Recover cells from your Cell-Mate3D™ cultures. Enzymes break down the Cell-Mate3D™ matrix at a physiological pH, releasing cells for downstream applications.

Quick and Easy Cell Collection

• 15 minute digestion time
• Perform at room temperature
• Up to 85% viability

Maintain and Analyze Your Cultures

• Passage your 3D cultures
• Perform Flow Cytometry
• Sort specific cell populations

Exposure to Cell Retrieval Enzymes Preserves Extracellular Markers

Cancer Stem Cell Markers

HeLa cells were retrieved from Cell-Mate3D™ culture,
stained with CD44-AlexaFluor 488 and CD24-Brilliant
Violet 421 antibodies and analyzed by Flow Cytometry.

Immune Markers

Mouse primary splenocytes were exposed to cell retrieval enzymes
and stained with CD45 FITC (leukocytes) CD3 PE (T-lymphocytes) &
CD19 APC (B cells) antibodies and analyzed by Flow Cytometry.

Viability Staining Post Cell Retrieval

Viability will vary depending on cell type and duration of culture. HeLa cells were isolated from Cell-Mate3D™ culture, stained with Calcein AM (live-green) and Ethidium Homodimer 1 (dead-red) and imaged using confocal fluorescent microscopy. In this case, viability was greater than 80%.

Contents:

Enzyme Blend: Hyaluronidase (CAS# 37326-33-3) and
Chitosanase (CAS# 51570-20-8).

Storage/Handling:

Store at -20°C for up to 1 year.

Safety:

Use appropriate personal protective equipment when
handling enzymes.

Stability:

Once reconstituted, enzymes are unstable and must be used
immediately. Reconstituted enzymes cannot be frozen and
thawed for later use. Vials are single use only.

Assessing Viability:

Viability should be assessed using Calcein AM and Ethidium Homodimer 1. Trypan staining is not suitable for use after cell retrieval because Trypan stains trace amounts of matrix material on the cell membrane.

Materials And Reagents Required:

• 100?m cell strainer
• Conical tube suitable for use with cell strainer
• .22?m syringe filter and syringe (>5mL)
• 24-well cell culture plate
• P-1000 pipet and tips
• Phosphate buffered saline (PBS)

• Bovine Serum Albumin (BSA)
• Complete cell culture media specific to cell type being used
• 125?L cell embedded Cell-Mate3D™ matrix
• Viability Staining Kit. For example use Molecular Probes
• Live/Dead Cell Imaging Kit1 (Thermo Fisher, Cat# R37601)
  1) Formerly Invitrogen/Life Technologies brand.

Before You Begin:

Prepare the Enzyme Dilution Buffer: 1mg/ml BSA in PBS.

Protocol:

1. Reconstitute Enzyme Blend in 1mL of enzyme dilution buffer.
2. Add 200?L of reconstituted enzyme blend to 4.8mL of complete cell culture media. Filter-sterilize with a 0.22?m syringe
filter. Allow solution to equilibrate to room temperature.
3. In a 24-well plate, digest one 125?L matrix with 1mL of the sterilized enzyme solution in a 37°C CO2 incubator for a
total of 15 minutes:

• • After 3 minutes, gently break the matrix apart with the pipette tip. Repeat every 3-4 minutes.
  • After 15 minutes, pipet up and down repeatedly until the majority of the matrix goes into solution. Some small fragments may remain.

4. Place the cell strainer onto a conical tube and transfer the cell-matrix mixture onto the cell strainer.
5. Wash by pipetting 7mL of media or PBS over the mixture while moving the pipette back on forth on the cell strainer. Use
3 mL of media or PBS to wash the well of the 24 well plate and pass through the cell strainer.
6. Pellet the cells at 120G (or speed required for your cell type) for 5 minutes.
7. Remove the supernatant without disturbing the cell pellet. Culture or process cells as desired. If preparing cells for
Flow Cytometry, filter samples before analysis to prevent clogs.

Trouble Shooting Guide:

1. If experiencing excess cell death, shorten the incubation period to 10 minutes and/or use gentler pipetting methods.
2. If using a smaller Cell-Mate3D™ matrix piece, digest at a ratio of 1:8 matrix to diluted enzyme solution.
3. If complex, tissue-like structures have formed in your Cell-Mate3D™ cultures, those structures may not pass through the
filter and can be collected off of the filter top for further treatment.

• Cell Retrieval Protocol for Cell-Mate3D
• SDS (U.S.)
• SDS (Europe)
• SDS (French)