Patent Application
20250164468
The present invention relates to a method for high-throughput screening to determine cellular lifespan.
Besides generally reducing life activity, aging remains the greatest risk factor for the development of chronic diseases that, subsequently, compromise independent human life and finally lead to death. Since 1950, life expectancy has drastically increased from 47 years to 73 years. Despite the recent trend toward dwindling birth rates, the world population spiked from 2.9 billion in 1950 to 7.8 billion in 2020. This remarkable increase in human longevity results in a higher proportion of the population above 65 years of age, with almost one out of five in the most advanced countries. However, the increase in the number of years lived is not a representation of the health status of the elderly. There is a remarkable gap of almost ten years between the healthspan, which is the period essentially free from diseases, and the actual lifespan. Elders tend to have costly chronic diseases such as cancer, neurodegenerative and cardiovascular diseases and other metabolic syndromes, which severely hamper their quality of life and ability to work. As such, this puts tremendous pressure on the healthcare system and the working population. The current medical approaches to prevent age-related pathologies are recommendations for a healthy lifestyle, including exercise and diet. However, these interventions alone are not sufficient to prevent the onset of age-related diseases.
Aging is characterized by a progressive loss of physiological integrity, efficiency in cellular functions, and metabolic signalling. Increasing efforts are directed to understand cellular aging processes affecting the highly interconnected and functionally redundant gene and protein interactions network. Despite the complexity of aging, recent research in different model systems, including mammals, has demonstrated that delayed aging and increased healthspan are feasible by anti-aging interventions such as rapamycin drug administration and calorie (glucose) restriction.
Recent research has shown that molecular mechanisms observed in human aging are conserved in different organisms, including single-cell yeast. The budding yeast Saccharomyces cerevisiae is one of the most studied model organisms for uncovering the biological processes involved in cellular aging. The benefits of studying aging in yeast include a short generation time, a tractable lifespan, and its amenability to high-throughput assays. Therefore, this organism became a powerful tool for the identification of anti-aging interventions. Yeast is commonly used to study aging in two distinct ways: replicative lifespan (RLS) and chronological lifespan (CLS). The RLS measures the number of times an individual cell divides, an aging model for mitotic cells such as stem cells. The CLS measures the length of time a non-dividing cell remains viable in the stationary phase, an aging model for post-mitotic cells such as neurons and muscle cells. The viability of aging yeast cells under nutrient-deprived stationary phase conditions decreases, and they eventually die.
CLS has been traditionally measured by estimating colony-forming units (CFUs) counts on the agar plate in an outgrowth assay. Yeast cells in a flask with total media (≥20 mL) are exposed to a single chemical compound. At different time points (e.g. after 2, 5, and 8 days), small aliquots of the chronological aging culture from the flask are serially diluted, plated onto nutrient agar plates, and incubated for 2 to 3 days for colonies formation and counting. The survival fraction is determined from the CFU for each chronological age-point relative to day 1 (considered 100% cell survival). The CFU approach is quantitative but associated with many downsides. It requires multiple serial dilutions, plating steps, and incubation periods for the colonies to appear. It is dependent on the ability of the post-mitotic cells to re-enter into the mitotic phase. Colony counting is performed either by manual or expensive instruments. Moreover, this method is unsuitable and too expensive for high-throughput screening (HTS) of thousands of compounds because it requires substantial amounts of the drugs tested, large volume flask cultures, and many agar plates. Unfortunately, the CFU method and all recent variants for measuring CLS are critically dependent on the ability of the post-mitotic cells to re-enter into the mitotic phase. In principle, this methodology cannot distinguish yeast in mitotic arrest from dead cells.
Currently available CFU-derived methods measure cell survival and growth based on the outgrowth of drug-exposed stationary phase yeast in nutrient-rich culture either in liquid medium or by a spotting assay on an agar plate. After 24 hours, the outgrowth of aged cells in liquid medium is measured by the absorbance at OD600 nm, whereas in the spotting assay, the outgrowth of spotted aged culture onto agar medium is visually identified after 48 hours. Furthermore, for pooled tagged yeast deletion strains, flow cytometry may be necessary to quantify the individual strain viability.
Conventionally, CLS of human cells is measured by entering the human cells in a post-mitotic stage by nutrient deprivation under selected anti-aging experimental conditions. Survival of post-mitotic cells is then measured by outgrowth in a fresh medium. That is, exhausted medium (together with floating dead cells) is aspirated. Attached cells are trypsinized and transferred to 6-well plates with fresh medium. They are incubated for seven days before determining the cell viability via counting the number of grown colonies using crystal violet staining. This method has previously been applied to prove the anti-aging effect of rapamycin as well as the flavonoid 4,4′-dimethoxychalcone to promote CLS in cells of several species including human.
However, this method requires a long experiment time. At the outgrowth stage, the 6-well plates need seven days to reach sufficient cell count to be detected by the crystal violet assay. If primary fibroblasts were used instead of immortal cell lines, it would require an even longer growing period. Further, the method is not suitable for cells with a low adherent ability to the culture plate. The rinsing of cells before trypsinization and crystal violet staining would result in varying levels of cell loss due to detachment, leading to variations in the results. This method is also not suitable for high-throughput screening as the number of compounds and concentrations that can be tested are restricted by the plate capacity. The crystal violet assay produces image data for qualitative visual comparison. Thus, the qualitative nature of the data fails to capture minor changes in cell lifespan induced by attempted anti-aging interventions.
In view of the above, there is a need to develop a new quantification method to determine the lifespan of the aged cell that overcomes or at least ameliorates, one or more of the limitations described above.
In an aspect, there is provided a method for high-throughput screening to determine cellular lifespan, the method comprising the steps of:
In another aspect, there is provided a method for high-throughput screening to determine cellular lifespan, the method comprising the steps of:
Advantageously, the method may provide a cost- and time-efficient way to quantitatively screen the viability and chronological lifespan of cells. The method may be used in any kind of screening of cell viability following treatment with conditions that modulate the lifespan of cells to directly quantify cell survival. Advantageously, the method may not require an outgrowth assay. More advantageously, the method as defined above may not require additional steps of aspiration, washing, trypsinization of transferring of cells to multiple new plates. Therefore, the method may provide an alternative method for screening the lifespan of cells to the methods that are conventionally available, thereby circumventing excessive handling, time-consuming procedures and resource-intensive steps.
Advantageously, the method may only require equipment and reagents that are cheap and readily available. Moreover, the method may facilitate high-throughput screening by using multi-well plates, whereby the output of fluorescence and optical density may be determined immediately one after another within a matter of seconds or minutes, using a plate reader. Further advantageously, by eliminating a conventional out-growth step, the method may be a high-throughput method, which may enable the testing of cell viability in numerous samples at the same time, significantly reducing assay time and use of resources. Further advantageously, the entire method from step a) to e) may be performed within a matter of minutes.
Advantageously, the method may be a plate-based method to determine the chronological lifespan of cells based on (i) the ratio of dead/surviving cells and (ii) the total amount of cells, which may be derived simultaneously from the same plate using a microplate reader using fluorescence and optical density measurements. Further advantageously, by treating the cells in a first multi-well plate and transferring them to a second multi-well plate for the screening, it may allow screening of different lifespan-modulating conditions at different time points from the same sample. This may facilitate consistent screening from the same sample and minimize the errors involved in having to compare viability of cells treated with the lifespan modulating conditions in different plates.
