SARTORIUS MSC NutriStem XF Basal Medium Instructions

Instructions for Use
MSC NutriStem®XF
A Defined, Xeno-Free (XF), Serum-Free (SF) Medium,
Designed to Support the Growth of hMSCwww.sartorius.com

A Defined, Xeno-Free (XF), Serum-Free (SF) Medium, Designed to Support the Growth of hMSC

MSC NutriStem®XFBasal Medium| MSC NutriStem®XFSupplement Mix| MSC NutriStem®XFBasal Medium,
without Phenol Red
---|---|---
05-200-1| 05-201-1| 05-202-1
2 – 8°C| -10°C to -20°C| v

Product Description and Important Notes

MSC NutriStem ®XF Medium is a serum-free, xeno-free medium formulation developed for the growth and expansion of human mesenchymal stem cells after being isolated from a variety of sources ( i.e., bone marrow, adipose tissue, umbilical cord tissue, and Dental pulp; BM-hMSC, AT-hMSC, UCT-hMSC, DP-hMSC). No adaptation is required for the transition from serum-containing medium to the MSC NutriStem®XF Medium.
MSC NutriStem®XF Medium is recommended for use with MSC Attachment Solution (Cat. No. 05-752-1) for optimal attachment and spreading of cells. MSC NutriStem®XF Medium supports the long-term growth of hMSC while maintaining its self-renewal and multi-lineage differentiation potential. For optimal cell passage and long-term culture of the cells, it is recommended to use Recombinant Trypsin Solution (03-078-1 or 03-079-1).
Isolation
For the initial isolation of hMSC, it is recommended to add 2 – 2.5% human AB serum to the complete medium to facilitate cell attachment and growth. (The requirement of human AB serum may be varied between different hMSC sources). The pre-coated step with MSC Attachment Solution is also required for the initial isolation of hMSC (with and without the addition of human AB serum.)
Alternatives to the pre-coating step
MSC NutriStem®XF complete medium may promote hMSC proliferation without a pre- coating step using advanced surface-treated cultureware (e.g. Corning CellBIND).
In addition, MSC NutriStem®XF complete medium supplemented with 5% human Platelet lysate (hPL) may also enable hMSC culturing w/o the pre-coating procedure.

Medium Components and Storage

MSC NutriStem ® XF Basal Medium,
without Phenol Red| 2 – 8°C| 05-202-1| 1 × 500 mL

NOTE

• No additional additives are required for the complete, ready-to-use medium.
• Contains L-glutamine.
• Does not contain antibiotics.
• Components are not sold separately.

Precaution and Disclaimer

• Do not use if a visible precipitate is observed in the medium.
• Do not use MSC NutriStem®XF Medium beyond the expiration date indicated on the product label.
• Please refer to the Safety Data Sheet (SDS) for hazard information.
• Do not use it if the liquid is present in the cap of the bottle.

Features

• Serum-free (SF), xeno-free (XF) medium: all components are defined and of non-xenogenic origin, including proteins.
• Enables culture of hMSC from different sources.
• Supports long-term growth of hMSC, retaining the fibroblast-like cell structure.
• No background differentiation.
• Maintains hMSC self-renewal and multi-lineage differentiation potential (e.g., osteocytes, adipocytes, and chondrocytes).
• Human MSC cultured with MSC NutriStem® XF express a high percentage of MSC surface markers and do not express hematopoietic markers.

 Adaptation of hMSC to MSCNutriSte

®XF MediumhMSC can be transferred directly to MSC NutriSystem ®XF Medium, without prior adaptation from any other culture media (including serum- containing medium).

