SARTORIUS NutriFreez D10 Cryopreservation Medium Instruction Manual

NutriFreez D10 Cryopreservation Medium
Instruction Manual

Instructions for Use
NutriFreez® D10 Cryopreservation Medium
Animal Component-Free (ACF), chemically defined, protein-free, cryopreservation medium with 10% DMSO

| 05-713-1| 05-714-1 (Phenol red free version)
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| 2 – 8°C|

Product Description

NutriFreez® D10 Cryopreservation Medium is a unique animal component-free, chemically defined, serum-free, protein-free, cryopreservation formulation containing Methylcellulose and 10% Dimethyl Sulfoxide (DMSO). NutriFreez® D10 Cryopreservation Medium is designed to maintain a multitude of cell types, even extremely sensitive cells, in ultra-low temperatures (-196°C) by providing a defined, protective environment. NutriFreez® D10 Cryopreservation Medium ensures high viability, recovery rates and performance with a variety of cells, including primary human cells, and mesenchymal stem cells.
NutriFreez® D10 Cryopreservation Medium is optimally formulated and does not contain any antibiotics, antimycotics, hormones, growth factors, serum, or protein.

Cell Type Applications

• Human Mesenchymal Stem Cells (hMSC), freshly isolated-expanded and
  – commercial from various sources:
  – Bone Marrow (BM-MSC)
  – Adipose Tissue (AT-MSC)
  – Umbilical Cord Tissue (UC-MSC)
  – Dental Pulp Tissue (DP-hMSC)

• Human Embryonic Stem Cells (hESC), Induced Pluripotent Stem Cells (iPSC)

• Human Peripheral Blood Mononuclear Cells (PBMC)

• Human Endothelial Cells (EC)

• T cells, including Chimeric Antigen Receptors (CAR-T) cells and TumorInfiltrating Lymphocytes (TIL)

• Neurons, Astrocytes

• Hybridomas

• CHO cells

• Vero cells

• Multiple mammalian cell lines: MRC-5, HEK-293, HepG2, HeLa, BSC-1, BGM, 3T3, MA-10, BHK-21 as well as other extremely sensitive cell types

Precaution and Disclaimer

• Do not use if a visible precipitate is observed in the freezing medium
• Do not use beyond the expiration date indicated on the product label
• Store at 2­8°C
• Avoid exposure to light
• Please refer to the Material Safety Data Sheet (MSDS) for hazard information
• Maintain aseptic work conditions
• Caution: not for direct administration into humans or animals

NOTE Precipitates might occur when using NutriFreez® D10 Cryopreservation
Medium in combination with Trypan Blue.

Storage and Stability

NutriFreez® D10 Cryopreservation Medium should be stored at 2 ­ 8°C.

Features

• Ready-to-use
• Chemically defined, serum-free, protein-free, animal component-free
• High post-thaw viability and recovery of cells
• Maintains stem cell pluripotency and expansion capabilities

Instructions for use

I . General Instructions for Use for the Cryopreservation of Mammalian Cells
NOTE

• For freezing human Induced Pluripotent and Embryonic Stem Cells- (hPSC) see section

• For freezing human Mesenchymal Stem Cells (hMSC) see section III.
  – Keep NutriFreez® D10 Cryopreservation Medium on the ice at all times during – use.

• For freezing adherent cells, detach cells using a dissociation solution according to the manual instructions.

Freezing procedure

1. To maintain aseptic work conditions, wipe the outer packaging with a cloth moistened in 70% Ethanol/70% Isopropanol before opening the NutriFreez® D10 Cryopreservation Medium.

2. Centrifuge cells to obtain a cell pellet, 300 ­ 400 xg for 4 ­ 5 minutes, then aseptically decant supernatant without affecting the cell pellet.

3. Suspend the pellet in cold (2 ­ 8°C) NutriFreez® D10 Cryopreservation Medium, mix thoroughly, and transfer the suspension to a cryovial (e.g., 1.0 mL of suspension in a 1.5 mL cryovial).
   NOTE If freezing multiple cryovials, keep the cells on ice at all times. Gently mix the resuspended cell solution frequently to ensure even distribution throughout the vials. Immediately transfer filled cryovials to ice before aliquotting the remaining cell solution.