More advantageously, the method as defined above may be performed independently of the ability of the cells to proliferate. In addition, the method as defined above may advantageously be performed on a variety of cell types, including fungal, bacterial and animal cells, regardless of whether they are primary cells or immortalised cells.
The following words and terms used herein shall have the meaning indicated:
The phrase “high-throughput screening”, in the context of the present disclosure, refers to the use of automated equipment to rapidly test the specific biological activity of chemical and/or biological compounds. High-throughput screening allows for the rapid testing of thousands to millions of chemical and/or biological compounds within a short time frame.
The phrase “chronological lifespan” in the context of the present disclosure, refers to the period of time that non-dividing cells remain alive in stationary phase or in nutrient deprived conditions.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
Unless specified otherwise, the terms “comprising” and “comprise”, and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements.
As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means +/−5% of the stated value, more typically +/−4% of the stated value, more typically +/−3% of the stated value, more typically, +/−2% of the stated value, even more typically +/−1% of the stated value, and even more typically +/−0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
The abbreviation CLS stands for chronological lifespan, which is defined as the number of days cells remain viable in a stationary phase.
There is provided a method for high-throughput screening to determine cellular lifespan, the method comprising the steps of:
The method may be for measuring the chronological lifespan of the cells, for screening anti-aging agents, for screening senolytic compounds, for screening anticancer agents, or for screening antifungal agents. The method may be for measuring the viability of cells.
The method may comprise a step (a) of treating the cells with a condition that modulates lifespan of cells, in a well of a multi-well plate.
Advantageously, the method may be used to screen the viability of cells following treatment with any kind of condition that may modulate the lifespan of cells.
The condition that modulates the lifespan of cells may be exposure to a lifespan modulating agent, or exposure to environmental conditions that modulate the lifespan of cells.
The lifespan modulating agent may be a chemical or biological reagent. The lifespan modulating agent may be selected from the group consisting of an anti-aging agent, anti-microbial agent, anti-fungal agent, anti-bacterial agent, anti-cancer agent, pharmaceutical drug, genotoxic agents, toxic chemical agent, and any mixture thereof.
The lifespan modulating agent may be an anti-aging agent. The anti-aging agent may be selected from the group consisting of rapamycin, metformin, resveratrol, caloric restriction, 2,5-anhydro-D-mannitol, aucubin, D-glucuronic acid, β-lapachone, isocytosine, rifamycin (sodium), crypotochlorogenic acid, isochlorogenic acid A, rhynchophylline, mupirocin. 3,5-dihydroxybenzoic acid, ethylparaben, nudifloramide, 4-methoxycinnamic acid, cefoselis (sulfate), DL-serine, avibactam (sodium), (S)-2-hydroxy-3-phenylpropanoic acid, vincristine (sulfate), N-acetyl-L-tyrosine, rubitecan, Royal Jelly acid, β-estradiol 17-acetate, 3,4-dimethoxycinnamic acid, 2-naphthol, N2,N2-dimethylguanosine, proxyphylline, amarogentin, lappaconitine (hydrobromide), 4-ethylphenol, purine, (S)-indoximod, actinonin, cirsimaritin, 6-biopterin, hederagenin, pentadecanoic acid, staurosporine, xanthoxylin, D-(+)-cellobiose, escin, 5α-cholestan-3-one, 20(S)-hydroxycholesterol, anthraquinone-2-carboxylic acid, L-kynurenine, quinine (hydrochloride dihydrate), DL-3-phenyllactic acid, retinoic acid, schisantherin B, narciclasine, doripenem (monohydrate), diflorasone, chlorogenic acid, glucosamine (hydrochloride), gardenoside, aloin B, 4-methylumbelliferone, lanolin, (R)-pyrrolidine-2-carboxylic acid, glycodeoxycholic acid (monohydrate), (−)-epigallocatechin, 2′-deoxyadenosine, wogonin, schisandrol B, 2,4-dihydroxybenzoic acid, hederacoside C, noricaritin, canrenone, glycoursodeoxycholic acid, glycodeoxycholic acid, cilastatin, emetine (dihydrochloride hydrate), norgestrel, heterophyllin B, hispidin, cinnamic acid, catechin, [6]-gingerol, epiberberine (chloride), terconazole, isoalantolactone, 2-furoic acid, vanillic acid, 5-acetylsalicylic acid, guggulsterone, thymol, homovanillic acid, uridine, gramicidin, gossypol, cyanidin-3-O-galactoside (chloride), kaempferol 3-O-β-D-glucuronide, L-homoserine, D-ribose (mixture of isomers), harmane, geraniol, 3,4-dimethoxyphenylacetic acid, 2-phenylpropionic acid, deltonin, 2-oxovaleric acid, estrone sulfate (potassium), (R)-citronellol, euphorbia factor L2, β-aminopropionitrile, bronopol, lincomycin (hydrochloride monohydrate), tridecanedioic acid, calcifediol, melanin, methyl acetylacetate, L-cysteine (hydrochloride hydrate), impulsin, formamide, 3-chloro-L-tyrosine, tetramethylpyrazine, esculin, DL-panthenol, baimaside, benzyl acetate, gentiopicroside, guaiazulene, engeletin, acivicin, tenofovir (hydrate), metyrosine, ripasudil, diphenmanil (methylsulfate), ambroxol (hydrochloride), VAL-083, aliskiren (hemifumarate), selenomethionine, cisatracurium (besylate), DL-methionine methylsulfonium (chloride), biapenem, dipotassium glycyrrhizinate, L-ornithine (hydrochloride), fosphenytoin (disodium), (R)-baclofen, trapidil, xanthinol nicotinate, cerivastatin (sodium), aprotinin, flavin adenine dinucleotide (disodium salt), biperiden (hydrochloride), streptomycin (sulfate), cefadroxil, diclofenac (sodium), estramustine (phosphate sodium), amifostine, quetiapine sulfoxide (dihydrochloride), minocycline (hydrochloride), L-selenomethionine, cangrelor (tetrasodium), chondroitin (sulfate), hyaluronidase, heparin (lithium salt), heparin (sodium salt) (MW 15 kDa), gentamicin (sulfate), polidocanol, plicamycin, nedaplatin, copanlisib dihydrochloride, hemin, 5-fluorouracil, methotrexate, atorvastatin and any mixture thereof.
The calorie restriction may be performed by restricting energy sources such as sugar. The sugar may be selected from the group consisting of sucrose, fructose, glucose and any mixture thereof.
The conditions that can modulate the lifespan of cells may include nutrients such as carbohydrates such as monosaccharides, disaccharides, oligosaccharides and polysaccharides, proteins such as contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins, antibodies and transport proteins and lipids such as fatty acids, triglycerides, phospholipids, sterols, sphingolipids, other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid-soluble vitamins, and hormones, carbon, nitrogen, amino acids, phosphate, nucleosides, nucleotides, nutrient starvation, osmotic condition, oxidative stress, temperature, pH, oxygen concentration, and any mixture thereof.
