Stem cell expansion is crucial for medical treatments because it allows scientists to grow large numbers of these powerful cells from a small initial sample.
This is important because many stem cell therapies require millions of cells to be effective, far more than can be obtained directly from a patient or donor.
By expanding stem cells in the lab, researchers and doctors can produce enough cells to treat patients with various diseases and injuries, potentially offering new hope for conditions that were previously difficult or impossible to cure.
Mesenchymal Stem Cell Expansion
Mesenchymal stem cell (MSC) expansion refers to the process of increasing the number of MSCs in vitro to obtain large quantities needed for clinical applications.
This is crucial because MSCs occur in relatively low numbers in tissues, and millions to hundreds of millions of cells are typically required per patient for therapeutic use.
Key aspects of MSC expansion include:
- Cell source: MSCs can be isolated from various tissues, with bone marrow, adipose tissue, and umbilical cord (Wharton's jelly) being the most common sources.
- Culture conditions: MSCs are expanded in specialized growth media, often supplemented with fetal bovine serum (FBS), human platelet lysate (HPL), or defined serum-free formulations.
- Bioprocessing strategies: Large-scale expansion employs methods like:
- Multilayered flasks: Provide increased surface area for 2D cell growth.
- Bioreactors: Allow for controlled 3D culture environments with better nutrient distribution.
- Spinner flasks: Enable suspension culture with microcarriers.
- Roller bottles: Provide continuous mixing for improved nutrient access.
- Optimization factors: Key parameters include cell seeding density, oxygen levels, pH, temperature, and agitation speed (for dynamic cultures).
- Quality control: Expanded MSCs must maintain their characteristic phenotype, differentiation potential, and therapeutic properties like immunomodulation.
- Scale-up considerations: The expansion process must be reproducible, cost-effective, and compliant with good manufacturing practices (GMP) for clinical applications.
Yes, we know the above was quite technical but in summary; the main goal of MSC expansion is to generate sufficient numbers of high-quality cells while preserving their therapeutic potential, ensuring safety, and minimizing the time and resources required for production.
Why Cell Expansion is Important
Increasing cell numbers is crucial in a clinical setting for several important reasons:
- Effective dosage: Many cell-based therapies require millions or even hundreds of millions of cells per treatment to be effective. The human body simply doesn't have enough of these specialized cells readily available, so they need to be expanded in the lab to reach therapeutic levels.
- Multiple treatments: Some conditions may require repeated treatments over time. Having a large supply of cells allows for multiple doses without needing to harvest more cells from the patient or donor each time.
- Consistency: By expanding cells in controlled laboratory conditions, doctors can ensure a more consistent and predictable treatment. This is important for both safety and effectiveness.
- Overcoming cell loss: When cells are injected or implanted in the body, many don't survive the process. Starting with a larger number of cells increases the chances that enough will survive to have the desired therapeutic effect.
- Broader application: With more cells available, treatments can potentially be given to more patients or used for a wider range of conditions.
- Research and development: Having larger quantities of cells also allows scientists to conduct more extensive research, potentially leading to new treatments or improvements in existing ones.
Increasing cell numbers through laboratory expansion is critical for improving therapeutic outcomes in cell-based therapies. This process allows clinicians to achieve effective dosages, enhance cell survival rates after implantation, enable multiple treatments over time, and ensure consistency and predictability of treatments. Furthermore, expanding cells overcomes the limitation of low natural quantities of therapeutic cell types in the body, broadens the application of treatments to more patients and conditions, and ultimately results in more potent and widely applicable cell-based therapies.
Factors that Influence Efficiency of Expansion
Several key factors influence the efficiency of mesenchymal stem cell (MSC) expansion:
- Culture medium and supplements: The choice between fetal bovine serum (FBS), human platelet lysate (HPL), or defined serum-free media significantly impacts expansion efficiency. HPL and defined media generally outperform FBS in promoting MSC proliferation.
- Bioprocessing strategy: The review compared four main strategies - bioreactors, spinner flasks, roller bottles, and multilayered flasks. Bioreactors and multilayered flasks generally achieved higher expansion ratios compared to other methods.
