Human Mesenchymal Stem Cells and Growth/Differentiation Media

These products are available in the US, Canada, and select European countries.

• Primary Human Mesenchymal Stem Cell Culture System
• Protocols for Human Mesenchymal Stem Cell Cultivation
• Human Mesenchymal Stem Cell Culture System Quality Control
• Materials for Human Mesenchymal Cell Culture

Primary Human Mesenchymal Stem Cell Culture System

Mesenchymal Stem Cells (MSC), also called Mesenchymal Stromal Cells, are self-renewing multipotent cells that can differentiate into diverse cell types. MSC have been shown to differentiate in vitro into adipocytes, chondrocytes, osteoblasts, myocytes, and ß-pancreatic islet cells. MSC can also transdifferentiate into neuronal cells and hepatocytes.

PromoCell^® Mesenchymal Stem Cells are produced from normal human tissues of varied origin including bone marrow, umbilical cord matrix, and adipose tissue. Differentiation of MSC into adipocytes, osteoblasts, chondrocytes, and neuronal lineages can be performed using the Mesenchymal Stem Cell Differentiation Media. Shortly after isolation, all Mesenchymal Stem Cells are cryopreserved using proprietary, serum-free freezing medium, Cryo-SFM. Thawing and seeding the cells constitutes passage 2 (P2). Each cryovial contains at least 500,000 viable cells when thawed. Human Mesenchymal Stem Cells are also available as proliferating cell cultures generated from cryopreserved cells that have been thawed and cultured for three days.

Mesenchymal Stem Cell Media was developed for the in vitro expansion and directed differentiation of mesenchymal stem cells (MSC) isolated from bone marrow, the umbilical cord matrix (Wharton´s Jelly), or adipose tissue. Mesenchymal Stem Cell Media is available as Medium (ready-to-use) and consists of a bottle of Basal Medium and one vial of SupplementMix. Adding the SupplementMix to the Basal Medium constitutes the complete Medium.

MSC Expansion Media	MSC Differentiation Media
Mesenchymal Stem Cell Growth Medium 2 (optimized low-serum formulation) Mesenchymal Stem Cell Growth Medium DXF (defined, xeno-free formulation, which requires fibronectin-coated vessels)	Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 Mesenchymal Stem Cell Osteogenic Differentiation Medium Mesenchymal Stem Cell Chondrogenic Differentiation Medium Mesenchymal Stem Cell Chondrogenic Differentiation Medium w/o Inducers Mesenchymal Stem Cell Neurogenic Differentiation Medium

Mesenchymal Stem Cell Media Supplementation Details

Mesenchymal Stem Cell Media contains all of the growth factors and supplements needed for the optimal expansion and directed differentiation of human mesenchymal stem cells. The Mesenchymal Stem Cell Growth Medium DXF, the MSC Chondrogenic Differentiation Medium, the MSC Chondrogenic Differentiation Medium w/o Inducers, and the MSC Neurogenic Differentiation Medium are all defined and serum-free. Mesenchymal Stem Cell Media do not contain antibiotics or antimycotics and are formulated for use in an incubator with an atmosphere of 5% CO₂.

Note: The MSC Chondrogenic Differentiation Medium w/o Inducers must be supplemented by the user with adequate chondrogenic inducers.

Culture vessel surface coating for MSC culture

The MSC Growth Medium DXF does not contain attachment and spreading factors. Therefore, culture vessels used with MSC Growth Medium DXF must be precoated with 10 μg/mL human fibronectin according to the manufacturers protocol. Alternatively, bovine fibronectin may be used.

Preparing Supplemented Media for MSC Culture

• Thaw the SupplementMix in a 37°C water or thermal bath with occasional swirling. Important note: Do not incubate longer than necessary. In case of visible precipitates after complete thawing, mix gently until all precipitates have re-dissolved.
• Transfer the entire contents of each supplement to the Basal Medium.
• Close the bottle and swirl gently until a homogeneous mixture is formed.