The treating step (a) may be performed in a first multi-well plate, wherein the first multi-well plate of step (a) is different to the second multi-well plate of step (c).
The treating step (a) may comprise a step (a1) of exposing the cells to a condition that modulates lifespan of the cells.
The exposing step (a1) may comprise the step of treating the cells with a lifespan modulating agent.
The cells may be treated with the lifespan modulating agent at a range of various concentrations. The cells may be treated with the lifespan modulating agent at serially double-diluted concentrations. The cells may be treated with the lifespan modulating agent at a concentration in the range of about 0 nM to about 2 M, more than 0 nM to about 2 M, or about 0.0001 nM to about 2 nM. The cells may be treated with the lifespan modulating agent at a concentration in the range of 0 nM to about 2 M, 0 nM to about 0.0001 nM, 0 nM to about 0.001 nM, 0 nM to about 0.01 nM, 0 nM to about 0.1 nM, 0 nM to about 1 nM, 0 nM to about 10 nM, 0 nM to about 100 nM, 0 nM to about 1 μM, 0 nM to about 10 μM, 0 nM to about 100 μM, 0 nM to about 1 mM, 0 nM to about 10 mM, 0 nM to about 100 mM, 0 nM to about 1M, about 0.0001 nM to about 0.001 nM, about 0.0001 nM to about 0.01 nM, about 0.0001 nM to about 0.1 nM, about 0.0001 nM to about 1 nM, about 0.0001 nM to about 10 nM, about 0.0001 nM to about 100 nM, about 0.0001 nM to about 1 μM, about 0.0001 nM to about 10 μM, about 0.0001 nM to about 100 μM, about 0.0001 nM to about 1 mM, about 0.0001 nM to about 10 mM, about 0.0001 nM to about 100 mM, about 0.0001 nM to about 1M, about 0.0001 nM to about 2 M, about 1 μM to about 10 μM, about 1 μM to about 100 M, about 1 μM to about 1 mM, about 1 μM to about 10 mM, about 1 μM to about 100 mM, about 1 μM to about 1M, about 1 μM to about 2 M, about 10 UM to about 100 μM, about 10 μM to about 1 mM, about 10 μM to about 10 mM, about 10 UM to about 100 mM, about 10 μM to about 1M, about 10 μM to about 2 M, about 100 μM to about 1 mM, about 100 μM to about 10 mM, about 100 μM to about 100 mM, about 100 μM to about 1M, about 100 μM to about 2 M, about 1 mM to about 10 mM, about 1 mM to about 100 mM, about 1 mM to about 1M, about 1 mM to about 2 M, about 10 mM to about 100 mM, about 10 mM to about 1M, about 10 mM to about 2 M, about 100 mM to about 1M, about 100 mM to about 2 M or about 1 M to about 2 M.
The treating step (a) may further comprise a step (a2) of exposing the cells to a condition that modulates cellular lifespan for a various duration. The treating step (a) may further comprise a step (a2) of exposing the cells to a condition that modulates cellular lifespan for a duration in the range of about 12 hours to about 90 days, about 12 hours to about 24 hours, about 12 hours to about 48 hours, about 12 hours to about 72 hours, about 12 hours to about 7 days, about 12 hours to about 14 days, about 12 hours to about 21 days, about 12 hours to about 30 days, about 12 hours to about 60 days, about 24 hours to about 48 hours, about 24 hours to about 72 hours, about 24 hours to about 7 days, about 24 hours to about 14 days, about 24 hours to about 21 days, about 24 hours to about 30 days, about 24 hours to about 60 days, about 24 hours to about 90 days, about 48 hours to about 72 hours, about 48 hours to about 7 days, about 48 hours to about 14 days, about 48 hours to about 21 days, about 48 hours to about 30 days, about 48 hours to about 60 days, about 48 hours to about 90 days, about 72 hours to about 7 days, about 72 hours to about 14 days, about 72 hours to about 21 days, about 72 hours to about 30 days, about 72 hours to about 60 days, about 72 hours to about 90 days, about 7 days to about 14 days, about 7 days to about 21 days, about 7 days to about 30 days, about 7 days to about 60 days, about 7 days to about 90 days, about 14 days to about 21 days, about 14 days to about 30 days, about 14 days to about 60 days, about 14 days to about 90 days, about 21 days to about 30 days, about 21 days to about 60 days, about 21 days to about 90 days, about 30 days to about 40 days, about 40 days to about 50 days, about 50 days to about 60 days or about 60 days to about 90 days. The treating step (a) may further comprise a step (a2) of exposing the cells to a condition that modulates cellular lifespan for a duration of more than 12 hours, more than 24 hours, more than 72 hours, more than 7 days, more than 14 days, more than 21 days, more than 30 days, more than 60 days or more than 90 days.
The method may comprise a step (b) of transferring a portion of the cells in the well of the multi-well plate of step (a) into the well of a second multi-well plate.
The transferring step (b) may comprise the step (b1) of shaking the first multi-well plate to form a cell suspension. The cells in the first multi-well plate may be adherent to the bottom of the well in the first multi-well plate. The shaking step (b1) may cause the cells to detach from the bottom of the well of the first multi-well plate and create a cell suspension in the well of the first microwell plate.
The shaking step (b1) may be performed at a frequency in a range of about 800 rpm to about 1200 rpm, about 800 rpm to about 1000 rpm or about 1000 rpm to about 1200 rpm.
The shaking step (b1) may be performed for a duration in the range of about 10 seconds to about 15 seconds, about 10 seconds to about 12 seconds or about 12 seconds to about 15 seconds.
The shaking step (b1) may be performed using a thermomixer.
Advantageously, the shaking step (b1) may achieve relatively fast and uniform detachment of cells from the well of the first microwell plate. Further advantageously, unlike conventional methods for detaching cells done by up-and-down pipetting, the shaking step may result in less bubble formation that may compromise accurate transfer of the cells from the first microwell plate to the second microwell plate. Typically, when bubbles are formed during pipetting, it is necessary to wait for the bubbles to disappear, or to manually burst the bubbles before taking the next step, which is not conducive to high-throughput screening. By using shaking instead of up-and-down pipetting, cell detachment may be achieved faster and more efficiently. In addition, by using shaking instead of pipetting, it may circumvent the need to use multiple pipette tips, thereby contributing to cost reduction, the process may be safer and most importantly, the risk of inaccurate transfer of cells may be avoided.
The transferring step (b) may be performed by transferring the portion of the cells, in the form of the cell suspension, using a pipette.
The multi-well plate may be a 6-well plate. 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate or a 1536-well plate.
The multi-well plate may be a 96-well plate. Advantageously, 96-well plates may facilitate screening of limited chemical or genome-wide deletion strains.
The multi-well plate may be a clear plate or an opaque-walled plate. The opaque-wall plate may have white walls or black walls.
Advantageously, the method may yield similar results regardless of whether a clear plate or a black-walled plate is used. As black-walled plates may be more expensive, the ability to use clear plates may facilitate more cost-effective high-throughput screening.
The sample containing the cells may comprise cell culture media or phosphate buffered saline.
