisfy your needs!
Is inomatrix 3D cell culture plates sterilized?
Yes, our 3D cell culture plates are sterilized. However, you can also add another step of sterilization using ethanol. To do so, please refer to our video guide by clicking this link.
Does inomatrix 3D cell culture plates needs a pre-treatment prior to cell seeding to enhance cell attachment?
It really depends on case by case basis. Our 3D cell culture scaffold is made from poly(ε-caprolactone), which means it has slightly negative surface charge. If this suits your application, then you can do cell seeding directly. Otherwise, you can do surface coating as if you are preparing cell seeding for 2D cell culture. Feel free to contact us if you want to discuss your specific protocol for cell attachment.
Can I remove the cells from the 3D cell culture?
Certainly. You can remove the cells using typical trypsinization protocol. However, keep in mind that for a 3D cell culture, you may want to prolong the incubation time for the trypsinization step compared to when you are using a 2D cell culture.
How can I stain live/Dead cells?
You can apply general protocol for 2D cell culture in our 3D cell culture too. To stain your live cells, BCECF-AM can be used while Propidium iodide might be a good choice to stain dead cells.
How can I characterize the morphology? Do I need to take the scaffold out of the plates?
You can use SEM or Confocal Microscopy to check the morphology of your samples. Also, yes, in most cases you need to take out the scaffold out of the plates for both techniques.
Is InoMATRIX compatible with MTT/ MTS assays ?
InoMATRIX products are compatible with both assays. However, we strongly recomend MTS assay. MTT results in formation of formazane crystals which need additional solubilization stem and fibers may be damaged during this step. For more deails please check our Protocols.
What about thickness of the scaffold /membrane ?
The standard thickness of InoMATRIX membrane is 200 µm. However, we offer custom fabrication of scaffolds with other thickness.
What's approximately pore size of various InoMatrix scaffold?
In case of nanofibrous scaffolds (NF) the pore size is about 1.5 µm. In case of microfibrous scaffolds (MF) it is about 6 µm. In case of nano/microfibrous scaffolds it is about 3 µm. In case of porous scaffolds (PF) the interfibrillar pores have size about 3 µm.However, the fibers are flexible and have high porosity (>80%) and enable cell penetration across the scaffold.
Is there any possibility to combine it with hydrogel? / We use hydrogels for cell growing ,can we use your product InoMatrix ?
InoMATRIX products (especially Inserts) are very compatible with hydrogels. The hydrogel can be formed on surface of fibers and support the cells. Our Insert membranes are having biomimicking structure and high porosity (80% for InoMATRIX compared to 10% in case of track-etched membranes). You can use them as replacement of standard inserts with non-biomimetic surface.
Why use InoMatrix instead classical 2D plastic / polystyrene / TCP surface for cell culturing?
InoMATRIX provides optimized 3D environment for your cells. During classical 2D cultures the cells are growing in monolayers and their morphology, expression of genes and behaviour is altered. This may alter reliability of 2D cell culture results.
The key advantages of InoMATRIX are:
- Fibrous morphology mimicking ECM – InoMATRIX Morphlology kit allows you to select optimal morphology of fibers for your cells.
- High specific surface enables efficient cell adhesion.
- Porous structure allows diffusion of nutrients – InoMATRIX has porosity >80% which keeps your cells in contact with environment.
- Flexble structure with inteconnected pores – InoMATRIX has total pore interconnection enabling cells to penetrate into the fibrous mesh.
- Easy manipulation – scaffolds have sufficient mechanical properties for manipulation during experiments.
- Large number of formats – InoMATRIX comes in form of standard microplates, inserts or custum shapes.
- Compatibility with standard methods.
Is possible cover InoMatrix scaffold with ECM proteins?
Polycaprolactone has negative zeta potential and enables adhesion of ECM proteins including collagen, laminin and fibronectin.
Is there any possibility to reach your InoMatrix scaffold in another format?
We are offering custom scaffolds in shape based on your expectations. InoMATRIX is formed by needleless electrospinning and could be obtained in large formats (up to 500 mm wide scaffolds).
Is possible to deliver InoMATRIX for organ-on-chip or tissue-on-chip applications?
Yes. InoCure has 3D printing and CNC milling capacities for construction of organ-on-chip systems with InoMATRIX.
InoMatrix plates are sterilized using gamma irradiation and are ready to use. In the case that you have non-sterile plates, or you need to re-sterilized them, you can use ethanol sterilization:
- Place test-plates in flow box and fill the wells with 70% ethanol
- Leave the plates with ethanol for 30 min at room temperature
- After incubation remove the ethanol from test-plates
- Wash the test-plates three-times with sterile phosphate buffer saline (PBS)
If necessary, cell can be remove from InoMatrix scaffold by enzymatic reaction – trypsinisation. Generously, incubation time has to prolongated compare to tissue plastic culturing.
- Work in sterile flow box and with sterile solutions for further cultivation of cells
- Wash the InoMatrix scaffolds with PBS
- Add 1x concentrated Trypsin-EDTA solution (30µl/96 well, 100µl/cm2)
- Incubate for 10 min in CO2 incubator at 37°C
- Shake the InoMatrix plate rigorously
- Stop the reaction with fetal bovine serum or medium with at least 10% FBS
- Transfer the solution to sterile centrifugal tube and centrifuge at 300x g for 5 min
- Aspirate the solution and resuspend the pellet of cells with fresh medium or required solution
Scaffold removal is needed to characterize your sample in SEM or Confocal Microscopy. If you are using insert, you can cut the scaffold and simply take it out for testing. If you are using a general well plate, you can use tweezer to remove the scaffold. You can follow the instruction on our protocol for step-by-step instruction on how to do morphology characterization.
Cell seeding 3D scaffold
Sterile InoMatrix test plates are ready to use. You can seed the cells based on your common protocol or as follow:
- Work in sterile conditions in a flow box
- Pre-wet InoMatrix scaffolds with culture media: add culture media in to the InoMatrix well plates and incubate them for at least 30 min. Remove the medium.
- Prepare cell suspension in required density in medium recommended for the concrete cell types. Cell seeding density depends on the cell type and the purpose of the study (usual seeding density of the chosen cell types are listed in the table below). We recommend seeding the cells in lower amount of media (30 – 50 µl) to facilitate the cell adhesion.
- Put the cell suspension into the wells and leave the cells to adhere for 1 – 2 hours in CO2 incubator
- Refill the wells with culture media
- Changed the cultivation media every 3 – 4 days or as recommended
Note: cell seeding density should be higher compared to cultivation on tissue culture plastic.
Cell seeding 3D scaffold
Table: Usual cell seeding density (per well of InoMatrix 96-well plate and 1 cm2)
|Cell type||Number of cells per well of 96-InoMatrix plate||Number of cells per cm2|
|Mesenchymal stem cells (MSCs)||25,000 – 30,000||75,000 – 90,000|
|Osteocytes (MG-63, SaOS)||10,000||30,000|
|3T3 fibroblast||3,000 – 5,000||9,000 – 15,000|
|Melanocytes||3,000 – 5,000||9,000 – 15,000|
|Keratynocytes||3,000 – 5,000||9,000 – 15,000|
|Type of well-plate||Growth Surface||Volume||Internal Ø||Dimensions|
|6||9.026||15.53||33.9||128 x 86 x 22|
|12||3.466||5.96||21.00||128 x 86 x 22|
|24||1.864||3.18||15.40||128 x 86 x 22|
|48||0.875||1.49||10.6||128 x 86 x 22|
|96||0.322||0.36||6.40||128 x 86 x 17|