Complete Ready-To-Use Medium Preparation

• The frozen MSC NutriSystem ®XF Supplement Mix should be thawed at 2 – 8°C or at room temperature. Avoid repeated freeze-thaw cycles (up to two times).
• For a complete medium, aseptically add 3 mL of MSC NutriStem®XF Supplement Mix to 500 mL of MSC NutriStem®XF Basal Medium. (Alternatively, aseptically add 0.6 mL of MSC NutriStem®XF Supplement Mix to 100 mL of MSC NutriStem®XF Basal Medium).
• MSC NutriStem®XF Basal Medium contains L-glutamine.
• Store at 2 – 8°C, protected from light.
• The complete MSC NutriStem®XF Medium is stable at 2 – 8°C for up to 30 days, protected from light.

Preparation of Pre-Coated

Cultureware with MSC Attachment Solution
(Cat. No. 05-752-1)

1. Dilute MSC Attachment Solution 1:100 using sterile DPBS (without Ca++ and Mg++ (Cat. No. 02-023-1) and gently mix using a pipette.
2. Add the diluted MSC Attachment Solution to the cultureware. Gently agitate the coated cultureware and verify the complete covering of the surface. Use Table 1 for recommended volumes.
3. Incubate the coated cultureware for at least 30 minutes in a humidified CO₂ incubator (37°C).
4. Following 30 minutes incubation:
   For immediate use:
   − Gently wash the cultureware with DPBS (For T-25 use at least 5 mL).
   − Seed cells immediately.
   It is critical that the coating does not dry out.
   For later use:
   − Wrap the coated cultureware with Parafilm® and incubate at 2 – 8°C. Coated cultureware stored under sterile conditions at 2 – 8°C is stable for 1 week.
   − Gently wash the cultureware with DPBS.
   − Seed cells immediately.
   It is critical that the coating does not dry out.
   Table 1: Recommended volume for the coating pocedure

Cultureware| Surface area cm²| Volume of 1:100 MSC Attachment Solution
---|---|---
96-well| 0.34| 0.1 mL
24-well| 1.9| 0.4 mL
12-well| 3.9| 0.8 mL
6-well/35 mm ware| 9.6| 2 mL
6 cm/T25 Flask| 25| 5 mL
T75 Flask| 75| 15 mL

Culturing of hMSC in complete MSC NutriStem® XF Medium

A . Recovery of Cryopreserved hMSC

1. Pre-warm 5 – 10 mL of complete MSC NutriStem ® XF Medium in a 50 mL conical tube.
2. Rapidly thaw a frozen vial of hMSC in a 37°C water bath, with agitation until a small amount of ice remains.
3. Slowly add (drop by drop while gently swirling) the cells into the pre-warmed complete MSC NutriStem ® XF Medium.
4. Centrifuge cells at 300 – 400 xg for 4 – 5 minutes at room temperature.
5. Remove supernatant and re-suspend cell pellet in 0.5 – 1 mL of complete MSC NutriStem ® XF Medium.
6. Perform a viable cell count (e.g., using Trypan Blue Exclusion Assay)
7. Add the desired volume of complete MSC NutriStem ® XF Medium.
8. Transfer the cells into MSC Attachment Solution pre-coated cultureware (see above). Seeding densities should be calculated (see table 2).
9. Incubate in a humidified CO₂ incubator (37°C).

NOTE
It is possible to avoid the centrifugation step after thawing. In this case, skip steps 4 – 5 and transfer the thawed cells (from Step 3) directly into the pre-coated culture flask using MSC Attachment Solution (Cat. No. 05-752-1) with the required volume of the complete MSCNutriStem® XF Medium, at a ratio of at least 1:10 (for the dilution of the DMSO).
B. Subculturing hMSC
MSC NutriStem® XF Medium was developed for optimal proliferation of MSC from a variety of sources (BM-hMSC, AT-hMSC, UCT-hMSC). The various sources and the variability of donors may influence hMSC proliferation rate. For the optimal proliferation of hMSC in MSC NutriStem ®XF Medium, it is recommended to seed hMSC at a concentration of 6000 – 5000 cells/cm² (Table 2), re-feed cells with fresh warmed complete MSC NutriStem® XF Medium every 2 – 3 days and subculture when the cells reach up to 80% confluence (usually 4 – 3 days post seeding). Avoid overgrown culture, as it leads to cell’s maturation and senescence.