If a 96-well plate is used, then the sample containing cells may comprise about 30 μL to about 250 μL of cell culture media or phosphate buffered saline. The volume of the cell culture media or phosphate buffered saline in the sample may be in the range of about 30 μL to about 60 μL, about 30 μL to about 90 μL, about 30 μL to about 120 μL, about 30 μL to about 150 μL, about 30 μL to about 200 μL, about 30 μL to about 250 μL, about 60 μL to about 90 μL, about 60 μL to about 120 μL, about 60 μL to about 150 μL, about 60 μL to about 200 μL, about 60 μL to about 250 μL, about 90 μL to about 120 μL, about 90 μL to about 150 μL, about 90 μL to about 200 μL, about 90 μL to about 250 μL, about 120 μL to about 150 μL, about 120 μL to about 200 μL, about 120 μL to about 250 μL, about 150 μL to about 200 μL, about 150 μL to about 250 μL, or about 200 μL to about 250 μL.
The cells may be fungal cells, bacterial cells or animal cells. The fungal cells may be yeast cells. The yeast cells may be cells of yeast selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris and any mixture thereof.
The animal cells may be mammalian cells. The mammalian cells may be human cells. The human cells may be primary cells or immortal cells. The human cell may be a cancer cell. The human cell may be selected from the group consisting of a kidney cell, liver cell, muscle cell, lung cell, neuron, retinal cell, skin cell and any mixture thereof. The cells may be human embryonic kidney 293 (HEK293) cells or lung epithelial (A549 cells) or fibroblast (IMR90) cells.
Advantageously, the method may be used for fungal cells, bacterial cells and/or animal cells. Although there are critical differences in aging of yeast and animals, the method may advantageously be used in both yeast and animals, and in different cell types within each category.
The method may comprise a step (c) of mixing, in a well of the second multi-well plate, a sample containing the portion of cells with a fluorescent viability dye to stain the cells.
The mixing step (c) may comprise a step (c1) of shaking the second multi-well plate.
The shaking step (c1) may be performed at a frequency in a range of about 800 rpm to about 1200 rpm, about 800 rpm to about 1000 rpm or about 1000 rpm to about 1200 rpm.
The shaking step (c1) may be performed for a duration in the range of about 10 seconds to about 15 seconds, about 10 seconds to about 12 seconds or about 12 seconds to about 15 seconds.
The shaking step (c1) may be performed using a thermomixer.
Advantageously, the shaking step (c1) may achieve relatively fast and uniform mixing of the cells with the fluorescent viability dye. Further advantageously, unlike conventional methods for mixing done by up-and-down pipetting, the shaking step may result in less bubble formation that may compromise fluorescence and optical density reading. Bubbles that form in conventional pipetting techniques may skew the fluorescence and optical density readings and may give a false result. Typically, when bubbles are formed during pipetting, it is necessary to wait for the bubbles to disappear, or to manually burst the bubbles before taking any readings, which is not conducive to high-throughput screening. By using shaking instead of pipetting, the mixing may be performed faster and more efficiently. In addition, by using shaking instead of pipetting, it may circumvent the need to use multiple pipette tips, thereby contributing to cost reduction, the process may be safer and most importantly, the risk of an artificial or false reading may be avoided.
The fluorescent viability dye may quantitatively differentiate between dead cells and viable cells.
The fluorescent viability dye may emit fluorescence only when it is contacted with dead or damaged cells, or the fluorescent viability dye may emit fluorescence only when it is contacted with living cells.
The fluorescent viability dye may have excitation and emission wavelengths that do not affect optical density measurements.
The fluorescent viability dye may be selected from the group consisting of propidium iodide, PrestoBlue™, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT), alamarBlue reagent, 7-hydroxy-10-oxidophenoxazin-10-ium-3-one (resazurin), SYTO 9, 2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide (Fun 1), disodium 2,2′-ethene-1,2-diylbis [5-({4-anilino-6-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate] (Calcofluor White), 3′,6′-Bis(acetyloxy)spiro[isobenzofuran-1 (3H), 9′-[9H]xanthen]-3-one (fluorescein diacetate), N,N,N′,N′-Tetramethyl-3,6-acridinediamine (acridine orange), 2′-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride (Hoechst 33342), and any combination thereof.
The fluorescent viability dye may be propidium iodide. Propidium iodide (PI) is a nucleic acid intercalating fluorescent dye that only enters the dead cells and, thus, may be a powerful marker for direct quantification of cell viability. PI may fluoresce only when in contact with dead or damaged cells. It may therefore used to detect necrotic or apoptotic cells. It may be impermeable to live cells with intact membranes, but may be able to penetrate dead or cells with damaged membranes to bind to DNA and RNA. Upon intercalating between the bases, the quantum signal of PI increases drastically, which can then be detected by the microplate reader. Previously, a PI-based approach was used for measuring CLS and quantifying cell viability by analyzing the images acquired by a fluorescent cell counter, UV transilluminator, or flow cytometry. However, multiple sample processing, imaging, and software requirements for data analysis were expensive and time-consuming, which hindered these methods in high-throughput applications.
In the mixing step (c), the propidium iodide may be present at a concentration in the range of about 3 μg/mL to about 7 μg/mL in phosphate buffered saline. The concentration of propidium iodide may be in the range of about 3 μg/mL to about 4 μg/mL, about 3 μg/mL to about 5 μg/mL, about 3 μg/mL to about 6 μg/mL, about 4 μg/mL to about 5 μg/mL, about 4 μg/mL to about 6 μg/mL, about 4 μg/mL to about 7 μg/mL, about 5 μg/mL to about 6 μg/mL, about 5 μg/mL to about 7 μg/mL, or about 6 μg/mL to about 7 μg/mL.
The cells may be mixed with the propidium iodide for a duration in the range of about 10 minutes to about 20 minutes, about 10 minutes to about 15 minutes or about 15 minutes to about 20 minutes. The mixing step (c) may be performed in the dark.
The fluorescence of propidium iodide may be measured at an excitation wavelength of about 535 nm and emission wavelength of 617 nm.
The optical density may be measured at about 600 nm (OD600). The optical density value may directly correlate with the number, density or concentration of cells.
The plate reader may be capable of measuring the absorbance, fluorescence, luminescence, time-resolved fluorescence, fluorescence polarization, light scattering and/or nephelometry of each well of a multi-well plate.
The method may comprise a step (x1) of washing the cells after the transfer step (b) and before the mixing step (c).
The method may comprise a step (x2) of washing the cells after the mixing step (c) and before the measuring step (d).
The washing steps (x1) and (x2) may independently comprise the steps of:
If a 96-well plate is used, then the resuspension step (y3) may be performed in phosphate buffered saline in a volume in the range of about 30 μL to about 200 μL of. The resuspension step (y3) may be performed in a volume of phosphate buffered saline in the range of about 30 μL to about 60 μL, about 30 μL to about 90 μL, about 30 μL to about 120 μL, about 30 μL to about 150 μL, about 60 μL to about 90 μL, about 60 μL to about 120 μL, about 60 μL to about 150 μL, about 60 μL to about 200 μL, about 90 μL to about 120 μL, about 90 μL to about 150 μL, about 90 μL to about 200 L, about 120 μL to about 150 μL, about 120 μL to about 200 μL, or about 150 μL to about 200 μL.
