Stem Cell Isolation and Maintenance

Though stem cells have been a source of intense study for decades, today’s researchers face many challenges, options, and opportunities. There are more types of stem cells available these days, compared to the time of their discovery in the early 1960s. Now, researchers can harvest different stem cell types from various tissues within the human body. In addition, there is a wealth of information and experiences to learn from regarding different methods of isolating and maintaining them. This article covers common methods of stem cell isolation and maintenance, with examples of specific cell types.

A variety of challenges

The variety of stem cells available today—whether derived from embryonic or adult cells—present a range of isolation challenges. Embryonic stem (ES) cells are derived from a cell mass inside human blastocysts a few days post-conception. The outer cell layers, the trophoectoderm, must be physically removed to access the inner cell mass using methods such as mechanical dissection, microdissection, laser dissection, or immunosurgery. A method called minimized trophoblast cell proliferation may also be used to limit trophoectoderm growth so that it’s easier to remove.

Adult-derived, or somatic, stem cells, on the other hand, are isolated with various protocols depending on their tissue source. Somatic stem cells include induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs; e.g., harvested from bone marrow, perinatal, adipose, and other tissues), and hematopoietic stem cells (HSCs; e.g., harvested from bone marrow, or cord blood). MSCs and HSCs are naturally pluripotent in adults. Depending on the tissue and cell type, isolating these cells may begin with blood collection and fractionation, bone marrow aspiration, or tissue biopsy, followed by cell sorting with flow cytometry to enrich the desired cell type. For example, cells expressing a unique set of markers can be targeted by fluorescently tagged antibodies, and isolated by fluorescence activated cell sorting (FACS).

In contrast, iPSCs must be re-programmed to become pluripotent; in this in vitro process, somatic cells become de-differentiated using Yamanaka factors. The resulting iPSCs “must be fully characterized by checking the expression of pluripotency markers and their differentiation potential into all three germ layers,” says Kit Man Tsang, a Senior Scientist at ATCC. “The successfully reprogrammed iPSCs can then be differentiated into different cell types for different research purposes.”

Careful isolation from tissue matrix

Unlike collecting blood cells, which are more easily filtered from whole blood, isolating stem cells that are embedded in tissue matrix is more complicated. This process may include mechanical dissociation, explant cultures, and/or enzymatic digestion—there is no best option, because it depends on the stem cell type, and the nature of the tissue. Whitney Cary Wilson, Field Application Scientist at Corning Life Sciences, who works with MSCs, notes that both the explant and enzymatic methods have advantages and drawbacks for these cells. “With explant culture, your time to cell line establishment tends to be longer and initial cell yield is lower, but the cell health and viability is higher, and population doubling time is shorter given that there are no deleterious effects from enzymatic digestion,” says Cary Wilson. In contrast, enzymatic isolation can produce more cells quickly, but can interfere with markers expressed on the cell surface. “If you need to produce more cells quickly (i.e., for an autologous application), then enzymatic isolation may be preferred,” says Cary Wilson.

In contrast, Alexander Schlacht, Product Manager in Epithelial Biology at STEMCELL Technologies, works with adult tissue-derived stem cells and lineage-specific progenitor cells isolated from tissue biopsies. He uses mechanical, non-enzymatic dissociation methods because these cause less stress to the cells, allowing them to establish cultures more quickly. “Effectiveness often depends on the tissue type and the quality of the starting material,” he says. “For fragile tissues or clinical biopsies, gentle methods preserve the tissue architecture and are often more effective than enzymatic methods for maintaining cell health and viability.” He also emphasizes the importance of using high-quality tissue samples for success isolating stem cells.

When working with damaged or low-input samples, Schlacht suggests adding inhibitors of the Rho-associated, coiled-coil containing protein kinase (ROCK) pathway right after stem cell dissociation; “ROCK inhibitors during the initial plating can help to boost survival” by preventing dissociation-induced apoptosis.

