Technical

Guidelines for 3-D Cell Encapsulation in Extracel–X™ Hydrogels for Xenograft Applications

For research use only

Important Notices

  • These guidelines describe how to prepare Extracel-X™ hydrogels for encapsulation of cancer cells and injection of this suspension into experimental animals for research purposes only.
  • Researchers are responsible for obtaining a valid Institutional Animal Care and Use Committee (IACUC) protocol prior to initiation of any experiments (if applicable). The guidelines below only pertain to the operational use of the Extracel-X™ product in order to assist in preparing an IACUC protocol.
  • Glycosan recommends conducting a bench-top study with Extracel-X™ to confirm the Extracel-X™ characteristics prior to initiating animal experiments in order to gain familiarity with handling and timing of use. The gelation time and final hydrogel properties are highly dependent upon the medium used, the extent of hydrogel dilution, and the final hydrogel pH (see “Bench-Top Study” below).
  • Glycosan strongly urges researchers to conduct pilot animal studies to optimize experimental conditions and familiarize the researcher with the handling of Extracel-X™ prior to doing large-scale animal testing. The pilot study will provide important information on the time course for tumor growth from a given cell line or primary tumor source, optimal injection size, cell concentration, and Extracel-X™ dilution (see “Pilot Study” below).

Basis for Xenograft Guidelines

  • This protocol assumes that a suspension of 50 or 100 µl of Extracel-X™ + cells will be injected into nine research animals.
  • The cell loading and amount of injected Extracel-X™ hydrogel used depends upon your application. The amounts discussed in these guidelines are based on published tumor xenograft experiments[1,2] where a cell concentration of 5×107 cells/ml was employed. Lower concentrations may also be effective; however, they will require longer tumor-formation times. Please inquire with Glycosan Technical Services (801-583-8212), as experiments with additional injection volumes, cell concentrations, and cell lines are ongoing.
  •  A pilot animal study is strongly recommended to determine optimal cell loading for a given cell source.

Key Points

  • The exact time for the hydrogel to become viscous and gel depends on:
    • Medium used
    • Amount of Extracel-X™ dilution with medium
    • Hydrogel-solution pH
  • The medium used to dilute the Extracel-X™ and the amount of dilution can profoundly affect the gelation time. The greater the medium dilution, the longer the gelation time. See theExtracel-X™ Hydrogel Kit Product Data Sheet for details or call Glycosan Technical Services (801-583-8212).
  • The pH of Extracel-X™ is controlled to ~7.4 prior to cell encapsulation and medium dilution. However, the cell-culture medium used can increase or decrease the pH and change the gelation time. For Extracel-X™, the higher the pH, the faster the gelation time.
  • If stiffer hydrogels are required, increase the amount of Extralink™ used from a 1:4 ratio of Extralink™ to Glycosil™ + Gelin-S™ to a 1:3 ratio or decrease the medium dilution.
  • Extracel-X™ forms a hydrogel by reacting thiols in Glycosil™ and Gelin-S™ with the thiol-reactive crosslinker, Extralink™. Both Glycosil™ and Gelin-S™ can form hydrogels in the absence of Extralink™ via disulfide bond formation; however, this reaction is normally very slow (hours instead of minutes).

Bench-Top Study

Design a pilot animal experiment based on the guidelines below (“Suggested Pilot Study”). Using the same conditions that will be used in the pilot animal study, perform gelation tests with Extracel-X™ as follows:

  1. Prepare cells in medium at the proper cell density/densities.
  2. Make up the Extracel-X™ Hydrogel Kit per instructions (see the Extracel-X™ Hydrogel Kit Product Data Sheet). Freeze unused solutions at -20 °C, unless they will be used in less than four hours.
  3. For each condition, add 0.25 mL of Glycosil™ and 0.25 mL of Gelin-S™ to a small glass vial. Pipette back and forth to mix.
  4. Add the appropriate amount of cells in medium to the Glycosil™ + Gelin-S™ mix (0.05 mL for 10% dilution, 0.50 mL of 50% dilution). Pipette to mix.
  5. Add 0.125 mL of Extralink™ to the vial. Record the time. Pipette to mix. The initial solution will be low viscosity (similar to medium).
  6. Every few minutes, invert the vial. As the hydrogel forms, the liquid will become more viscous. Record the time at which the hydrogel no longer flows when the vial is inverted.
  7. The gelation time is the difference between the two recorded times. This establishes the maximum length of time you will have to use Extracel-X™ after the Extralink™ is added.

Note: If you are uncertain about the type of medium or dilution, please determine the gelation time for all of the most likely conditions.