The washing steps (x1) and (x2) may independently be repeated multiple times. The washing steps (s1) and (x2) may independently be repeated 1, 2, 3, 4 or 5 times.
The method steps (c) to (f) may be performed within a duration of less than about 45 minutes, less than about 30 minutes, less than about 20 minutes. The method steps (c) to (f) may be performed within a duration in the range of about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes or about 5 minutes to about 45 minutes.
The method may further comprise a step (g) of comparing the normalized fluorescence intensity value of cells treated with the lifespan modulating condition with the fluorescence intensity value of positive control cells and negative control cells.
The fluorescence intensity value of the positive control cells may be measured by measuring the fluorescence intensity of a sample containing cells with a fluorescent viability dye to stain the cells, whereby:
The fluorescence intensity of the negative control cells may be measured by measuring the fluorescence intensity of a sample containing cells without a fluorescent viability dye, whereby:
The comparing step (g) may further comprise a step (g1) of calculating % cell survival.
The method may be for high-throughput screening of the viability of yeast cells following treatment with an anti-aging agent.
Advantageously, the method may be useful in screening numerous anti-aging agents to determine how effective the anti-aging agent is effective in improving cell viability or extending chronological lifespan.
Further advantageously, the method may circumvent the need to perform an outgrowth assay. Circumventing the need to perform an outgrowth assay may result in significant cost- and time-savings. In a typical assay for the viability of yeast cells following treatment with an anti-aging agent, the yeast cells would have to undergo an outgrowth assay on agar to manually count the colony forming units (CFU), which is not only more expensive but also time-consuming given that it must be performed over days before a result may be obtained. In conventional assays, even if fluorescence markers are used to determine cell viability, the results may have to be manually processed, and the results may only be qualitative. Therefore, conventional assays for viability of yeast cells may not be conducive for high-throughput screening. In contrast, the method as defined herein may circumvent the need for an outgrowth assay or manual analysis of the results, thereby allowing high-throughput screening of the viability of the cells in a quantitative manner and in a matter of minutes rather than days. In addition, by circumventing the need to perform an outgrowth assay, the method as defined herein may be able to detect the viability of the cells more accurately at the correct stage in the cell cycle.
If the method is for high-throughput screening of the viability of yeast cells following treatment with an anti-agent agent, the cell culture medium may be a YPD medium comprising 1% Bacto yeast extract, 2% Bacto peptone and 2% glucose or may be a synthetic defined (SD) medium contain 6.7 g/L yeast nitrogen base with ammonium sulfate without amino acids and 2% glucose or may be a synthetic defined (SD) medium contain 6.7 g/L yeast nitrogen base with ammonium sulfate and amino acids and 2% glucose or may be a synthetic defined (SD) medium contain 6.7 g/L yeast nitrogen base without ammonium sulfate and amino acids+CSM (complete supplement mixture) and 2% glucose.
In an example, there is provided a method for high-throughput screening of anti-aging agent in cells, the method comprising the steps of:
There is also provided a method for high-throughput screening to determine cellular lifespan, the method comprising the steps of:
If a 96-well plate is used, then the sample containing cells may comprise cells in a range of about 1,000 cells to about 200,000 cells, about 1,000 cells to about 2,000 cells, about 1.000 cells to about 10,000 cells, about 1,000 cells to about 25,000 cells, about 1,000 cells to about 50,000 cells, about 1,000 cells to about 75,000 cells, about 1,000 cells to about 100,000 cells, about 1.000 cells to about 150,000 cells, about 2,000 cells to about 10,000 cells, about 2,000 cells to about 25,000 cells, 2,000 cells to about 50,000 cells, 2,000 cells to about 75,000 cells, 2,000 cells to about 100,000 cells, 2,000 cells to about 150,000 cells, about 2,000 cells to about 200,000 cells, about 10,000 cells to about 25,000 cells, 10,000 cells to about 50,000 cells. 10,000 cells to about 75,000 cells, 10.000 cells to about 100,000 cells, 10,000 cells to about 150,000 cells, about 10,000 to about 200.000 cells, about 25.000 cells to about 50.000 cells, about 25.000 cells to about 75,000 cells, about 25,000 cells to about 100,000 cells, about 25,000 cells to about 150,000 cells, about 25,000 cells to about 200,000 cells, about 50,000 cells to about 75,000 cells, about 50,000 cells to about 100.000 cells, about 50,000 cells to about 150,000 cells, about 50,000 cells to about 200,000 cells, about 75,000 cells to about 100,000 cells, about 75,000 cells to about 150,000 cells, about 75,000 cells to about 200,000 cells, about 100,000 cells to about 150,000 cells, about 100,000 cells to about 200,000 cells or about 150,000 cells to about 200,000 cells.
The accompanying drawings illustrate a disclosed embodiment and explain the principles of the disclosed embodiment. However, the drawings are designed for illustration purposes only, and not as a definition of the limits of the invention.
Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
The prototrophic wild-type Saccharomyces cerevisiae CEN.PK113-7D yeast strain genetic background (Euroscarf, Köhlerweg, Oberursel, Germany), and the Saccharomyces cerevisiae BY4743 strain (Euroscarf. Köhlerweg, Oberursel. Germany) were used. The CEN.PK113-7D snf1Δ deletion strain was constructed. Standard rich Yeast Extract-Peptone-Dextrose (YPD) medium (containing 1% w/v Bacto yeast extract, 2% w/v Bacto peptone, and 2% w/v glucose) (BD, Franklin Lakes, New Jersey, United States of America) YPD agar (with 2.5% w/v Bacto agar) (BD, Franklin Lakes, New Jersey, United States of America), and synthetic defined (SD) medium (BD, Franklin Lakes, New Jersey, United States of America) contained 6.7 g/L yeast nitrogen base with ammonium sulfate without amino acids from BD DIFCO™ (BD, Franklin Lakes, New Jersey, United States of America) and 2% w/v glucose. Rapamycin (Enzo, Farmingdale, New York, United States of America) stock solution was prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Missouri, United States of America). The maximum amount of DMSO used in any assay was 1%. 2,5-anhydro-D-mannitol (Santa Cruz Biotechnology, Texas, United States) and D-glucose (Sigma-Aldrich, St. Louis, Missouri, USA) were freshly prepared by direct dissolution of the compound in medium and further filtered under sterilized conditions.
The various compounds tested in Table 1 were obtained from Sigma-Aldrich (St. Louis, Missouri, United States of America), MedChemExpress (Monmouth Junction, New Jersey, United States of America), Enzo Life Sciences, Inc. (Farmingdale, New York, United States of America), Santa Cruz Biotechnology (Dallas, Texas, United States of America), or Biosynth Carbosynth (Compton, Berkshire, United Kingdom).
Yeast strain was recovered from frozen glycerol stock at 30° C. on YPD agar medium. Yeast was grown in SD medium overnight at 30° C. with shaking at 220 revolutions per minute (rpm). Cells grown overnight were diluted to OD600 nm˜0.2 in fresh SD medium to initiate the chronological lifespan experiments.