Storage and Stability of MSC Products

• Upon arrival, immediately store the Basal Medium at 4 – 8°C in the dark; do not freeze the Basal Medium. Store the SupplementMix at -20°C.
• If stored as directed, the products are stable until the stated expiration date.
• Once supplements are added to the Basal Medium, the shelf life of the complete medium is six weeks at 4 – 8°C. Do not freeze the complete medium.
• For use, aliquot and prewarm just the volume of the complete medium required for single use, and maintain the remaining medium refrigerated at 4 – 8°C.
• Upon delivery, cryopreserved MSCs should be seeded directly, or immediately stored in liquid nitrogen.
• Proliferating MSCs must be processed immediately.

Protocols for human mesenchymal stem cell cultivation

Protocol for Handling Cryopreserved Mesenchymal Stem Cells

Important Notes:

• Immediately after arrival, store the cryopreserved cells in liquid nitrogen, or seed them immediately. Storage at -80°C is not sufficient for cell preservation and causes irreversible cell damage.
• Handle using aseptic techniques and a laminar flow biosafety cabinet
• Fibronectin-coated cultureware is required for use with MSC Growth Medium DXF (see Medium Preparation step 1b).

1. Prepare the medium
   
   a.) For MSC Growth Medium 2
   
   Calculate the required culture surface area according to the recommended plating density (see table below) and the lot-specific cell numbers stated on the certificate of analysis. Fill with the appropriate volume of PromoCell® Growth Medium (at least 9 mL per vial of cells) in cell culture vessels. Place the vessels in an incubator (37°C, 5% CO₂) for 30 minutes.
   
   b.) For MSC Growth Medium DXF
   
   Calculate the culture surface area needed according to the recommended plating density (see table below) and the lot-specific cell numbers stated on the certificate of analysis. Coat the culture vessels with 10 µg/cm² human or bovine fibronectin according to the manufacturer’s instructions. Fill precoated vessels with the appropriate volume of PromoCell Growth Medium (at least 9 mL per vial of cells). Place the vessels in an incubator (37°C, 5% CO₂) for 30 minutes.
   
   
2. Thaw the cells
   Remove the cryovial from the liquid nitrogen container and immediately place it on dry ice — even for brief transportation. Under a laminar flow biosafety cabinet, briefly twist the cap a quarter turn to relieve pressure, then re-tighten. Immerse the vial into a water bath (37°C) just up to the screw cap for 2 minutes. Ensure that no water enters the thread of the screw cap.
   
   
3. Disinfect the vial and seed the cells
   Thoroughly rinse the cryovial with 70% ethanol under a laminar flow biosafety cabinet. Then, aspirate the excess ethanol from the thread area of the screw cap. Open the vial and transfer the cells to a cell culture vessel containing the prewarmed medium from step 1.
   
4. Incubate the cells
   Place the vessel in an incubator (37°C, 5% CO₂) for cell attachment. Replace the medium after 3-4 hours and every two to three days thereafter. The cells should be sub-cultured according to the subcultivation protocol (see below) once they have reached 70 - 90% confluency.

Protocol for Handling Proliferating Human Mesenchymal Stem Cells

Important notes:

• Start immediately after delivery of the cells.
• Use aseptic techniques and a laminar flow biosafety cabinet.
• For culture with MSC Growth Medium DXF, fibronectin-coated plasticware is needed (see Protocol for Cryopreserved Cells, step 1b).

1. Incubate the cells
   Unpack the culture vessel, do not open the cap, and immediately place it in an incubator (37°C, 5% CO₂) for 3 hours to allow the cells to recover from the transportation.
   
   
2. Replace the transport medium
   Carefully open the vessel, rinse the inner side of the lid with 70% ethanol, and let air dry. Aspirate the transport medium (Mesenchymal Stem Cell Growth Medium 2) from the vessel. Add 10 mL of the appropriate PromoCell^® Cell Growth Medium.
   
   
3. Check and incubate the cells
   Check the cell density. Open the cap half a turn to vent, or use flasks with vented caps, and place the vessel in an incubator (37°C, 5% CO₂). Change the medium every two to three days. The cells should be subcultured according to the subcultivation protocol below once they have attained >70% confluency.