- Cell seeding density: Lower initial seeding densities often resulted in higher expansion ratios.
- Oxygen concentration: Hypoxic conditions were found to enhance MSC proliferation in some studies, though the effects on differentiation potential varied.
- Agitation speed: For dynamic culture systems like bioreactors and spinner flasks, optimizing the agitation speed is crucial to reduce shear stress while maintaining efficient nutrient transfer.
- Microcarrier selection: For 3D culture systems, the choice of microcarriers affects cell attachment and growth.
- Culture duration: Longer culture periods generally increased total cell yield but could potentially affect cell quality.
- Cell source: MSCs from different tissue sources (e.g., bone marrow, adipose tissue, Wharton's jelly) may have varying expansion potentials.
- Medium composition and feeding strategy: Optimizing nutrient availability and waste removal is important for maintaining high proliferation rates.
- pH and temperature control: Maintaining optimal environmental conditions is crucial, especially in large-scale bioreactor systems.
These factors need to be carefully optimized to achieve efficient large-scale expansion of MSCs while maintaining their characteristic phenotype, differentiation potential, and therapeutic properties.
Culture Period May Influence Quality
The culture (expansion) period can significantly influence the quality of expanded mesenchymal stem cells (MSCs) in several ways:
- Cell yield: Longer culture periods generally increased total cell yield, allowing for greater expansion ratios. However, this needs to be balanced against potential negative effects on cell quality.
- Stem cell characteristics: Extended culture times may lead to a loss of stem cell properties. The review notes that "MSCs expanded in vitro for a long period of time may lose their stem cell characteristics."
- Proliferation and differentiation potential: Previous studies reported that "MSC proliferation and differentiation potential decreased when they reached a higher passage number." This suggests longer culture periods could negatively impact these key properties.
- Surface marker expression: Some studies found that extended culture in bioreactors or spinner flasks led to decreased expression of important MSC surface markers like CD90 and CD105. This could affect the cells' therapeutic potential.
- Immunomodulatory properties: Changes in surface marker expression, particularly loss of CD90, have been associated with weaker immunosuppressive activity in MSCs.
- Differentiation bias: Prolonged culture may alter the differentiation potential of MSCs, potentially enhancing certain lineages while suppressing others.
Culturing is the process of growing cells in a controlled laboratory environment, allowing them to multiply and increase in number. For mesenchymal stem cells (MSCs), this expansion process is crucial because it enables scientists to produce large quantities of these valuable cells from a small initial sample, making it possible to generate enough cells for therapeutic applications.
Safe Expansion Techniques
Expanding stem cells too many times can indeed have negative effects on cell quality and therapeutic potential. This concept is related to the number of "passages" or subcultures the cells undergo.
- Passage number and cell quality: As mesenchymal stem cells (MSCs) are expanded in culture, they undergo multiple rounds of cell division. Each time the cells are harvested and reseeded into new culture vessels, it's considered a "passage." The review notes that "MSCs expanded in vitro for a long period of time may lose their stem cell characteristics."
- Proliferation and differentiation potential: Previous studies reported that "MSC proliferation and differentiation potential decreased when they reached a higher passage number." This suggests that as cells are passaged multiple times, they may lose some of their key stem cell properties.
- Surface marker expression: Extended culture periods and higher passage numbers can lead to changes in the expression of important MSC surface markers. For example, some studies found decreased expression of CD90 and CD105 in MSCs expanded in bioreactors or spinner flasks for prolonged periods.
- Immunomodulatory properties: Changes in surface marker expression, particularly loss of CD90, have been associated with weaker immunosuppressive activity in MSCs. This could potentially reduce their therapeutic efficacy in certain applications.
- Genetic stability: With increasing passages, there's a higher risk of genetic abnormalities accumulating in the cells, which could potentially lead to safety concerns for clinical applications.
- Senescence: As cells are passaged multiple times, they may approach their replicative limit (Hayflick limit), leading to cellular senescence and reduced functionality.
