Mesenchymal Stem Cells (MSC) Serum-free Medium (GMP Grade) 2.0
Newly upgraded formula, with defined chemical composition and perfectly combines cell isolation, subculture and exosome harvesting.
Description
GMP Mesenchymal Stem Cell Serum-Free Medium 2.0 is a high-grade medium specifically designed for the culture of human mesenchymal stem cells (MSCs). It uses a completely serum-free, animal-derived component-free, chemically defined formulation that meets GMP standards for production. This medium is ideal for primary isolation, cell bank establishment, and continuous passage of MSCs from various sources such as bone marrow and umbilical cord.
By eliminating the need for serum or serum substitutes, it simplifies the culture process and enhances the efficiency and quality of stem cell culture and exosome harvesting. In short, this product helps researchers maintain high-quality cells while simplifying the process, making it an ideal choice for clinical research applications.
| Product Name | Cat. No. | Packaging Spec | Storage Conditions | Shelf Life |
| Mesenchymal Stem Cells Serum-free Medium (GMP Grade) 2.0 | NC0107 | 500 mL/bottle | 2-8℃, protect from light | 12 months |
| Mesenchymal Stem Cells Serum-free Medium Supplement (GMP Grade) | NC0106.S | 5 mL/bottle | -20℃ , protect from light | 12 months |
| Gentle Digestive Enzymes for MSCs | NC1004.1 | 500 mL/bottle | 2-8℃, protect from light | 12 months |
Product Advantages
Serum-Free & Animal-Free: Ideal for Clinical Use
All proteins are artificially recombinant, free from human or animal sources, making them suitable for clinical applications.
Enhanced Cell Adhesion and Adaptation
Significantly improves cell attachment and adaptability, supporting lower seeding densities and higher expansion rates.
Continuous Culture Without Medium Change
No need to change the culture medium; efficient passaging up to P20 without cell aging.
Low Background Exosomes for Large-Scale Cultivation
Extremely low background exosome content, compatible with 2D and 3D large-scale cultivation systems like cell factories, microcarriers, and bioreactors, facilitating higher exosome yield.
Simplified Usage Instructions
Even if users have limited experience with stem cell culture, they can easily follow these steps to culture MSCs and harvest exosomes:
Medium Preparation:
Remove the medium base (500 mL bottle) and additives (5 mL bottle) from refrigeration. Thaw the additive at 2–8°C and mix it with the medium base as per the instructions. After mixing, the complete medium is ready for use. No additional serum or growth factors are required as the medium already contains all necessary components to support MSC growth.
Cell Seeding:
Seed MSCs in culture flasks or dishes at the recommended density (approximately 8,000–10,000 cells/cm²) to achieve optimal growth. The medium is suitable for use with standard tissue culture-treated vessels without any additional coating required (Tip: Ensure the culture surface is suitable for MSC adhesion for optimal results).
Culture Conditions:
Place the flasks in a CO₂ incubator at 37°C with 5% CO₂. Change the medium after 24–48 hours to remove residual digestion enzymes and cell debris. Change the medium every 2–3 days to maintain adequate nutrient supply and remove waste products.
Passaging & Expansion:
When cells reach approximately 80–90% confluence (typically 3–4 days post-seeding), digest the cells using the provided gentle stem cell digestion enzyme . This enzyme protects cell viability better than traditional trypsin. After digestion, reseed the cells at the recommended density for the next passage. Repeat the process to expand the required number of cells.
Exosome Harvesting:
If exosome harvesting is the goal, replace the medium with fresh complete medium when cells reach the exponential growth phase (around 50–70% confluence). Continue to culture for approximately 48 hours, allowing cells to secrete sufficient exosomes. No serum adding is required—this serum-free medium supports exosome production without additional treatments. Afterward, collect the supernatant and use ultracentrifugation or other methods to isolate exosomes. It is recommended to use methods like ExtraPEG for efficient exosome enrichment .
Related recommendation:
Performance Display
1. Cell passaging performance
Thawing of cryopreserved P2 cells and continuous serial subculture
| Passage number | culture vessel | number of cells harvested per vessel | expansion multiple | seeding density/cm² | Days |
| P3 | 150 mm dish | 1.50E+07 | 12.52 | 8000 | 3 days |
| P4 | 1.68E+07 | 14.00 | |||
| P5 | 1.59E+07 | 13.22 | |||
| P6 | 1.33E+07 | 11.07 | 10000 | ||
| P7 | 1.34E+07 | 11.13 |
Isolation and serial passage of primary umbilical cord MSCs
| Passage number | culture vessel | number of cells harvested per vessel | expansion multiple | seeding density/cm² | Days |
| P0 | 150 mm dish | 3.68E+06 | 0 | 14-16 days | |
| P1 | 1.50E+07 | 10 | 8000 | 3 days | |
| P2 | 1.36E+07 | 11.29 | |||
| P3 | 1.53E+07 | 12.75 | |||
| P4 | 1.39E+07 | 77.55 | |||
| P5 | 1.21E+07 | 10.08 | |||
| P6 | 1.54E+07 | 10.28 | 10000 | ||
| P7 | 1.75E+07 | 11.64 | |||
| P8 | 1.50E+07 | 10.00 | |||
| P9 | 1.73E+07 | 11.56 | |||
| P10 | 1.45E+07 | 9.64 |
2. Exosome culture performance
2.1 This product is used to produce exosomes in cell factories.
P2 cells were revived and continuously passaged to P3 cells before being inoculated into cell factories. A total of 2.52E+12 exosomes were harvested in 15 days.