Subculturing Protocol

1. Remove culture medium and gently wash once with DPBS w/o Ca, Mg (Cat. No. 02-023-1).
2. For T25 culture flask add 1 – 3 mL of Recombinant Trypsin Solution with or without EDTA (Cat. No. 03-078-1, 03-079-1). (For any other cultureware, the appropriate volume should be adjusted). NOTE The more the culture is confluence, the slower the detachment will be and the higher volume is recommended.
3. Incubate for 2 – 10 minutes at room temperature and verify cell detachment using an inverted microscope. (Incubation at 37°C will not accelerate detachment). Usually, within 2 – 5 minutes (at R.T.) the cells will dissociate by gently tapping the flask.
4. Following detachment, add 5 – 10 mL of pre-warmed MSC NutriStem ® XF. Alternatively use diluted (1:50, in DPBS) Soybean Trypsin Inhibitor (SBTI) (Cat. No. 03-048-1). Collect cell suspension into a sterile tube and re-wash the cultureware as necessary to collect the entire cells.
5. Centrifuge cells for 4 – 5 minutes at 300 – 400 xg at room temperature. Carefully discard the supernatant.
6. Re-suspend cell pellet in a minimal volume of pre-warmed complete MSC NutriStem ® XF Medium. Take sample volume to perform a viable cell count. For cryopreservation continue with section C.
7. Re-seed cells into pre-coated cultureware (see above). Seeding densities and the required volume of complete MSC NutriStem ® XF Medium to be added should be calculated (see Table 2).
8. Incubate in a humidified CO₂ incubator (37°C).
9. Re-feed cells with fresh warmed complete MSC NutriStem® XF Medium every 2 – 3 days.

Table 2: Recommended seeding densities (approximately 5000 – 6000 cells/cm2)

6 mL/T-25
Recommended| 1.8 – 2.1 × 10⁴| 4.3 – 5.3 × 10⁴| 11 – 14 × 10⁴
seeding densities| cells/well| cells/well| cells/well

C . Cryopreservation of hMSC

1. Rapidly re-suspend hMSC pellet with cold NutriFreez ® D10 Cryopreservation Medium (Cat. No. 05-713-1) (recommended between 0.5 – 1 × 10⁶ cells/mL, 1 mL/vial).
2. Immediately place the cryovials in an appropriate freezing container (e.g., “Mr. Frosty”) and place at -80°C for overnight.
3. Transfer the cryovials into liquid nitrogen.

Quality Control

MSC NutriStem® XF Medium performance is tested for optimal maintenance and expansion of undifferentiated hMSC, while maintaining their multilineage differentiation potential. Additional tests are pH, osmolality, endotoxins, and sterility tests. For full specifications please check the lot-specific Certificate of Analysis (CoA).

Quality Assurance

• For in vitro diagnostic use, research use, or for use as ancillary material in manufacturing cell, gene, and tissue-based products.

• Listed in Europe under CE IVD class I, thus complying with European In-Vitro Diagnostic Devices Directive (98/79/EC) requirements.

• Listed in the US FDA under IVD Class I.

• Manufactured under ISO 13485 QMS and in compliance with applicable cGMP guidelines.

• Manufactured under controlled environments and processes in accordance with:
  1. ISO 13408 – Aseptic Processing of Health Care Products;
  2. ISO 14644 – Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones.

• Submitted under US FDA MF (Master File) and Health Canada MF (Master File).

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

10.1 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.

Auxiliary 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 all genders.
Copyright notice:
These instructions, including all components, are 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.

© 2021
Biological Industries Israel Beit Haemek Ltd.
2511500 Kibbutz Beit Haemek Israel
AM | DIR: 2740208-000-01
Revision 02

References

• Sartorius | Biopharma, Laboratory, Applied & Life Sciences

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