Chronological lifespan (CLS) experiments were conducted in 96-well plates with 200 μL yeast culture. The prototrophic wild-type yeast strain (CEN.PK113-7D) and snf1Δ deletion strains were grown in the SD medium in 96-well plates at 30° C. Saccharomyces cerevisiae BY4743 strain was grown in the SD medium supplemented with histidine (40 mg/L), leucine (160 mg/L), and uracil (40 mg/L) in 96-well plates at 30° C. For assays testing the effect of rapamycin, cellular inoculum was transferred into 96-well plates containing serially double-diluted concentrations (0 to 10 nM) of rapamycin. For calorie restriction assay, cellular inoculums were prepared in SD medium containing 2% w/v, 0.5% w/v, and 0.25% w/v glucose and transferred to 96-well plates. Likewise, cellular inoculum was transferred into the 96-well plates containing serially double-diluted concentrations (0-8 mM) of D-fructose, D-mannitol, D-maltose, and D-sorbitol. Cells were incubated at 30° C., and the growth was measured at different time points. The growth time 72 hours was considered as day 1 for the CLS assay. Cell survival was quantified at various age time points by three different approaches: (i) propidium iodide fluorescence-based method, (ii) outgrowth in YPD liquid medium and (iii) spotting assay (
40 μL yeast cells on different age time points were transferred into a 96-well plate different to the 96-well plate in which the CLS analysis was performed. Cells were washed and incubated in 100 μL 1×PBS with PI (5 μg/mL) for 15 minutes in the dark. Positive and negative sample control were included for quantitative analysis. Positive control (cells boiled at 100° C. for 15 min) was PI-stained and processed in the same 96-well plate. Samples without PI-stained cells served as the negative control. After incubation, cells were washed and resuspended in 100 μL PBS. The fluorescence reading of the samples (excitation at 535 nm, emission at 617 nm) and OD600 nm were measured by a microplate reader (BioTek, Winooski, Vermont, United States). The fluorescence intensity of each sample was normalized with OD600 nm. The normalized fluorescence intensity of each sample was subtracted from the background signal of the unstained negative sample. The obtained fluorescence intensity of the positive control sample (boiled dead cells) was considered 0% cell survival. Cell death was confirmed by allowing the boiled dead cells to grow in the medium. Cell survival of different age time point samples were calculated by normalizing the fluorescence intensity with positive control sample (boiled cells).
Hence, cell survival was calculated using the formula:
Yeast stationary culture (3 μL) of different age time points were transferred to a second 96-well plate containing 200 μL YPD medium and incubated for 24 hours at 30° C. Outgrowth (OD600 nm) of aged cells was measured by the microplate reader. Quantification of cell survival for each age point was determined relative to day 1 (considered 100% cell survival). This quantification was performed by using the formula,
Yeast stationary culture (3 μL) of different age time points were spotted onto the YPD agar plate and incubated for 48 hours at 30° C. The outgrowth of aged cells on the YPD agar plate was photographed using the BioRad GelDoc imaging system (Bio-Rad, Hercules, California, United States).
The prototrophic yeast strain (CEN.PK113-7D) was grown in the synthetic defined medium with indicated concentrations of rapamycin in 96-well plates at 30° C. A total of 30 samples of individual rapamycin concentration was tested with DMSO control (0 nM rapamycin) for Z-factor analysis. Cell growth OD600 nm was measured at 72 hours using a microplate reader and a graph is plotted against different concentrations of rapamycin (
High-throughput screening of various chemicals was tested using the same protocol for the propidium iodide fluorescence-based method as described above. The prototrophic yeast strain (CEN.PK113-7D) was grown in the synthetic defined medium with different chemical reagents with a single testing concentration (Table 1) in addition to eight replicates of DMSO and water controls in 96-well plates at 30° C.
Cell survival at age point day 7 was quantified, and the growth time point 72 hours was considered day 1 (
The CLS of the aged cells was further determined by the yeast outgrowth in YPD liquid medium method as described above. The growth time point 72 hours was considered as day 1. At chronological age point day 14, 3-μL of culture were transferred to a second 96-well plate containing 200 μL YPD medium. Outgrowth OD600 nm in YPD liquid medium was measured after incubation for 24 hours at 30° C. using a microplate reader (
Human embryonic kidney 293 (HEK293) cells (ATCC, Manassas, Virginia, United States of America), lung epithelial (A549) cells (ATCC, Manassas, Virginia, United States of America) and fibroblast (IMR90) cells (Coriell Institute, Camden, New Jersey, United States of America) were cultured in the standard D10 medium, consisting of high-glucose DMEM (HyClone, Cytiva, Marlborough, Massachusetts, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco™, ThermoFisher Scientific, Waltham, Massachusetts, USA) and 1% Penicillin Streptomycin Solution (Gibco™, ThermoFisher Scientific, Waltham, Massachusetts, USA). All cells were cultured in a humidified incubator with 5% CO2 at 37° C.
The stock solution of rapamycin was prepared in dimethyl sulfoxide (DMSO), followed by serial dilution to form various concentrations used in the experiment. The final DMSO concentration was kept at 0.05% in all experiments. The stock solution of 2,5-anhydro-D-mannitol was prepared in D10 medium. The chemical library used in screening were tested at 1 μM concentrations. Controls used were the same as the solvent applied, DMSO or water.
80,000 cells were seeded in a 96-well culture plate with or without compound treatment. On Day 4, Day 6 and Day 8 after seeding, floating dead cells were aspirated with exhausted D10 medium. Then, attached cells were gently washed with 1× Phosphate-buffered saline (PBS) to remove D10 medium residue and treated with 0.2 mL of 0.25% trypsin. Cells were gently mixed by pipetting and a 2% aliquot (4 μL) was transferred to 6-well experiment plates with fresh D10 medium. After seven days of growth in a 37° C. incubator with 5% CO2, the crystal violet (CV) assay was performed on the test plate to determine the viability of cultured cells. For the CV assay, the media was first aspirated, before the cells were stained with 0.05% w/v CV and left to shake with 20 oscillation speed for 20 minutes. The media with CV was subsequently aspirated and the cells were washed once with water. The water was then aspirated and the plate inverted. Residual water was removed by tapping the plate dry and the plate was subsequently left to dry in air for at least 2 hours. Once dry, the plates were imaged using a scanner (Perfection V700 photo, Epson, Singapore).
The seeding, washing and trypsinization process was performed similarly to that for the crystal violet assay. 80.000 cells were seeded in a 96-well culture plate with or without compound treatment. At different age time points after seeding, floating dead cells were aspirated with exhausted D10 medium, cells were gently washed with 1×PBS to remove D10 medium residue and treated with 0.2 mL of 0.25% trypsin. Cells were gently mixed by pipetting and a 5% aliquot (10 μL) or 10% aliquot (20 μL) were transferred to 96-well experiment plates with fresh D10 medium. After one day of growth in a 37° C. incubator with 5% CO2, the cell number was assessed with PrestoBlue™ Cell Viability Reagent (Invitrogen™, Waltham, Massachusetts, USA) according to the manufacturer's protocol. In brief, the cell viability reagent was diluted 10-fold with growth D10 medium. The D10 medium was aspirated from the 96-well plate and replaced with the diluted PrestoBlue reagent. After incubation in a 37° C. incubator with 5% CO2 for 3 to 4 hours, reading was taken with a Synergy MX microplate reader at 560 nm excitation and 590 nm emission (BioTek, Winooski, Vermont, United States).