4. Freeze the cells gradually (1 ­ 2°C per minute) by using a controlled rate freezing system and store the vials in liquid nitrogen (vapor phase). Alternatively, place the vials in an appropriate freezing container (e.g. Mr. Frosty) and transfer them to -80°C overnight. The following day transfer cryovials into liquid nitrogen (vapor phase recommended).
   NOTE Long-term storage at -80°C is not recommended.

5. It is recommended to determine the efficiency of cryopreservation by thawing one vial after 24 hours of storage in liquid nitrogen and following the thawing procedure outlined below.

Thawing of cryopreserved cells:

1. They briefly warm the culture medium of choice in a 37°C water bath.
2. Rapidly thaw the cryovial of cells in a 37°C water bath by gently shaking the vial and removing the vial when only a small frozen cell pellet remains. Do not vortex cells.
3. Disinfect the vial by wiping it down with a cloth moistened with 70% Ethanol or Isopropanol.
4. Suspend the cells in a warmed growth culture medium at a ratio of at least 1:10 (cell suspension to culture medium).
5. Centrifuge cells to obtain a cell pellet, 300 ­ 400 xg for 4 ­ 5 minutes, then aseptically decant supernatant without affecting the cell pellet and resuspend in growth medium as desired.
6. Culture the cells according to the recommended seeding density.

II . Instructions for Use for the Cryopreservation of Human Induced
Pluripotent and Embryonic Stem Cells (hPSC)
NOTE

• hPSC may be frozen as clumps or single cells with high viability and minimal differentiation post-thaw.
• The single cells can be thawed onto recombinant Laminin coated culture ware without the addition of ROCK inhibitors. In the case of using other matrices (e.g. MatrigelTM), a Rock inhibitor is required. Keep NutriFreez® D10 Cryopreservation Medium on the ice at all times during use.

The procedure describes the cryopreservation of cells cultured in a 6-well plate.

1. Using a vacuum aspirator and a sterile aspirator pipette, remove the hPSC culture medium from the cultureware or well(s) to be harvested for cryopreservation.

2. Rinse wells with Dulbecco’s PBS w/o Ca & Mg (Cat. No. 02-023-1), using approximately 2 mL of DPBS per 10cm² culture surface area, then aspirate out the DPBS.

3. Determine the desired viable cell density and calculate the required volume of NutriFreez® D10 Cryopreservation Medium needed for a concentration of approximately 1 × 10 viable cells/mL.

4. Add dissociation solution as desired. Cells may be detached using the enzyme and method that the culture has been routinely passaged with. In case of using Collagenase, Dispase or EDTA, incubate at 37°C or at room temperature until the edges of the colonies begin to loosen from the plate.
   NOTE Incubation times may vary between cell lines, colony size and the detachment solution used. Begin checking the culture after 3 minutes.

5. Cells cultured on Laminin may be detached using Recombinant Trypsin EDTA Solution (Cat. No. 03-079-1) to yield a single cell suspension.
   NOTE Once the cells are detached from the surface, neutralize the enzyme by adding 2 ­ 4 volumes of pre-warmed complete medium to the volume of the trypsin solution used. Alternatively, 1X Soybean Trypsin Inhibitor (SBTI) solution (Cat. No. 03-048-1) diluted in DPBS can be used to neutralize the trypsin.

6. Transfer the clumps or cell suspension to a centrifuge tube.

7. Centrifuge at 200 x g for 5 minutes at room temperature then aseptically decant supernatant without affecting the cell pellet.

8. Resuspend the cell pellet in the pre-determined volume of cold NutriFreez® D10 Cryopreservation Medium on ice (1mL for every 1 × 10 viable cells). In the case of aggregates; do not break up cell masses any more than necessary, two or three gentle pipetting motions are usually sufficient.

9. Dispense aliquots of this suspension into cryo vials (e.g., 1.0 mL of suspension in a 1.5 mL cryovial)
   NOTE If freezing multiple cryovials, keep the cells on ice at all times. Gently mix the resuspended cell solution frequently to ensure even distribution throughout the vials. Immediately transfer filled cryovials to ice before aliquotting the remaining cell solution.

10. Freeze the cells gradually (1 ­ 2°C per minute) by using a controlled rate freezing system and store the vials in liquid nitrogen (vapor phase). Alternatively, place the vials in an appropriate freezing container (e.g. Mr. Frosty) and transfer to -80°C overnight.

