35 Cell Culture Research
Musculoskeletal diseases are increasingly important in all age groups with respect to injuries and degenerative diseases. The latter are particularly relevant in developed and aging societies, where mobility is an essential part of independence, especially in higher age. Degenerative diseases of bone, joints, and muscle like osteoporosis, osteoarthritis, and sarcopenia are common in the elderly and are main causes of dependence and disability. The burden of disease is extremely high and is second after mental and behavioral disorders worldwide with respect to years lived with disability. Injuries and degenerative conditions of tendons and ligaments are also increasing both in sport and in age-associated degenerative diseases and here regenerative therapeutic strategies are even less developed.
Jargon Simplified: Cell Line
A cell line is a cell culture that will proliferate indefinitely given appropriate fresh medium and space. It is immortalized and thereby escapes the Hayflick limit of population doublings. Cell lines may be looked upon as resembling neoplastic cells. In contrast, primary cells have a limited lifespan and undergo replicative senescence if they reach the Hayflick limit of population doublings.
Cell culture of mesenchymal cells has become an indispensable research tool. Transformed cell lines are commercially available and can be used as reproducible models for various scenarios. Primary cells can be isolated from various sources such as bone marrow, trabecular bone, cartilage, tendons, ligaments, and muscle. Being closer to physiology compared to cell lines derived from tumorous tissues, their disadvantage is their limited number and lifespan in culture. Their donor-dependent variability may both be an advantage and a disadvantage with respect to the reproducibility of experimental settings and their potential to get insights for individualized medicine, respectively. Immortalized and reprogrammed primary cells are coming into focus based on new technologies that are developed for genetic engineering and reprogramming.
The last three decades have seen an incredible increase in cell culture techniques and research with mesenchymal stem cells (MSC) from various sources to describe their potency, differentiation, and plasticity, and to develop stem cells as a tool for cell-based therapies. The techniques developed have reached a basis of state-of-the-art routine and offer a huge potential for future improvement. The transition from two-dimensional to three-dimensional culture is key for many questions to be addressed. In order to mimic the physiological environment of the cells in vivo with respect to their microenvironment in terms of, for example, extracellular matrix (ECM), oxygen tension, and mechanical conditions, respective techniques will have to be refined and co-cultures with other cell types will have to be established. Reprogramming techniques will be helpful to avoid senescence and to get closer to individualized medicine. Enhanced bioreactor techniques will allow for reconstitution of a physiological environment in vitro to set up complex new experiments.
35.1 Mesenchymal Stem Cells
MSCs are adult stem cells scattered all over the organism. They reside in protected niche microenvironments and give rise to regenerative populations of precursors, capable of forming repair tissues upon injury or degeneration. By asymmetric cell division, the pool of stem cells ideally is unchanged, while daughter cells are amplified and form new tissue, together with precursors for vascularization where needed. The sequential phases of repair—blood clot–derived growth factors and precursor amplification, inflammation, modeling, and remodeling—are quite uniform throughout various tissues and for both regeneration and healing. 1, 2
MSCs are the most important source for tissue regeneration in mesenchymal tissues and as a support for nonmesenchymal tissues. 3, 4 MSCs are multipotent precursor cells that can give rise to osteoblasts, chondrocytes, adipocytes, and other mesenchymal offspring upon specific stimuli for lineage-specific commitment. The nature and homogeneity of cell populations assigned as MSCs has been discussed since their first description. 5, 6 The biology of commitment and differentiation for bone, fat, and cartilage formation has been largely unraveled, whereas the way from MSCs to muscle, tendon, and ligament cells is less well characterized but is under intensive research. As demonstrated by Sacchetti and coworkers, 7 CD146 + MSCs could establish a complete bone plus bone marrow microenvironment in vivo, indicating that the multipotency of at least some distinct MSC populations goes beyond a simple trilineage capacity of forming bone, cartilage, and fat.
What we culture and amplify is not the stem cell itself but the offspring (so called transient amplifying pool) generated by asymmetric cell division, which is prone to commitment and ages in culture.
35.2 Primary Cultures of Mesenchymal Stem Cells and Related Precursor Cell Populations
35.2.1 Core Definition of Mesenchymal Stem Cells: How to Establish a Mesenchymal Stem Cell Culture
Primary mesenchymal precursor populations can be established from outgrowth populations of bone marrow aspirates, mashed pieces of trabecular bone (bone chips), adipose tissue, pieces of tendon and ligaments, and almost any other tissue. The “gold standard” of MSC populations are those retrieved from bone marrow, whereas other populations are very similar but also significantly different in their transcriptome signature and multipotency.