The assay was performed by preparing the cells with PI (2 μg/mL or 5 μg/mL) in a D10 medium. 200 μL culture (8×104 cells) was seeded per well in a 96-well plate with different concentrations of the anti-aging drug (rapamycin or 2,5-AM) and no treatment control (DMSO). The culture plate was kept away from light throughout the experiment and incubated at 37° C. with 5% CO2. At different age time points, reading was taken with a Synergy MX microplate reader at 535 nm excitation and 617 nm emission (BioTek, Winooski, Vermont, United States).
Statistical analysis of all the results such as mean value, standard deviations, correlation (R2), significance and graphing were performed using the GraphPad Prism v.9.3.1 software. The results were statistically compared using the ordinary one-way Analysis of Variance (ANOVA) and two-way ANOVA, followed by multiple comparisons by Dunnett's post hoc test or Sidak's post hoc test. In all the graph plots, P values shown as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 were considered statistically significant, while n.s refers to non-significant.
A new protocol was developed where wild-type yeast cells or their mutated variants were added to 96-well plates with media enriched with nutrients and specified amounts of chemical agents of interest. Propidium iodide (PI) was utilized as the marker for cell death since this fluorescent dye only permeates through dead cells. Quantification of cell survival in the stationary phase after a specific number of days was done by reading the PI fluorescence data in a microplate reader, which then generated an electronically readable spreadsheet for data analysis. The total amount of cells was quantified by measuring the OD600 nm optical density from the same plate. A microplate reader is a low-cost device readily available in most laboratories, providing fast and efficient readout (within about 15 minutes) of absorbance and fluorescence.
It was first shown that carrying out the CLS study in microplate wells conveyed the same results as that in traditional methods using cuvettes, since the microplate reader is adequately sensitive to detect cell survival data via the fluorescence intensity measured. It was also necessary to determine if PI was an appropriate dye for staining the yeast cells. To test this, yeast cell cultures were grown in the glass flask and boiled for 15 minutes at 100° C., before both the live and dead cells were washed and incubated in 1×PBS under the presence and absence of PI (5 μg/mL) for 15 minutes. After incubation, the cells were washed and resuspended in PBS. Microscopic visualization of the cells was performed, and microplate readings of the fluorescence intensity were recorded (
Next, PI-stained dead cells were resuspended in PBS to the final 48 OD600 nm measured in a cuvette using a spectrophotometer. The cells were then serially diluted in PBS (OD600 nm 48 to 0.05) before being transferred to a black 96-well plate (Costar 3603, Corning, Cambridge, Massachusetts, United States of America). Black microplates are four times more costly than clear plates and less easily available, but are more suitable for fluorescence measurements due to dampening of the autofluorescence originating from the samples and microplate surfaces. With a GelDoc imaging system, the degree of incorporation of PI staining in the dead yeast cells at various dilutions was visualized (
It was further necessary to identify the optimal range of cell density quantified by OD600 nm, so that cell density could be directly measured in 96-well plates instead of cuvettes. Using the black microplates with dead yeast cells as described above, the correlation of OD600 nm measurements taken from the cuvettes and from the microplate directly, was determined (
Although black microplates are recommended for fluorescence-based assays due to the ability to quench background fluorescence, their high cost and low availability called for cheaper and readily available alternatives. Comparisons were made between similar experiments carried out in both black and clear microplates (Costar 3596, Corning. Cambridge, Massachusetts, United States of America). PI-stained cells in clear microplates were visualized with a GelDoc imaging system to confirm sample staining (
Rapamycin is one of the well-known anti-aging agent that demonstrates the extension of lifespan of several model organisms, including yeast, nematodes, fruit flies and mice, by inhibiting the nutrient-sensing complex TORC1 (target of rapamycin complex 1). TORC1 is a conserved multi-subunit protein complex in eukaryotic cells that couples nutrients in the environment with cell growth and proliferation.
The effect of rapamycin on the CLS of a yeast strain was studied to examine the validity of the inventive protocol. Prototrophic yeast strain (CEN.PK113-7D) was utilized to circumvent the strong effects of amino acid auxotrophy on cell survival in stationary phase culture. Cells were aged with different concentrations of rapamycin in the SD medium and their respective growth at different time points (24 hours, 48 hours and 72 hours) were measured. It was found that cell growth reached saturation approximately 24 hours after incubating with 5 nM or less rapamycin (
By utilizing PI fluorescence to determine cell survival at various chronological age time points, the CLS of aging cells grown in different concentrations of rapamycin was evaluated. The 72 hour growth time point was taken as day 1. Based on the survival graph plotted (
The inventive protocol was validated by directly comparing it with two traditional outgrowth assays that measures the CLS of yeast. The outgrowth assay was performed in liquid medium to assess the effect of rapamycin on CLS. The survival of chronologically aging cells was monitored at various age points by transferring a 3 μL cell culture into 200 μL of YPD medium in a fresh 96-well plate. Outgrowth correlated with the number of viable cells in the inoculum (
Another method to assess the outgrowth of aged cells was to perform a spotting assay on the agar medium. After spotting 3 μL aging cell culture on the YPD agar plate and incubating for 48 hours, the GelDoc imaging system was utilized to visualize the outgrowth of the aged cells (
The inventive protocol was found to parallel the trends of CLS extension by rapamycin observed for both the YPD liquid and agar outgrowth assays.
These results validated the reproducibility of the protocol and the feasibility in using the inventive protocol for high-throughput screening. Furthermore, the Z-factor of the protocol was assessed to determine the quality of the inventive screening protocol. Generally, Z-factors of 0.5 to 1.0 suggest an excellent high-throughput assay. Hence, the Z-factor was evaluated for various concentrations of rapamycin with PI-stained aged cells (
The inventive protocol was also tested on the BY4743 yeast strain, to determine whether it was still effective in a different yeast genetic background. The effect of rapamycin was tested on the CLS of BY4743 strain. Saccharomyces cerevisiae BY4743 strain is an auxotrophic for histidine, leucine and uracil. The BY4743 strain was grown in different concentrations of rapamycin in the SD medium supplemented with auxotrophic constituents (
Since calorie restriction has been known to slow aging and extend CLS of model organisms, including yeast and mammalian cells, the effect of calorie restriction on CLS of yeast was also investigated, to test the validity of the inventive protocol. This was done by restricting glucose content in the culture medium, where cells were cultured in three different SD medium conditions containing 0.25%, 0.5%, and 2% glucose. Cell growth was measured at various time points (24 hours, 48 hours, and 72 hours). It was observed that all cell cultures achieved growth saturation after 24 hours, but cell growth was notably lower in 0.25% and 0.5% glucose in comparison with that in 2% glucose (
As a proof of principle, hundreds of chemical agents were screened for the identification of anti-aging compounds (
The validity of the inventive protocol was also tested on human cells. Similar to previous examples, the fluorescence readings of the human cell line samples were measured by a microplate reader at 535 nm excitation and 617 nm emission on day 1, day 4, day 6, and day 8. Cell survival was determined by normalization of fluorescence intensity at different rapamycin concentrations with untreated control (DMSO) for each result on the respective days. Based on the results obtained, it was observed that rapamycin had increased the chronological lifespan of both HEK293 and A549 cells (
In a further investigation using the PI assay, the anti-aging effect of 2,5-AM in the human lung primary fibroblast cells, IMR90, was also tested. The primary fibroblast cells are widely used in aging research as they have a finite lifespan, and they better mimic the organism's aging process compared to immortal cell lines. 2,5-AM treatment significantly increased the cellular lifespan of IMR90 cells, resulting in a lower percentage of cell death. Both the 2.5 mM and the 5 mM treatment groups showed 32.33% and 48.08% more live cells compared to the untreated control, respectively (
It was also found that 2,5-AM increased cellular lifespan in the HEK293 cells, compared to the untreated group (
The inventive protocol did not require any outgrowth procedures, including aspiration, washing, trypsinization or transferring of cells to a new plate. As such, the inventive protocol was found to be more economical and reduced variations introduced in the outgrowth methods such as those described in the Comparative Examples, as it required one plate per experiment only. The assay was easy to perform as no additional steps were required after the assay plate had been cultured, and readings could be taken at multiple age time points with the same samples on a microplate reader. The PI assay measured the amount of cell death. Hence, it was independent of the ability of the cells to proliferate. Therefore, it was also found to be suitable for compounds or procedural interventions that suspend cell proliferation.