11. The following day, transfer vials to liquid nitrogen storage (vapor phase).
    NOTE Long term storage at -80°C is not recommended

Thawing of Cryopreserved hPSC

1. Briefly warm NutriStem® hPSC XF Medium, or other growth culture media of choice, in a 37°C water bath.
2. Add 9 mL of warmed NutriStem® hPSC XF Medium, or other growth culture media, into a centrifuge tube.
3. Rapidly thaw the cryovial of cells in a 37°C water bath by gently shaking the vial and removing the vial when only a small frozen cell pellet remains. Do not vortex cells.
4. Disinfect the vial by wiping it down with a cloth moistened with 70% Ethanol or Isopropanol.
5. In a sterile biological safety cabinet, transfer the contents of the cryovial drop by drop into the 9 mL of culture medium in the previously prepared centrifuge tube. Gently rock to continually mix the cells as the new cell droplets are added to the tube.
6. Centrifuge the cells at 200 xg for 5 minutes. Remove and discard the supernatant.
7. Gently resuspend the cell pellet in NutriStem® hPSC XF Medium (Cat. No. 05-100-1) or other growth culture media, and plate as desired.
8. Refresh culture medium 48 hrs after plating.

III . instructions for Use for Cryopreservation of Human Mesenchymal Stem Cells (hMSC)
-NOTE
Keep NutriFreez® D10 Cryopreservation Medium on the ice at all times during use
Freezing procedure of hMSC

1. Using a vacuum aspirator and a sterile aspirator pipette, remove the hMSC culture medium from the cultureware or well(s) to be harvested for cryopreservation.

2. Rinse wells with Dulbecco’s PBS w/o Ca & Mg (Cat. No. 02-023-1), using approximately 2 mL of DPBS per 10 cm culture surface area, then aspirate out the DPBS.

3. Detach adherent hMSC using a sufficient volume of Recombinant Trypsin Solution (Cat. No. 03-078-1) to cover the entire cell culture surface and incubate the cells at room temperature or 37°C for 3 to 5 minutes.
   NOTE Recombinant Trypsin-EDTA Solution (Cat. No. 03-079-1) can be used if the cells are over-confluent or are difficult to detach after a short incubation with Recombinant Trypsin Solution.

4. Observe the cells under a microscope. If less than 90% of the cells are detached from the culture surface, continue incubating and observe again at 1-minute intervals to check for complete detachment.
   NOTE Incubation times will vary between cells and confluency levels. Begin checking the cultures after 3 minutes. Do not over-incubate the culture, as MSC can be sensitive to enzymatic stress. Tap the vessel periodically to expedite cell detachment and monitor the progress of the enzyme solution.

5. Once the cells are detached from the surface, neutralize the action of the trypsin enzyme by adding a volume of pre-warmed complete medium that is 2 to 4 times the volume of the trypsin solution used.
   NOTE Alternatively, 1X Soybean Trypsin Inhibitor (SBTI) solution (Cat. No. 03-048-1) diluted in DPBS can be used to neutralize the trypsin.

6. Collect the cell suspension and transfer to a centrifuge tube. If needed, rinse the cultureware with additional media to collect any remaining cells, and transfer to the same tube.

7. Centrifuge at 300 xg for 5 minutes at room temperature, and then aseptically decant supernatant without affecting the cell pellet.

8. Resuspend the pellet in the amount of complete media (approximately 3 ­ 5 mL) required to perform a cell count to determine the total viable cell number (e.g, using Trypan Blue Exclusion Assay).

9. Centrifuge the cell suspension at 300 xg for 5 minutes at room temperature.

10. Determine the required volume of NutriFreez® D10 Cryopreservation Medium is needed for a concentration of approximately 1 × 10 viable cells/mL.

11. Remove the supernatant from the centrifuge tube and quickly but gently resuspend the pellet in cold NutriFreez® D10 Cryopreservation Medium according to the freezing volume determined in the previous step.

12. Dispense aliquots of this suspension into cryovials (e.g., 1.0 mL of suspension in a 1.5 mL cryovial).
    NOTE If freezing multiple cryovials, keep the cells on ice at all times. Gently mix the resuspended cell solution frequently to ensure even distribution throughout the vials. Immediately transfer filled cryovials to ice before aliquotting the remaining cell solution.

13. Freeze the cells gradually (1 ­ 2°C per minute) by using a controlled rate freezing system and store the vials in liquid nitrogen (vapor phase). Alternatively, place the vials in an appropriate freezing container (e.g. Mr. Frosty) and transfer to -80°C for overnight.