Current literature emphasizes the importance of optimizing culture conditions to "obtain the huge number of cells in a short period of time and in a cost-effective manner without compromising the cell quality." This suggests that achieving the necessary cell numbers with fewer passages is preferable for maintaining MSC quality and therapeutic potential.In practice, many researchers and clinicians aim to use MSCs at lower passage numbers (typically below passage 5-7) to ensure optimal cell quality and therapeutic efficacy. However, the exact optimal passage number can vary depending on the specific cell source, culture conditions, and intended application.
Mesenchymal Stem Cell Media
Mesenchymal stem cell (MSC) media are specialized culture media designed for the in vitro expansion and differentiation of mesenchymal stem cells. Here are the key points about MSC media:
- Composition: MSC media typically consist of a basal medium supplemented with growth factors and other components essential for MSC growth and maintenance.
- Types:
- Growth media: Designed for expanding MSCs while maintaining their multipotency.
- Differentiation media: Formulated to induce differentiation of MSCs into specific cell types like adipocytes, osteoblasts, or chondrocytes.
- Serum options:
- Traditional media contain fetal bovine serum (FBS).
- Serum-free media (SFM) are available, offering more defined conditions and avoiding animal-derived components.
- Benefits of serum-free media:
- More stable population doubling time
- Lower cellular senescence
- Lower immunogenicity
- Higher genetic stability
- Faster cell growth rates
- Commercial options: Companies like PromoCell and Sigma-Aldrich offer ready-to-use MSC media, including both growth and differentiation formulations.
- Customization: Some media allow for user supplementation with specific inducers, such as chondrogenic factors.
- Quality control: Rigorous testing ensures that the media support MSC growth, maintain multipotency, and induce proper differentiation when required.
- Applications: These media are crucial for various research areas, including regenerative medicine, tissue engineering, and cell therapy studies.
- Cell sources: MSC media are designed to support cells derived from various sources, including bone marrow, adipose tissue, and umbilical cord matrix.
- Alternative options: Some researchers have explored using more common media like Dulbecco's Modified Eagle's Medium (DMEM) as a cost-effective alternative to specialized MSC media, though results may vary.
For the stem cell startup: When selecting MSC media, it's important to consider factors such as the specific research goals, cell source, and whether serum-free conditions are required for the intended application.
Conclusion
In conclusion, stem cell expansion is a crucial process for making cell therapies available to patients with various conditions, including degenerative diseases. Here are the key takeaways for patients and consumers interested in stem cell treatments:
- Expansion allows scientists to grow large numbers of high-quality stem cells from a small initial sample, making treatments possible.
- Different methods like bioreactors and specialized flasks are used to grow stem cells efficiently while maintaining their therapeutic properties.
- The quality of expanded stem cells is carefully monitored to ensure they remain safe and effective for treatment.
- Newer techniques using human platelet lysate instead of animal-derived products may produce better results and reduce risks.
- When considering stem cell therapies, look for clinics and trials that use well-established, controlled expansion methods and follow strict quality standards.
- Always consult with qualified medical professionals and seek treatments from reputable, accredited facilities that use properly expanded and tested stem cells.
Remember, stem cell research is advancing rapidly, so stay informed about the latest developments and approved treatments for your specific condition.
References
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(2) Balint R, Richardson SM, Cartmell SH. Low-density subculture: a technical note on the importance of avoiding cell-to-cell contact during mesenchymal stromal cell expansion. J Tissue Eng Regen Med. 2015 Oct;9(10):1200-3. doi: 10.1002/term.2051. Epub 2015 Jul 7. PMID: 26153119; PMCID: PMC4858810.
(3) Sprangers K, Avramova V, Beemster GT. Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves. J Vis Exp. 2016 Dec 2;(118):54887. doi: 10.3791/54887. PMID: 28060300; PMCID: PMC5226352.
(4) Ripoll JJ, Zhu M, Brocke S, Hon CT, Yanofsky MF, Boudaoud A, Roeder AHK. Growth dynamics of the Arabidopsis fruit is mediated by cell expansion. Proc Natl Acad Sci U S A. 2019 Dec 10;116(50):25333-25342. doi: 10.1073/pnas.1914096116. Epub 2019 Nov 22. PMID: 31757847; PMCID: PMC6911193.