Note: If you desire a longer gelation time, increase the amount of medium used to more than 40% dilution (see the Extracel-X™ Hydrogel Kit Product Data Sheet).

Note: If problems occur or if you have questions, please call Glycosan Technical Services at (801) 583-8212.

Suggested Pilot Study

Please use this pilot study to determine the optimal:

  • Extracel-X™ dilution with medium
  • Cell density
  • Hydrogel-injection volume
  •  Coordination of surgical or injection manipulations with hydrogel handling

This protocol is based on nine mice with four subcutaneous injections each (see Table 1 below), where each experimental condition (cell density and injection volume) has an “n=3” (see Table 2 below). Please adjust this protocol (cell density and injection volume especially) as required based on your experimental requirements and experience.

Table 1. Injections on each mouse

Inj 1: Control injection: Cells + PBS
Inj 2: 90% Extracel-X™ + 10% Cells + medium
Inj 3: 70% Extracel-X™ + 30% Cells + medium
Inj 4: 50% Extracel-X™ + 50% Cells + medium

Table 2. Pilot Study Conditions

  Cell density, cells/mL Injection volume, L
Mice 1-3 1 x 107 100
Mice 4-6 1 x 107 50
Mice 7-9 1 x 106 50

 

Prepare the Extracel-X™ hydrogels as follows:

  1. Prepare Extracel-X™ Hydrogel Kit components under aseptic conditions as directed by the instructions in the Extracel-X™ Hydrogel Kit Product Data Sheet.
  2. Prepare cells for encapsulation with Extracel-X™ by resuspending them in the relevant sterile cell-culture medium to the appropriate cell density and volume (1 x 10 7 cells/mL for 50 and 100 µL injections and 1 x 10 6 cells/mL for 50 µL injections).
  3. Prepare the research animals for surgery as dictated by your approved IACUC protocol and sterilize the sites for surgery with iodine and alcohol swabs.
  4. Add the appropriate volume of cells + medium to the appropriate amount of Glycosil™ + Gelin-S™. Mix the resulting suspension by gently vortexing or pipetting. Note: Freeze unused Glycosil™ and Gelin-S™ solutions at -20 °C for future use as soon as the lyophilized pellets are dissolved.

    For three 100-µL injections (all volumes are in µL):
      Glycosil™ Gelin-S™ Extralink™ Cells + medium Cells only Total Volume
    Inj 1: Control injection: Cells + PBS n/a n/a n/a n/a 300 300
    Inj 2: 90% Extracel-X™ + 10% Cells + medium 150 150 75 30 n/a 405
    Inj 3: 70% Extracel-X™ + 30% Cells + medium 100 100 50 140 n/a 390
    Inj 4: 50% Extracel-X™ + 50% Cells + medium 100 100 50 200 n/a 450
    For three 50-µL injections (all volumes are in µL):
      Glycosil™ Gelin-S™ Extralink™ Cells + medium Cells only Total Volume
    Inj 1: Control injection: Cells + PBS n/a n/a n/a n/a 150 150
    Inj 2: 90% Extracel-X™ + 10% Cells + medium 75 75 38 15 n/a 203
    Inj 3: 70% Extracel-X™ + 30% Cells + medium 50 50 25 70 n/a 195
    Inj 4: 50% Extracel-X™ + 50% Cells + medium 50 50 25 100 n/a 225
  5. When the animals are ready for injection of the hydrogel, add the appropriate amount of Extralink™ to the cells + Glycosil™ + Gelin-S™. Mix the resulting suspension by gently vortexing or pipetting. Note: Once the Extralink™ is added to the Glycosil™ + Gelin-S™ + cells, you have between 20 minutes and an hour before the hydrogel forms. Prepare accordingly. If you cannot inject all the animals within this amount of time, consider aliquoting the cells + Glycosil™ + Gelin-S™ into individual injection amounts and adding the Extralink™ just prior to injection into each animal.
  6. Draw the Extracel-X™ + cells into a sterile syringe with a 20-gauge needle.
  7. Inject the required amount of hydrogel into the research animal at the desired location.
  8. After injection, properly care for the research animals.

References

1 Liu, Y., Shu, X. Z., Prestwich, G. D., “Tumor engineering: orthotopic cancer models in mice using cell-loaded, injectable, cross-linked hyaluronan-derived hydrogels”, Tissue Engineering 13(5), 1091-1101 (2007).
2 Prestwich, G. D., Liu, Y., Yu, B., Shu, X. Z., Scott, A., “3-D culture in synthetic extracellular matrices: New tissue models for drug toxicology and cancer drug discovery,” in press in Advances in Enzyme Regulation (2007).