Product Information

Catalog. No. Product Name Format Stock Conc. Storage on Arrival
ax494319 Hydrogel 5 ml syringe 5 mL N/A Follow the protocol
ax494319 Hydrogel 10 ml syringe 10 mL N/A Follow the protocol
ax494341 Cell Recovery Enzyme 2.5 ml 2.5 mL N/A Follow the protocol

Hydrogel

Axol’s biocompatible hydrogel is extracted from birch (Betula sp.), sourced from sustainable and responsibly managed forests. The Hydrogel consists of micro and nanosized cellulose fibrils, the length of fibrils may be several micrometers while the diameter is on the nanometer scale (4-100 nm). The product is supplied ready to use in a syringe format at a working concentration of 1.5% cellulose and 98.5% ultra-pure water. It is not a concentrate. The hydrogel is not auto fluorescent, is pH neutral and is compatible with automated liquid handling systems, high content imagers and standard microscopes set ups.

The hydrogel supports culture of a variety of cells including iPS, ES, cancer, retinal, neural cells as well as patient derived cells and biopsies and used for 3D organoids and spheroid culture, supporting cell growth and differentiation with consistent results. Tested applications range from 3D cell culture and organ-on-a-chip models, through to drug release studies and 3D printing.

Eliminating the need for temperature control on the bench, Axol’s hydrogel is stored, shipped and suitable for use at room temperature, reducing complexity and cost. There is no batch to batch variability.

Do not store the product below 0°C as freezing will result in destabilization of the product, rendering it unusable.

Storage Instructions

The product should be stored in the dark 5-22°C for optimum performance. Once opened it is recommended that the product is stored undiluted at 2-8°C for a maximum of 3 months. If the product has been diluted, e.g. with culture media, then it should be stored at 2-8°C for a maximum of 7 days. If the media contains an unstable component, then storage time will be restricted to the shelf life of the given component.

NB: The product has been autoclaved at a temperature of 120°C. The product should be handled in accordance with good industrial hygiene and safety practices. Use protective gloves and clothes to avoid skin exposure. If exposed, wash the exposed skin with water. Use protective laboratory eyewear to avoid contact with the eyes. In its wet state the product does not form dust. However, if dried, avoid breathing the dust. Dust filters are recommended.

General Recommendations

  • Use a low retention pipette tips to avoid Hydrogel sticking to the tip.
  • Aspirating and dispensing Hydrogel should be performed slowly to avoid air bubbles and to ensure an accurate volume.
  • For an exact amount of undiluted Hydrogel, the product can be weighed before dilution.
  • Electric dispensing pipettes can be used for mixing to reduce user variability.
  • A multi-stepper pipette is recommended for repeated dispensing of Hydrogel into the well plates.
  • If Hydrogel has accumulated on the walls of the vial or water droplets are present, then the vial can be centrifuged briefly to concentrate the product at the bottom for easier recovery.
  • Microplates containing Hydrogel should be handled with care. Avoid shaking the plate when moving it between locations.
  • When culturing adherent cells, the use of low-attachment microplates or pre-coating with polyHEMA is recommended to prevent cells attaching to the bottom of the wells.
  • When changing the medium, Extra care should be taken not to disturb the top of the gel. If loss of Hydrogel occurs when changing medium, then it is recommended to exchange only half the amount of media at one time.

Recommended Procedure for Diluting and Mixing Hydrogel

The viscosity can be adjusted by diluting the product to a less viscous state. Dilution can be made with cell culture media, PBS, or ultra-pure water. Hydrogel concentrations of 0.2-1.0% are typically used for cell culture applications. The optimal concentration will depend on the cell type being used. We recommend a concentration of 0.5% of Hydrogel, per cell seeding density of 1000 cells/μl.

Example dilution protocols for 3D cell culture experiments

Working concentration required = 0.5 %. Final volume = 1 ml.

  • Pipette 567 μl culture medium into a test tube.
  • Add 333 μl Hydrogel and mix by pipetting up and down until the solution is homogenous by visual inspection.
  • Add 100 μl cell suspension to the diluted Hydrogel slowly and stir carefully using the pipette tip to evenly disperse the cells.
  • Hydrogel is now ready for use at a working concentration of 0.5 %.

NB: Pre-diluted Hydrogel without cells can be stored for 7 days at 2-8°C if no unstable components are present in the media

Dilution Table

Volume of Hydrogel, diluent and cell suspension required for the preparation of  1 ml of diluted Hydrogel for a variety of final working concentrations.

Final Hydrogel Concentration Total Volume Volume of Hydrogel Stock Solution 1.5% Diluent Cell Suspension
1% 1 ml 667μl 233μl 100μl
0.9% 1 ml 600μl 300μl 100μl
0.8% 1 ml 533μl 367μl 100μl
0.7% 1 ml 467μl 433μl 100μl
0.6% 1 ml 400μl 500μl 100μl
0.5% 1 ml 333μl 567μl 100μl

Cell Recovery Enzyme

Axol’s Cell Recovery Enzyme is a cellulase enzyme mixture in aqueous sodium acetate buffer solution, pH 5. The Enzyme is exclusively designed for the digestion of the bio-friendly Axol’ Hydrogel enabling recovery of cells, spheroids, organoids or tissue samples intact.

