GENERAL USER MANUAL (V.22.01)

3DProSeed Hydrogel Well Plate, 96-well black glass bottom

Catalog Number: ECT.PS1.001.096 (single pack)  and ECT.PS1.010.096 (Pack of 10 individually sealed plates)

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

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Product Description

The 3DProSeedTM hydrogel weil plate (a registered trademark of Ectica Techologies AG) consists of a 96-well imaging plate containing pre-assembled synthetic hydrogels, delivered hydrated ready for cell seeding. This product uniquely combines the advan­tages of three-dimensional (3D) hydrogel-based cul­tures with the workflow integration of standard 2D cultureware. Every user says that seeding cells and handling the 3DProSeed plate is comparable to any other plate and as a consequence the effort towards automation is minimal. The user can proceed with the seeding of cells with no hydrogel prepara­tion and cell-encapsulation steps.
The key innovation of this product is the patented engineered surface of the hydrogel. Moving towards its surface, the hydrogel gradually decreases in cros­slinking density becoming water ("in-depth density gradient"). Cells landing on the 3DProSeed hydrogel surface do not polarize and do not flatten out as they would typically do on a regular hydrogel or hard pla­stic surface. Cells remain round for a longer time as if they were still in suspension and they subsequent­ly start to actively migrate in-depth by adhering and degrading the synthetic gel, establishing a 3D cul­ture. Furthermore, the 3DProSeed hydrogel surface gives the user the possibility to establish advanced co-culture systems by seeding different cell popu­lations at different time-points in the same hydro­gel ("sequential seeding"). The sequential seeding of cell populations is particularly interesting for the establishment of an artificial model of the stromal environment, to which tumour or immune cells can be subsequently added. 

The plate

The plate is a polystyrene 96-well black plate with a 180-µm glass bottom. The geometry of the well is designed to avoid mechanical stress to the hydrogel during pipetting. An inner well contains the optically transparent gel, protecting it from the manipulation with pipets. The footprint respects industry standards, and technical drawings of the plate are available upon request.
The diameter of the inner well (and hydrogel) is 4 mm. The height of the inner well is 1 mm. The effective thi­ckness of the hydrogel is approximately 500 µm. The refractive index of the hydrogel is the same as the one of water, making it ideal for transmission and fluore­scence microscopy. Videos can be found the end of this page.

The hydrogel composition

The hydrogel is synthetic, based on state-of-the-art poly(ethylene glycol) (PEG) bioconjugate technology. The PEG backbone is conjugated with cell adhesion peptide motives and metalloproteinase (MMP) clea­vage sites to support cell-mediated degradation. The standard formulation can contain traces of a medi­cal-grade transglutaminase enzyme of human plasma origin. This product is free from any animal-derived component. A GMP version is available upon request where this transglutaminase enzyme is produced re­combinantly making the product fully synthetic and GMP-compatible.

Manufacturing standard

The product is produced in a highly controlled cle­an-room environment (clean room class ISO5-GMP-B). Every batch is tested for the absence of microbial con­tamination. A certificate of microbial load of the batch can be requested for each lot.

Disclaimer of warranty

The product is indicated for use in laboratory research only and it is prohibited to use the product and related information for animal or human studies and for dia­gnostic purposes.

Storage conditions

Upon arrival, store the product between 4 and 25 °C, away from direct sources of light and heat. Please refer to the EXP. DATE on the packaging for expiry. Do not freeze and do not store upside down.

3DProSeed GRAPHIC description.tif

Figure 1. Schematic representation of the 3DProSeed product. The synthetic hydrogels are contained in specially designed 96-well plates, with an inner well geometry that protects the gel from pipetting manipulations. The crosslinking density of the hydrogel is gradually reduced moving towards the surface, which enhances the penetration of cells that seeded on the hydrogel surface and supports the formation of 3D cultures. The optically gel and the glass-bottom make this product ideal for microscopy imaging.

General protocol for cell seeding and culture maintenance

Before starting, please verify the good status of the plate. The polyethylene (PE) pouch must be intact with no visible liquid inside. The plate must be intact inside the pouch. Open the PE pouch with scissors or a scalpel under the sterile laminal flow bench. Once removed from the pouch, the plate is still sealed on its top-side with a polypropylene (PP) adhesive foil, which is keeping the content inside each of the 96 wells. From the glass bottom side, verify that the glass is intact and that the gels look transparent, as if the wells were filled with water. Please report any anomaly before use. lf all is normal, proceed as described below:

1. Adjust the assay plate.  Make sure the plate is at room temperature for at least 30 min prior to use.

2. Prepare the cells and media to be used. Prepare the cells and media in accordance with your own protocols or specific protocols provided by Ectica Technologies.

3. Remove the sealing adhesive foil. Carefully peel off the sealing foil from the assay plate. We suggest to firmly hold the plate with the left hand on the table and peel off the sealing foil with the right hand, starting from the top-right corner of the plate and slowly moving to the bottom-left corner. A liquid meniscus may form on top of the wells due to the negative pressure applied by removing the foil, but it will pop and disappe­ar within seconds.

