Cell Senescence Culturing Methods

Summary

Development of therapeutic approaches that slow or ablate the adverse physiological and pathological changes associated with aging have been considered as an important goal for gerontological research. As cellular senescence is characterized as the basis for aging in organisms, culturing and subculturing of normal human diploid fibroblasts to mimic the in vivo aging processes have been developed as major methods to investigate molecular events involved in aging. It has been established that normal human diploid fibroblasts can proliferate in culture for only finite periods of time. There are many ways to study aging in vitro. In this chapter, we will discuss some of the basic laboratory procedures for cell senescence culturing methods.

1. Introduction

Aging can be impacted by both environmental and inherent factors. The outcome of cellular aging is characterized by replicative senescence. Specifically, accumulation of damage in both nuclear and mitochondria DNA can lead to cellular senescence which may affect aging processes in organisms (1) (Figure 1). It is generally accepted that human cells can only divide for a limited number of times. Gene mutations can accumulate during DNA replication, which ultimately lead to abnormal regulation of cell metabolism and physiology that contribute to the aging processes. Additionally, another well-known mechanism related to cell replicative senescence is the inability of normal somatic cells to fully maintain telomere DNA due to lack of telomerase (2).

Figure 1.

Cellular senescence will lead to dysfunction of tissue and organs and eventually aging of organisms.

Cell senescence is a final, common pathway for actively dividing cells. It can be achieved through oncogenic- and replication-induced senescence (3, 4). The cell culture model system has been widely used to study replicative cellular senescence. However, questions have been raised regarding whether such a procedure accurately represents aging in organisms. Cultivation requires that cells continuously undergo proliferation which may stress cells unnaturally, and does not occur in an organism (4). Moreover, cells in culture and those in an intact organism differ drastically in metabolic needs, growth conditions and many other factors. The conventional 10-fold dilution of serum in culture media contains a lower concentration of protein than is normally found in extracellular tissue fluids (5). In addition, it has been demonstrated that the needs of cultured cells differ from those of cells that are freshly isolated from intact tissue which require 13 amino acids for stable growth, whereas cells in an organism require 8–10 amino acids. Furthermore, many types of cells need to be maintained in conditioned medium or seeded at a high population density (6). However, observations that the propagative lifespan of skin fibroblasts in cell culture declines as a function of donor age suggest a critical role of replication-mediated cellular senescence in aging (3, 7, 8). Thus, the limited proliferative capacity of somatic cells appears to be an appropriate system to study human aging, as long as correct correlations are made between observed phenomena and physiology of the entire organism. The culture system exhibits the gradual, stepwise changes that permanently alter the organism, not so much in discrete phases as in a continuum of events that mirror the time from the fusion of the gametes to the demise of the organism. Another strategy is to approach the question from a therapeutic perspective by focusing on the prevention and treatment of the ailments known to increase with age. These studies have concentrated on specific age-related conditions with unfavorable consequences or the processes underlying many age-related dysfunctions (9, 10). In this chapter, important concepts involved in aging cell culture as well as the detailed procedures involved with this process will be discussed. Specifically, in vitro signs of aging including morphological changes and aging biomarkers will be covered. Basic procedures involved in aging cell culture, such as subculturing, cell counting, cell freezing and thawing will be also discussed. Common aging cell culture models such as fetal lung fibroblasts WI-38, skin cells will be discussed. Major issues and experimental design considerations will also be evaluated.

1.1. In Vitro Signs of Aging

1.1.1. Morphological Changes during Cellular Aging

Characteristic morphological changes that accompany replicative senescence in cultured cells include increased cell size, nuclear size, nucleolar size, number of multinucleated cells, prominent Golgi apparati, increased number of vacuoles in the endoplasmic reticulum and cytoplasm, increased numbers of cytoplasmic microfilaments, and large lysosomal bodies (11). The increased cell and nuclear size and numbers of inclusion bodies observed in late-passage cells could be due to the increase of the intracellular content of RNA and proteins which might be caused by reduced protein degradation by proteosome-mediated pathways, decreased RNA turnover, the uncoupling of cell growth from cell division and putative block of senescent cells in late G1. Aging cells also seem to exhibit an increased sensitivity to cell contact (11), perhaps as a result of changes in interactions with the extra-cellular matrix (ECM) or expression of secreted proteins (4), resulting in reduced harvesting and saturation densities (12).