After several days of primary culture, mesenchymal precursors start to grow in the dishes; they tightly adhere to plastic, which is a simple and effective selection criterion for MSC. The populations obtained are predominantly spindle cell–like cultures that show self-renewal and can give rise to several mesenchymal lineages derived from mesenchyme (Fig. 35.1). Consensus conferences have been held (e.g., by The International Society for Cellular Therapy) that defined a minimum requirement for the definition of MSCs. 8 Cells must be plastic adherent when cultured under standard conditions and express the surface markers CD73, CD90, and CD105, and they should not express CD45, CD34, CD14, CD11b, CD79, or CD19 and HLA-DR. Finally, they must be capable of in vitro differentiation into osteoblasts, adipocytes, and chondrocytes, as described subsequently in the section of trilineage differentiation tests (Table 35.1).
Having met all these criteria, one can work with these cultures as accepted true MSC cultures (Fig. 35.1). However, these populations are still heterogeneous. The various cell types that grow in such cultures may be MSCs in variable stages of multipotency, lineage commitment, presenescence, or even senescence, or may be “contaminated” with, for example, endothelial precursors. Taking passage 1 of cultures is usually the time point where the plastic adherence criterion has worked out and other contaminating cells have been removed. Cell preparations from bone marrow in passage 0, for example, contain mature B cells that adhere to MSC but disappear after the first passage. Expression of immunoglobulin light or heavy chains is usually a reasonable criterion to proof if B cells have been removed from the cultures (our unpublished results).
35.2.2 Enhancing the Homogeneity of Mesenchymal Stem Cell Cultures
There is no single molecule that characterizes a true MSC and that would allow for highly specific sorting. Several groups have described MSC subpopulations with specific attitudes such as preferential osteogenic potential or high colony-forming capacity, but there is no accepted standard of selection that would allow for a standard operating procedure to get robust and homogeneous populations for defined purposes. In the future, the trend will be toward more defined MSC populations, which can be obtained by combined selection procedures (Table 35.1). Selection for CD146, platelet-derived growth factor receptor (CD140), and stem cell antigen 1 (LY6A/E) expression may enhance the quality of cultures and provide more homogenous populations with respect to their multipotency. Selection for low-affinity nerve growth factor receptor (CD271), THY-1 + + (CD90), and vascular cell adhesion molecule 1 (CD106) expression was reported to separate highly stable and clonogenic populations from senescence prone ones that accumulate deoxyribonucleic acid damage.
The classical way of selecting more homogenous cell populations is preparative fluorescence activated cell sorting. Analytical fluorescence activated cell sorting procedures allow for more extensive nonpreparative characterization of the sorted populations using, for example, multicolor applications.
Jargon Simplified: Fluorescence Activated Cell Sorting
Fluorescence activated cell sorting analysis is a fluorescent antibody–based flow cytometry technique that allows analysis of single cells as to their pattern of membrane-associated proteins (intracellular proteins may less frequently also apply for this measure) even in heterogenous mixtures. It is used both for diagnostic cell phenotyping and preparative sorting of specific cell populations.
Selection of cell subpopulations is based on targeting cell type–specific surface molecules, but may also perform as “negative selection,” which specifically removes contaminating populations. In addition, antibody-based immobilization procedures are commercially available such as magnetic bead–based applications, which do not require high tech equipment and are less expensive. 9 Another way to clonally select subpopulations of MSCs is the colony forming assay in several variations. This assay is based on picking single clones that arise from colonies in, for example, three-dimensional agarose environment and may also be used in combination with other methods of selection and characterization. 10
35.3 Media and Serum
The routine experimental setting is that we use commercially available media in combination with serum, the latter being usually fetal calf serum (FCS). If heading for translational settings for therapeutic strategies, the use of nonhuman materials and well-defined media is a major obstacle, which should be avoided from the beginning, and well-defined media are desired. Before buying larger batches of FCS, screening for the efficacy of respective batches is recommended. Commercially available media can be very variable and especially variably expensive, ranging from basic media to so called stem cell media, which often comprise unknown growth factors that considerably influence the cell biology and cell signatures. The decision for the experimental setup should be carefully discussed ahead of the start of cell cultures (see Table 35.2).
Serum-free media are being developed for MSC propagation, especially for cultures that are produced for therapeutic purposes. Standard operating procedures that meet the regulations for good medical practice are being developed for future cell-based clinical applications using MSC large-scale amplification methods. 11, 12 There is a research need about industrial production methods for MSC production and banking. 13
Related posts:
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