Axol’s Cell Recovery Enzyme is applied using a very simple process. The enzyme is added before incubation where cells or organoids are recovered which eliminates the damage caused by repeated pipetting steps. Cells once recovered can be replated or used in downstream DNA/RNA analysis without interference from the hydrogel and the enzyme, which are both from a plant source. Axol’s Cell Recovery Enzyme and Hydrogels do not impact cell survival or function.

The enzyme is mixed with the sample and incubated at 37°C until the Hydrogel has been fully reduced to glucose. Enzymatic removal allows cells to be recovered from the matrix efficiently and retain their 3D structure. The amount of Cell Recovery Enzyme required for cell recovery is dependent on the amount of Hydrogel (cellulose) present in the sample. It is recommended that the enzyme be used at a working concentration of 300 μg/mg. An equal volume of working concentration of Cell Recovery Enzyme to Hydrogel/cell matrix volume present in the sample should be used, e.g. if 100 μl of Hydrogel/cell matrix is present in the sample then 100 μl of Cell Recovery Enzyme (300 μg/mg) should be added to the sample.

NB: Hazard Statements: The Cell Recovery Enzyme may produce an allergic reaction.

Storage Instructions

The unopened product should be stored in the dark at 5-22°C for optimum performance. Once opened it is recommended that the product is stored at 2-8°C for a maximum of 6 months. If the product has been diluted, e.g. with culture media, then it should be used immediately. Any leftover diluted enzyme should be discarded.

Procedure for Removing Hydrogel by Enzymatic Degradation

  • Calculate the amount of Hydrogel present in the sample well using the following equation, where 100 μl of 1% Hydrogel = 1 mg.
    Sample well volume (μl) x % Hydrogel concentration / 100 = mg Hydrogel / sample well.
  • Calculate the amount of enzyme needed to degrade the Hydrogel in the sample well using the following equation, where 300μg Enzyme is required to degrade 1mg of Hydrogel. Amount (mg) Hydrogel / sample well x 300 = μg Cell Recovery Enzyme.
  • Calculate the volume of Cell Recovery Enzyme stock solution needed using the following equation: Cell Recovery Enzyme amount (μg) / Cell Recovery Enzyme stock concentration (10 μg/μl) = Volume (μl) of Cell Recovery Enzyme stock solution.
  • Prepare the working concentration of Cell Recovery Enzyme by diluting the stock solution with culture media as follows: Sample well volume (μl) – Volume (μl) of Cell Recovery Enzyme stock solution (10 mg/ml) = Volume of culture media for dilution.
  • Pipette the diluted Cell Recovery Enzyme onto the top of the sample in the microplate. Incubate the plate at 37°C for a minimum of 8 hours until the hydrogel has fully degraded. Recover the cells from the well using standard techniques.

Example Experimental Procedure

Materials: 80 μl of 0.9% Hydrogel/cell mix per well in a 96-well microplate, Cell Recovery Enzyme (10 mg/ml), Cell culture medium.

  •  Amount of Hydrogel present in the sample: 80 μl x 0.9 / 100 = 0.72 mg Hydrogel / sample well
  • Amount of Cell Recovery Enzyme required to degrade Hydrogel in sample: 0.72 mg x 300 μg/mg = 216 μg Cell Recovery Enzyme
  • Volume of Cell Recovery Enzyme stock solution needed: 216 μl / 10 μg/μl = 21.6 μl Cell Recovery Enzyme stock solution.
  • Prepare the working concentration of Cell Recovery Enzyme by diluting the stock solution (10 mg/ml) with culture media:
    80 l – 21.6 μl = 58.4 μl culture media for dilution.
  • Pipette the diluted Cell Recovery Enzyme onto the top of the sample in the microplate. Incubate the plate at 37°C until the hydrogel has fully degraded. Recover the cells from the well using standard techniques.

Cell Recovery Enzyme Dilution Table

Volume of Cell Recovery Enzyme and cell culture medium required for the preparation of 100 μl of Cell Recovery Enzyme working solution (300 μg/mg) for the degradation of 100 μl of Hydrogel.

Hydrogel Conc. In 100 μl of Sample Amount of Cell Recovery Enzyme Needed Volume of Cell Recovery Enzyme Stock Solution (10 mg/ml) Volume of Cell Culture Medium
1% 300 μg 30 μl 70 μl
0.9% 270 μg 27 μl 27 μl
0.8% 240 μg 24 μl 76 μl
0.7% 210 μg 21 μl 79 μl
0.6% 180 μg 18 μl 82 μl
0.5% 150 μg 15 μl 85 μl
0.4% 120 μg 12 μl 88 μl
0.3% 90 μg 9 μl 91 μl
Menu