1.1.2. Biomarkers of Aging

Cellular aging is characterised by altered cellular morphology as aforementioned above, expression changes of IGF-1, EGF, c-fos, increased activity for senescence-associated-β-galactosidase (SA-β-GAL), increased formation of senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PML NBs), permanent DNA damage, chromosomal instability and an inflammatory secretome (13–15) (Figure 2). They also display increased average cell cycle times, largely at the expense of longer G1 intervals. IGF-1 is produced by many cell types and plays an important role in regulation of cell proliferation. IGF-1 mRNA production is reduced to a undetectable level in senescent cells whereas IGF-1 receptor mRNA production remains at a detectable level (16). Epidermal growth factor (EGF) signaling has been postulated to impair downstream receptor binding in nonproliferating senescent human diploid fibroblasts (17). A loss of c-fos in senescent WI-38 cells has also been reported, suggesting that lack of proliferation in aging cells was due in part to selective repression of c-fos (18). Staining for β-galactosidase has been widely used in many studies to determine the amount of senescence achieved in culture due to the specific staining of senescent cells in situ (19–21). More recently, annexin A5 has been indicated to accumulate at the nuclear envelope during replicative senescence and drug-induced cellular senescence in primary human fibroblasts (22).

Figure 2.

Biomarkers for aging. Aging biomarkers include alteration of small molecules such as EGF, IGF, c-fos, senescence-associated-β-galactosidase (SA-β-GAL) and appearance of macromolecules such as senescence-associated heterochromatin foci (SAHF) and promyelocytic leukemia protein nuclear bodies (PMLNBs).

1.2. Quantification of Cellular Aging

Currently, a novel way to quantify cellular aging is through calculating population doublings that the cells undergo during the culturing process. Population doublings is defined as the number of times that the cell number is doubled. This is based on the observation that during cellular aging, the proliferative potential decreases. Generally, population doublings can be recorded by plating a specific number of cells and then counting those cells after a defined period of growth. If the population has doubled, for example a plating of 1.0 × 10⁶ cells is followed by a growth phase and a count of 2.0 × 10⁶; one population doubling is added to the current age of the cells. However, because even counts as specified above are almost never achieved, a more precise method is needed for accuracy. Figure 3 outlines an equation in the aging research field that takes into account all the cells counted and results in a more exact population doubling.

Figure 3.

Equation to determine population doubling of aging cells in culture. Where A equals no. of cells plated, B equals no. of cells counted after growth period, C equals old population doubling, D equals new population doubling and n equals the largest number that satisfies the equation A(2ⁿ) ≤ B .

1.3. Concepts in Culturing Aging Fibroblasts

For aging research, important terms have been introduced for convenience of study. These include cumulative population doublings (CPD), senescence and senesced. CPD refers to the number of times that the cell number has been doubled. It has been used to measure the total number of cell divisions and it can be affected by several biological factors including the maximum lifespan of the species, age of the donor, the site of the biopsy, and the culture conditions. If cultures fail to reach confluency in 1 week, the culture is termed as “senescence”. Cultures are considered to be “senescent” (at the end of their replicative lifespan) when they are unable to complete one population doubling during a 4-week period that includes 3 consecutive weeks of re-feeding with fresh medium containing 10% FBS. Generally, WI-38 cells with less than 30 population doublings are considered to be in early passage, and those at the end of their replicative lifespan are greater than 95% lifespan completed. This translates to a population doubling of about 58–65, depending upon the subline that is being cultured.

2. Materials

The following materials, which must be sterile, are used to culture cells in vitro. Although antimicrobial agents can be added to culture media to prevent contamination, certain antibiotic-resistant microbial organisms existing in the environment can cause serious contamination. Thus cell culturing should be performed under a laminar flow hood. For most aging cell types, an antibiotic/antimycotic solution of streptomycin, amphotericin B, and penicillin (Mediatech, Inc., http://www.cellgro.com) can be added to the medium at a final concentration of 1% to adequately hinder the growth of bacteria, fungi, and yeast. Cells are cultured in a humidified 37°C incubator with varied concentration of CO₂ depending on cell types. Medium, trypsin/EDTA solutions, and phosphate-buffered saline (PBS) should be warmed to 37°C in a water bath before use. Different cell types may be cultured in different medium.

1. Monolayer cultures of cells.

2. Trypsin/EDTA solution (see ^{Note 1}).

3. Complete medium with serum.

4. Sterile Pasteur pipets.

5. 70% (w/v) ethanol.

6. Sterile PBS without Ca²⁺and Mg²⁺.

7. Tissue culture plastic ware (pipets, flasks, plates, cryovials, 15- and 50-mL conical tubes), all sterile.

3. Culturing Aging Fibroblasts

3.1. Procedures for Culturing Aging Fibroblasts

Human fetal lung fibroblasts, WI-38 cells, have been widely used as a cellular model to study molecular events associated with the aging process. The WI-38 cell line was originated from lung tissue of a therapeutically aborted fetus at 3 months gestational age. Additionally, fibroblasts explanted from the skin have also been widely used as a model to study the aging process.

The methods outlined as follows describe the trypsinization and subcultivation of monolayer cells, the freezing of monolayer cells, and the thawing and recovery of frozen cells. Notes have been added to discuss specific tips for aging cell culture.

3.1.1. Trypsinization and Subcultivation

Cells should be subcultured upon approximately 90% confluence to avoid contact inhibition or transformation.

1. Remove media from primary culture with a sterile Pasteur pipet. Wash adherent cells with a small volume of sterile PBS to remove residual fetal bovine serum (FBS), which may inhibit trypsin.