In addition, the inventive protocol can be conducted with a large variety of human cell lines, including cells from various human tissues as well as cancer and senescence-induced cell lines. It may be useful to assess the anti-cancer or senolytic potential of large sets of compounds and interventions.
A schematic comparison of the PB outgrowth assay in Comparative Example 3 and the inventive protocol is shown in
These advantages made the inventive protocol highly compatible with high-throughput compound screening requirements. The protocol is not just restricted to the CLS measurement alone. As the inventive protocol is not limited to the type of compounds used, it may be used for senolytic compound screening with senescent-induced cells or as anti-cancer compound screening with cancer cell lines under nutrient-limiting conditions. Thus, there is more potential in this cell viability assay with the usage of different cell lines. The HTS method utilizing the inventive protocol may be potentially valuable in many additional ways for drug discovery and biopharmaceutical research.
In this approach, an outgrowth assay was used to determine the number of live cells in the 6-well experiment plate using crystal violet staining. Crystal violet is a triarylmethane dye that binds to ribose molecules such as DNA. The crystal violet-based outgrowth method relies on the detachment of adherent cells from the culture plate during cell death. Hence, dead cells would be washed away with the D10 medium and only live cells would remain attached to the culture plate. These live cells were then stained with crystal violet dye and assessed qualitatively.
The cell viability of 2,5-AM treated cells was examined at different concentrations using the crystal violet-based CLS assay. It was observed that after seven days of outgrowth. 2,5-AM was able to increase cellular lifespan in HEK293 cell line (
However, this assay required a long time for cellular outgrowth. It also showed that there was a risk of cell detachment during multiple washing steps in the staining. It was not suitable for high-throughput screening (HTS) analysis of multiple compounds, concentrations and/or treatments. It was also evident that the qualitative nature of the obtained data could obscure analysis, resulting in ambiguous or misleading outcomes at a low number of surviving cells.
In order to overcome some of the limitations mentioned in the crystal violet assay, a method that requires less time, has less experimental error and, most importantly, generates quantitative data for CLS analysis of human cells was explored. The anti-aging compound 2,5-AM, was used to validate the efficiency of this approach based on the outgrowth PrestoBlue™ assay. PrestoBlue (PB) is a resazurin-based reagent used to detect cell viability and traces of cytotoxicity in the medium. It produces results that can be detected both colorimetrically and fluorometrically. It is permeable and non-toxic to the cells, hence, enabling the measurement of cell viability over a period of time without cell lysis. The resazurin component is reduced to form resorufin by live cells, resulting in a visible change from blue colour to red colour in the reagent, at the same time developing fluorescence, which can be easily detected by a microplate reader. The PB reagent is highly sensitive; it detects even a small number of cells (a couple of hundreds). It allows conveniently fast cell detection and data collection. The PB assay was easy to perform as the reagent is a commercially available as ready-to-use solution. The method is quantitative, can be used in high-throughput analysis and has a fast output. It should also be noted that the PB can be replaced with other fluorescent viability dyes including propidium iodide, and various resazurins such as MTT, XTT, and alamarBlue, to obtain the same results.
In the outgrowth assay using human cell lines, 2,5-AM treatment significantly increased the cellular lifespan, which resulted in more live cells that were able to proliferate in the assay plate. For the HEK293 cell line, the 2.5 mM treatment group had 76.31% more viable cells compared to the untreated group (
The anti-aging effect of 2,5-AM in the human lung primary fibroblast cells, IMR90 was tested. An anti-aging effect similar to that for HEK293 was also observed for the IMR90 cells. The 2.5 mM treatment group had 59.05% more surviving cells and the 5 mM treatment group had 59.89% more viable cells compared to the untreated group (
The cellular outgrowth for 5% (10 μL) cells transferred to the assay plates for the HEK293 and IMR90 cell were also examined. A pattern of anti-aging activities similar to the results when 10% of cells was transferred (
The PB-based outgrowth method was further tested with rapamycin, an established anti-aging compound in multiple models, including the HT-p21-9 cell assay where it has been shown to increase cellular lifespan. It was found that rapamycin treatment significantly increased the lifespan of HEK293 cells (
This assay allowed determination of cellular lifespan in a short time with significant throughput capacity. The detection of viable cells using the PB reagent provided quantitative data, which can be computationally further evaluated. Notably, other resazurin-based reagents such as the MTT, XTT and alamarBlue reagent can replace PB as well as any efficient colorant that stains surviving cells.
However, the outgrowth assay required aspiration of medium, washing and trypsinization, similarly to the outgrowth crystal violet assay. These steps introduced multiple sources of errors to the experiment such as cell detachment during aspiration and washing, potential problems during trypsinization or the transfer of live cells. To note, the outgrowth assay depended on the ability of the cells to proliferate in the fresh medium after days of compound treatment. Hence, if the treatment caused a decline in cell proliferation, cells would not proliferate even if they were alive, giving a false negative result. For example, calorie restriction has been shown to increase cellular lifespan but reduce proliferation. Thus, this outgrowth assay was not suitable for the detection of cellular lifespan in this scenario. Moreover, inefficiency in detecting surviving cells with low metabolic activity was also an issue with the PB assay and other related assays that measured cytotoxicity via a NADH-dependent cellular oxidoreductase enzymes.
The method as defined above may be useful in high-throughput screening of a large number of potential chemical agents or culturing conditions which affect CLS in various types of eukaryotic cell models or human cell lines. The method may be useful in the quantification of CLS in said cell models. The method may be useful in high-throughput screening for the identification of geroprotective interventions or potential drugs within a short period of time at a considerably low cost. The method may be useful in the high-throughput screening of gene-drug interactions in an array of genetically modified strains. The method may be useful in the screening of gene-regulated age-dependent factors that affect CLS of cells. The method may be useful in identification of anti-aging compounds, toxic chemicals that accelerate aging and death, anti-fungal compounds, senolytics and anti-cancer agents.
It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202201534P | Feb 2022 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2023/050097 | 2/17/2023 | WO |