Maintenance considerations

Most standard cell culture advice is appropriate for stem cells too—passage the cells before overcrowding occurs to keep them happy and healthy, treat them gently and with sterile technique, and be vigilant for signs of contamination. In addition, optimize the media and supplements for cell health, which can “greatly affect the cells’ pluripotency and proliferative capacity,” says Tsang. Another important consideration is the cells’ growth matrix, such as Matrigel, fibronectin, and laminin. “The rigidity, engrained growth factors, and components of the matrix can significantly affect stem cell longevity,” she says. “ATCC has a cell basement membrane that is optimized for iPSC culture.”

Minimizing stress is particularly important “so that you do not induce senescence of the cell line prior to producing enough healthy cells for your application,” says Cary Wilson, who finds that seeding MSCs at a lower density (1,000–5,000 cells/cm²) often helps to keep them healthier for longer. Other ways to reduce stress on MSCs include passaging them at 80–90% confluence, and finding the best media. “The media formulation you use will have a strong impact on cell health as well, so it is important to screen different media types for your specific application and cell type to determine which will perform best for you,” she says.

Schlacht notes the importance of observing cultures carefully over time. “Long-term cultures can start to differentiate over time or decrease stem cell function,” he says. “We monitor growth kinetics and cell morphology to ensure that the culture retains its stem cell identity.”

More challenges

Isolating one type of stem cell from a tissue containing many different cell types is an ongoing challenge, says Tsang. Even with antibody labeling and FACS sorting, isolating a cell population remains difficult. “You must pick out a combination of surface markers unique to the stem cell type you’re looking to isolate,” she says. “Labeling CD34, for example, won’t be enough because multiple adult stem cell populations express this marker, and you won’t get a pure HSC population.” By using multiple antibodies, researchers can focus on their targeted cell type, through positive and/or negative selection for different cell surface markers. Stem cell researchers also face consistency challenges, according to Tsang, due to genetic heterogeneity among stem cell donors, and the generally long experimental timelines. “A good solution is to purchase cells from a vendor who has well-characterized lots of stem cells from various backgrounds,” she says.

Cary Wilson believes that one of the main challenges in working with MSCs is finding a platform for scaling up successfully, especially in allogeneic applications. “Once you start to expand MSCs at scale in the non-static tissue culture environments the bioreactors inherently create, you can introduce shear stress to the cells, which can become problematic for their health,” she says. “If you plan to expand MSCs in a perfusion or circulated environment within a bioreactor, it will be important to add an extracellular matrix substrate such as fibronectin to promote cell adherence and support cell health.” With future advances in scaled-up stem cell production, allogeneic stem cell applications such as immunotherapies hold the promise of worldwide benefits.

Best practices for isolating and maintaining stem cells

1 Select the Right Isolation Method for the Cell Type Choose isolation techniques suited to the specific stem cell type, such as mechanical dissection for ES cells or cell sorting with flow cytometry for MSCs.

2. Use Fluorescence-Activated Cell Sorting (FACS) FACS allows precise isolation of stem cells by sorting them based on surface markers using fluorescently tagged antibodies.

3. Consider Tissue-Specific Isolation Techniques Different tissues may require mechanical dissociation or enzymatic digestion, depending on the stem cell type and tissue structure.

4. Incorporate ROCK Inhibitors for Better Cell Survival Adding ROCK inhibitors can improve cell survival and prevent apoptosis after dissociation, especially from low-quality tissue.

5. Optimize Cell Culture Media for Stem Cell Health Tailor the culture media to the specific stem cell type to support its pluripotency and proliferative capacity.

6. Maintain Proper Growth Matrices for Longevity Use ECM components like Matrigel, fibronectin, or laminin to promote better cell adherence and long-term stem cell viability.

7. Passage Cells Before Overcrowding Occurs Regularly passage cells before overcrowding to maintain healthy growth and prevent stress-induced damage.

8. Seeding Cells at Lower Density for Better Health Seed stem cells at a lower density (1,000–5,000 cells/cm²) to reduce stress and improve overall cell health.

9. Monitor Cell Growth and Morphology Regularly check stem cell cultures for signs of differentiation or morphological changes to ensure they maintain their stem cell identity.

10. Be Mindful of Genetic Heterogeneity Genetic variability among stem cell donors can affect outcomes, so consider sourcing cells from well-characterized, consistent vendors.