4. Aspirate the storage buffer. Insert the pipet tip (ideally 200µL tip) in the well and descend along the side wall while aspirating the storage saline buffer, as shown in the schematic below. lt is possible to use multichannel pipets or pumps (lowest suction pressure) to accelerate the process. Avoid touching or aspirating right over the gel because of the risk of damaging it. lt is acceptable if some storage buffer remains in the well, since it is a Tris-based buffer which does not nega­tively affect the culture development. lt is normal to perceive a small suction resistance while aspirating the storage buffer. To minimize this effect gently retrieve the pipette tip and the liquid will be aspirated.

RemoveBuffer.jpg

5. Add cells and medium. Add the cell suspension with a maximum volume of 200 µL in each well, as shown in the schematic below. Transfer the plate to the incubator. Notice that residual trace activity of trypsin can result in digestion of the hydrogel. Especially when using serum-free media, ensure that following cell detach­ment, trypsin is inhibited by appropriate inhibitors or inactivating solutions (i.e. soybean). Minimize the time between steps 4 and 5 to reduce the risk of gel drying. The optimal seeding density depends on the culture type and readout. For specific suggestions refer to the cell culture protocols provided by Ectica Technologies. To better control the concentration of the medium components, it is possible to soak and rinse the hydrogel with the culture medium before the addition of the cells.

AddCellsandMedium.jpg

6. Establish the 3D cell culture. Maintain the cells in culture and change the culture medium every second day, applying similar pipetting techniques as in steps 4 and 5.

7. Proceed with sequential cell seeding (if applicable). At the desired time point, remove the culture medium and add a second cell population in the appropriate co-culture medium (maximum volume of 200 µL per well), as shown in the schematic below. Change the medium every other day.

SEQUENTIAL SEEDING.jpg

Sample handling after cell culture

Fixation and cell staining

  1. Remove the cell culture medium and wash with 200 µL/well PBS for 5 min at room temperature.

  2. Fix the cells with 200 µL/well of 4% formaldehyde in PBS for 30 min.

  3. Wash 3 times, each with 200 µL/well PBS for 5 min at room temperature.

  4. If cell permeabilization is necessary (intracellular epitopes), add 200 µL/well 0.3% TritonX-100 in PBS and incubate for 30 min at room temperature.

  5. Wash 3 times, each with 200 µL/well PBS for 5 min at room temperature.

  6. Block with 200 µL/well of PBS containing 5% donkey serum (blocking buffer) for at least 2h at room temperature. 

  7. Incubate overnight at 4°C with 100-150 µL/well of primary antibody solution in blocking buffer (for specific dilutions refer to the cell culture protocols provided by Ectica Technologies). We recommend performing a titration curve for each new antibody used. For most antibodies tested, higher dilution ratios gave better results.

  8. Add 200 µL/well of blocking buffer and wait 5 min at room temperature without shaking. Discard the solution, add again at least 200 µL/well of blocking buffer, and incubate for a minimum of 3h at room temperature without shaking.

  9. lncubate for 1h at room temperature with 100-150 µL/well of secondary antibody solution in blocking buffer (for specific dilutions refer to the cell culture protocols provided by Ectica Technologies). We recommend performing a titration curve for each new antibody used. For most antibodies tested, a dilution range of 1:200-1:500 resulted in better signal-to-noise ratios). Hoechst and phalloidin counterstaining can also be performed at this stage (1:500-1:1000 dilution).

  10. Repeat step 8.

Note: For extra-cellular matrix (ECM) protein staining, it is recommended to add the primary antibody to the cell culture medium prior to fixation and incubate for at least 4h at 37°C in a 5% CO2 humidified incubator. After this step the guidelines for fixation and staining outlined above can be followed.

Retrieval of the cellular and extracellular fractions

  1. Digest gels with 100 µL/well of a 0.25% trypsin/EDTA solution at 37 °C under continuous orbital shaking (at least 800 rpm). The time required for complete gel digestion may vary from 10 min to 1 h, depending on the cell type and cul­ture density. We recommend monitoring the gel dissolution microscopically to identify the optimal trypsinization time that leads to a single-cell dispersion, as shown in the figure below.

  2. Inhibit trypsin by adding 200 µ/well of FBS-containing medium or a trypsin inhibitor.

  3. Collect the digested samples in 1.5 mL Eppendorf tubes and centrifuge at 10,000xg for 15 min.

  4. Collect the supernatant, containing the extracellular fraction, for further proteomics analysis.

  5. The pellet, containing the cellular fraction, can used for FACS, DNA/RNA extraction, mass spectrometry, or other further appropriate assays.

Gel digestion.jpg

Figure 2. Hydrogel digestion process. An 11-day culture of adipose-derived MSCs on was subjected to digestion with 100 µUwell of a 0.25% Trypsin/EDTA solution at 37 °C under continuous orbital shaking at 800 rpm. Images were acquired at 4x magnification at the indicated time points to monitor the progression of the digestion. Shaking and imaging were performed on a Biotek Agilent Cytation 1. For such a high-density culture, complete digestion required 75 min of trypsinization. Scale bar= 1000 µm

Microscopy

Since the gels are transparent, cell seeding and growth can be easily fol­lowed with bright-field phase contrast microscopy.
Because the thickness of the gel is approximately 500 µm, imaging cultures up to the top of the gel using a confocal microscope requires the use of long-di­ stance objectives with a working distance of at least 0.7 mm.

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