14. The following day, transfer the cryovials into liquid nitrogen (vapor phase).
    NOTE long-term storage at -80°C is not recommended.

It is recommended to determine the efficiency of the cryo-preservation by thawing one vial after 24 hours of storage in liquid nitrogen and following the thawing procedure outlined below.
Thawing of Cryopreserved hMSCs

1. Briefly warm 5 ­ 10 mL of complete MSC NutriStem® XF Medium (Cat. No. 05-200-1 and 05-201-1) or other growth culture medium, in a 50 mL centrifuge tube.

2. Rapidly thaw a cryovial of hMSC in a 37°C water bath, by gently shaking the vial and removing the vial when only a small frozen cell pellet remains. Do not vortex cells.

3. Disinfect the vial by wiping it down with a cloth moistened with 70% Ethanol or Isopropanol.

4. In a sterile biological safety cabinet, transfer the contents of the cryovial drop by drop into the culture medium in the previously prepared centrifuge tube. Gently rock to continually mix the cells as the new cell droplets are added to the tube, then resuspend the cells by carefully pipetting up and down.

5. Centrifuge cells at 300 xg for 4 ­ 5 minutes at room temperature.
   NOTE It is possible to skip the centrifugation step after thawing by simply transferring the thawed cells directly onto a culture ware with the medium at a ratio of at least 1:10 (for the dilution of the DMSO).

6. Remove the supernatant and resuspend cell pellet in 0.5 ­ 1 mL of complete MSC NutriStem® XF Medium or another culture medium.

7. Perform a viable cell count (e.g, using Trypan Blue Exclusion Assay).

8. Add the desired volume of complete MSC NutriStem® XF Medium or another culture medium.

9. Culture cells as desired and incubate in a humidified CO incubator (37°C).

10. Refresh culture medium 48 hrs. after plating.

Quality Control

Each lot is tested for pH, sterility and recovery, and morphology of Vero cells, and human stem cells (hPSC and hMSC). For full specifications please check the lot-specific Certificate of Analysis (CoA).

Quality Assurance

• For in vitro diagnostic use, research use, or use as ancillary material in a manufacturing cell, gene or tissue-based products.
• Notified under US FDA IVD part 864.9225 Cell-freezing apparatus and reagents for in vitro diagnostic use.
• Listed in Europe under CE IVD class I, thus comply with European In-Vitro Diagnostic Devices Directive (98/79/EC) requirements.
• Manufactured under ISO 13485 QMS and in compliance with applicable cGMP guidelines.
• Manufactured under controlled environments and processes in accordance with:
  – ISO 13408 ­ Aseptic Processing of Health Care Products
  – ISO 14644 ­ Cleanrooms and associated controlled environments

Authorized Representative in the European Community:
MedNet GmbH
Borkstraße 10, 48163 Münster, Germany
Manufacturer
Biological Industries Israel Beit Haemek Ltd. Kibbutz Beit Haemek 2511500, Israel
8.1 Product Label Symbols

| Indicates the manufacturer’s catalog number so that the product can be identified.
---|---
| Indicates the manufacturer’s batch code so that the batch or lot can be identified.
NOTE Synonyms for batch code are lot number and batch number.
| Indicates the date after which the product is not to be used.
| Indicates the temperature limits to which the product can be safely exposed.
| Indicates a product that has been manufactured using accepted aseptic techniques.
****| Indicates that the product meets the requirements of the applicable EC directives
|  Indicates a product that is intended to be used as an in vitro diagnostic medical device.
|  Indicates the need for the user to consult the instructions for use.

Related Products

Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 11
37079 Goettingen, Germany
Phone: +49 551 308 0
www.sartorius.com
The information and figures contained in these instructions correspond to the version date specified below.
Sartorius reserves the right to make changes to the technology, features, specifications, and design of the equipment without notice.
Masculine or feminine forms are used to facilitate legibility in these instructions and always simultaneously denote the other gender as well.
Copyright notice:
This instruction manual, including all of its components, is protected by copyright.
Any use beyond the limits of the copyright law is not permitted without our approval.
This applies in particular to reprinting, translation, and editing irrespective of the type of media used.
© Sartorius Germany

Printed in the EU on paper bleached
without chlorine. | MB
DIR: 2740223-000-00
Revision 01

References

• Sartorius | Biopharma, Laboratory, Applied & Life Sciences

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