2. Add sufficient 37°C trypsin/EDTA solution (see ^{Note 1}) to cover the cell monolayer. Place in incubator for 1–2 min (see ^{Note 2}). Check cells with an inverted microscope to make sure cells are detached.

3. Add appropriate amount (2–6 mL) of warmed culture medium and disperse cells by gently pipetting up and down.

4. Add appropriate aliquots of the cell suspension to new culture vessels (see ^{Note 3}).

5. Incubate cultures, checking for adherence after 24 h.

6. Renew medium every 2–4 d.

3.1.2. Cell counting

Cell counting is essential for aging cell culture research. Here we will describe the method of counting cells with a hemacytometer (Improved Neubauer).

1. Split cell cultures as in 3.1.1.

2. To check for viability, 0.75 mL of cells is mixed with 0.25 mL of trypan blue.

3. Nine microliters of this suspension is loaded onto the hemacytometer with coverslip and viewed under the microscope (100× magnification). Viable cells remain unstained.

4. After counting, calculate the number of cells per milliliter:

   cells/ml = average count per square of grid x dilution factor × 10⁴.

3.1. 3. Freezing

Cells to be frozen should be in late log phase growth.

1. Dissociate monolayers with trypsin/EDTA (see ^{Note 1}) and resuspend cells in complete medium.

2. Count resuspended cells to determine viability and number.

3. Gently pellet cells for 5 min at approx 300–350g (1500 rpm in a swinging bucket or 45° fixed-angle rotor) and remove and discard supernatant above the pellet.

4. Resuspend cells in freezing medium (see ^{Note 4}) at a concentration of a 5 × 10⁶ cells/mL. Aliquot cell suspension into labeled cryovials and freeze immediately.

5. Cells can be frozen at −80°C for short-term storage. (Optional: place cryovials into a styrofoam container or slow freezer such as the Nalgene® Cryo Freezing Container filled with isopropanol, which has a repeatable −1°C/min cooling rate to prevent the formation of damaging ice crystals.)

6. Transfer cells in cryovials to liquid nitrogen (−196°C) after 24 h for long-term storage.

3.1.4. Thawing and recovery of frozen cells

1. Remove cryovial from liquid nitrogen and place in a 37°C water bath. Agitate gently until thawed.

2. Wipe vial with 70% ethanol before putting under hood and opening.

3. Transfer cells to a sterile 15 mL conical tube containing medium prewarmed to 37°C. Centrifuge 5–10 min at 150–200g (approx 1000 rpm in a swinging bucket or 45° fixed-angle rotor). Discard supernatant containing residual dimethylsulfoxide (DMSO).

4. Resuspend pellet in 1 mL complete medium and transfer to culture plate/flask containing the appropriate amount of medium. Place in incubator. Check cells for adherence after 24 h.

3.1.5. Monitoring aging process through aging biomarkers (β-galactosidase staining)

1. Monolayers at low cell density are placed in PBS containing 3% formaldehyde for 5 min.

2. After three washes with PBS, stain cells overnight at 37°C in staining solution (150 mM NaCl, 2 mM MgCl₂, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 40 mM citric acid, and 12 mM sodium phosphate, pH 6) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside.

3. Cells are then washed with PBS.

4. β-galactosidase activity is monitored visually by scoring blue precipitate in the cytoplasm and photographed at 200X.

3.2. Aging Cell Culture: Major Issues and Experimental Design Considerations

In this section, some major issues related to aging cell culture are discussed, which include the in vitro cell culture model to study in vivo senescence and experimental design in aging-related cell culture studies.

3.2.1. Effect of Culture Conditions

Effects of culture conditions other than cell proliferation itself have also been investigated. One obvious difference in terms of the environment between in vitro and in vivo cell growth is exposure to direct light when cultured in vitro. Fluorescent light can cause DNA double strands breaks (23, 24). Additionally, It has been shown that exposure to fluorescent light can damage photoactive components of cell culture medium (25). Several other conditions have also been reported to induce a premature senescent phenotype, such as hydrogen peroxide (26), tert-butylhydroperoxide (27), and ultraviolet (28) and gamma radiation (29). Although the trypsinization procedure to remove adherent cells from culture dishes may also cause cellular stress, weekly trypsinizations have been shown to not affect the proliferative capacity of the mass culture (30).

3.2.2. Timeline

Aging studies that attempt to mimic cellular senescence are often time-consuming since it generally requires numerous population doublings for cells to acquire critically-attenuated telomere lengths which triggers senescence. For studies in this regard, it is better to investigate one serially cultured sample at different ages than to examine a few different cultured samples at different time periods. It is important to freeze cells in liquid nitrogen at various ages while serially culturing aging cells. This can enable the investigator to restart the study at certain points if a problem arises with a culture. Many different cell lines used in aging research can be provided by the Coriell Institute (http://ccr.coriell.org/nia/).