Subcultivation Protocol for Mesenchymal Stem Cells

Important Notes:

• For culture with MSC Growth Medium DXF, fibronectin-coated cultureware is required (see Protocol for Cryopreserved Cells, step 1b).
• For routine subculture of human MSC, the use of Accutase^® dissociation reagent is highly recommended with all MSC Growth Media.
• Use aseptic techniques and a laminar flow biosafety cabinet.

1. Prepare the reagents and wash the cells
   Place the Accutase^® Solution at room temperature for at least 30 minutes to adjust the temperature of the reagents. Carefully aspirate the medium from the culture vessel. Add 100 µL HEPES BSS Solution per cm² of vessel surface to wash the cells, and agitate the vessel carefully for 15 seconds.
   
   
2. Detach the cells
   Carefully aspirate the HEPES BSS buffer from the culture vessel. Add 100 µL Accutase Solution per cm² of vessel surface and incubate for 2-4 minutes at room temperature. Close the vessel and examine the cells under a microscope. When the cells start to detach, gently tap the side of the vessel to loosen the remaining cells.
   
   
3. Harvest the cells
   Carefully aspirate the cell suspension and transfer it to a centrifugation tube. Spin down the cells for 3 minutes at 220 x g.
   
   
4. Incubate the cells
   Discard the supernatant, add 1 mL of the appropriate Cell Growth Medium, and resuspend the cells by gently pipetting up and down. Plate the cells according to the recommended seeding density in new sterile cell culture vessels containing prewarmed Cell Growth Medium. Place the vessels in an incubator (37°C, 5% CO₂) and change the media every two to three days.

Human Mesenchymal Stem Cell Culture System Quality Control

Human Mesenchymal Stem Cells Quality Control

Rigid quality control tests are performed for each lot of Human Mesenchymal Stem Cells.

• Tested for cell morphology, proliferation potential, adherence rate, and cell viability.
• Characterized by flow cytometric analysis of a comprehensive panel of markers including CD73/CD90/CD105 and CD14/CD19/CD34/CD45/HLA-DR as proposed by the ISCT [1].
• Differentiation assays into adipogenic, osteogenic, and chondrogenic lineages are performed for each lot under culture conditions without antibiotics and antimycotics.
• Tested for the absence of HIV-1, HIV-2, HBV, HCV, HTLV-1, HTLV-2 and microbial contaminants (fungi, bacteria, and mycoplasma).
• A detailed certificate of analysis (CoA) for each lot is available for download.

Human Mesenchymal Stem Cell Specifications

Product	Recommended Culture Media	Recommended Differentiation Media	Plating Density
Human Mesenchymal Stem Cells from Bone Marrow (hMSC-BM)	C-28009 C-28019	C-28016 C-28012 C-28013 C-28014 C-28015	4,000 cells per cm²
Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC)	C-28009 C-28019	C-28016 C-28012 C-28013 C-28014 C-28015	4,000 cells per cm²
Human Mesenchymal Stem Cells from Adipose Tissue (hMSC-AT)	C-28009 C-28019	C-28016 C-28012 C-28013 C-28014 C-28015	4,000 cells per cm²

Mesenchymal Stem Cell Media Quality Control

All lots of PromoCell^® Mesenchymal Stem Cell Media are subjected to comprehensive quality control tests using primary human mesenchymal stem cells. Each lot of MSC Growth Medium is validated for maintenance of multipotency, growth promoting activity, adherence rate, and typical morphology of the tested mesenchymal stem cells. Each lot of MSC Differentiation Media is tested for the capacity to induce directed differentiation of MSC to specific cell lineages. In addition, all lots of media have been tested for the absence of microbial contaminants (fungi, bacteria, mycoplasma).

Intended Use for Mesenchymal Stem Cells and Media System

These products are for in vitro use only and not for diagnostic or therapeutic procedures. For safety precautions please see appropriate MSDS.