| Sampling time point | Exosome concentration (pieces/mL) | Number of exosomes harvested (pieces) | Culture medium volume (mL) |
| Day 3 | 3.53E+09 | 4.24E+11 | 120 |
| Day 6 | 5.36E+09 | 6.43E+11 | 120 |
| Day 9 | 4.76E+09 | 5.71E+11 | 120 |
| Day 12 | 5.46E+09 | 6.55E+11 | 120 |
| Day 15 | 1.91E+09 | 2.29E+11 | 120 |
| Total: | 2.52E+12 | 600 | |
2.2 This product is used to harvest exosomes from microcarrier 3D cultured cells
The cycle is 15 days, 500mL of culture medium, and a total of 3.50E+12 exosomes were harvested.
| Sampling time point | Exosome concentration (pieces/mL) | Number of exosomes harvested (pieces) | Culture medium volume (mL) |
| Day 3 | 6.19E+09 | 6.19E+11 | 100 |
| Day 6 | 9.26E+09 | 9.26E+11 | 100 |
| Day 9 | 7.92E+09 | 7.92E+11 | 100 |
| Day 12 | 6.06E+09 | 6.06E+11 | 100 |
| Day 15 | 5.54E+09 | 5.54E+11 | 100 |
| Total: | 3.50E+12 | 500 | |
FAQ
Q: How can I optimize culture conditions for the best MSC proliferation and exosome yield?
A: We recommend seeding MSCs at the ideal density (around 10,000 cells/cm²) to balance initial adhesion and growth rate. Change the medium every 2–3 days to provide fresh nutrients. For exosome harvesting, replace the medium when the cells reach the exponential phase (approximately 50% confluence) and allow culture for an additional 48 hours to maximize exosome secretion. No serum starvation is needed since the serum-free medium already supports exosome production. Ensure that you harvest the supernatant when the cell confluence is around 80% to achieve high-quality exosomes .
Q: Is this medium suitable for all sources of mesenchymal stem cells?
A: Yes, this medium is suitable for MSCs s sources, including bone marrow, umbilical cord Wharton’s jelly, and adipose tissue . It performs particularly well with human-derived MSCs. For other sources or species (e.g., mouse-derived MSCs), adaptation may be require serum-free medium is designed to support the growth of human MSCs optimally.
Q: Do I need to add growth factors or serum substitutes to this serum-free medium?
A: No, this medium already contains all the necessary nutrients for MSC growth, including recombinant human proteins (such as albumin, transferrin, and insulin), and does not require any additional serum or substitutes . If your cells were previously cultured in serum-containing media, you can transition to this serum-free medium smoothly. In most cases, no special is required.
Q: If my cells were previously cultured in serum-containing media, what should I be aware of when switching to this serum-free medium?
A: In most cases, MSCs can transition directly from serum-containing culture conditions to this serum-free medium without complications. We recommend slightly increasing the cell density during the initial transition and monitoring cell attachment and morphology closely. Our medium includes recombinant human albumin and other proteins that promote cell attachment, so no extra coating is required. Some initial delay in cell proliferation may occur but should stabilize after 1–2 passages .
Q: What precautions should I take when scaling up from research-scale to large-scale manufacturing using this serum-free system?
A: When transitioning to large-scale production, pay attention to the following:
- Cell Expansion Strategy: Maintain optimal cell density and regularly monitor cell viability at each stage.
- Agitation/Aeration: In bioreactor-based processes, ensure that mixing and oxygen transfer rates are optimized for MSCs; excessive shear stress may reduce cell viability.
- Batch Consistency: Use the same lot of serum-free medium whenever possible to ensure consistent results.
- Validation: Perform pilot runs to validate parameters (e.g., growth rate, productivity, exosome yield) before full-scale manufacturing.
Q: If the supplement (additive) is only partially used, can the remaining portion be refrozen for future use?
A: Generally, we advise against multiple freeze-thaw cycles. If absolutely necessary, aliquot the supplement into sterile tubes upon the very first thaw—each aliquot should contain just enough for a single preparation of medium. This helps preserve activity and minimizes the risk of contamination. Any leftover supplement that has been thawed multiple times may show diminished performance and is not recommended for critical applications.
Q: Does this serum-free formulation specifically enhance exosome yield or purity compared to serum-based systems?
A: Because there is no serum-derived exosome or protein contamination, the exosomes collected from your culture supernatant are almost exclusively MSC-derived. This not only simplifies downstream purification but also improves the overall purity of your exosome preparation. In many cases, users report equal or higher yields of functional exosomes compared to serum-based media—especially when using optimized harvest timings and gentle handling techniques.
Q: Are there any specific recommendations for cryopreserving MSCs cultured in this serum-free medium?
A: Yes. We recommend using a dedicated, serum-free MSC cryopreservation solution (e.g., our GMP-grade cell cryopreservation medium) to protect cell viability during freezing. Once cells reach the desired passage and confluence:
- Harvest cells using the gentle stem cell digestion enzyme.
- Centrifuge to remove all remnants of the enzyme.
- Resuspend the cell pellet in the cryopreservation medium at the recommended density (commonly 1×10^6 to 5×10^6 cells/mL).
- Aliquot into cryovials and freeze rapidly—either by using a controlled-rate freezer or by placing the vials in a suitable freezing container at -80°C, then transferring them to liquid nitrogen after 12–24 hours.
