Protocol for Cloning Epithelial Stem Cell Variants from Human Lung
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Abstract:
The plurality of clonogenic cells derived from human lung includes a spectrum of diverse p63+ stem cells responsible for the regeneration of normal epithelial tissue and disease-associated metaplastic lesions. Here, we report protocols for the cloning, expansion, and characterization of these stem cell variants, which in general assist in analyses of stem cell heterogeneity, genome editing, drug screening, and regenerative medicine. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2011), Zuo et al. (2015), and Rao et al. (2020).Keywords:
Cloning (programming)
Clonogenic assay
Regenerative Medicine
Objective To compare the effect of growth and clonogenic assays in measurement of surviving tumor cells after radiation. Methods The surviving cell number and surviving fraction (SF) of MCF 7 and Colo 205 cell lines following irradiation with 2,4,and 8 Gy were measured by using Courtenay Mills clonogenic and growth assays. Results In the clonogenic assay group, the detected surviving tumors had a clonic function. There was no difference of radiosensitivities between Colo 205 cells living in suspension and on adherence. In growth assay group, the relative surviving index reached the lowest level at the 6 th to 9 th days after culture and the mean value was close to that by the clonogenic assay. Conclusion Two methods reflect different aspects of cell proliferation. Growth assay can provide information on early proliferation kinetic events. The great merit of Courtenay Mills assay is that it claims to quantify not only surviving cells but cells with clonogenic potential, which is presumably a clinically relevant end point.
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Cell counting
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Abstract The technique of thymidine (TdR) suicide has been used with the intestinal microcolony assay to demonstrate that in the middle of the light cycle, nearly all intestinal clonogenic cells, in the B6D2F 1 mice used in these experiments, were not in S phase. Doses of tritiated thymidine [ 3 H]TdR up to 1 mCi/mouse did not kill a significant fraction of those clonogenic cells which survived a test dose of 12 Gy γ‐rays. This finding supports some data in the literature, but conflicts with others. However, the suicide technique was found in the studies reported here to be very efficient in sterilizing clonogenic cells in the middle of the dark cycle, and also in a regenerating epithelium at day 3 after a dose of 9 Gy. This implies that the technique can discriminate well between populations of clonogenic cells which differ in their content of cells in S phase. the lack of a suicide effect in the middle of the light cycle indicates that the majority of proliferative epithelial cells are not clonogenic.
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Microfold cell
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SummaryThe radiation sensitivity of two small-cell lung carcinoma cell lines growing as multicellular spheroids in static culture was determined using clonogenic cell survival and growth delay as endpoints. Growth delay determination suggested that clonogenic cell kill was less than was obtained by direct assay of cell survival. Recovery from potentially lethal damage was assayed in one line (HC12) but was not demonstrable, and clonogenic cell survival decreased with time in treated spheroids with diameters greater than 300 µm which contained a hypoxic cell population. Microscopic examination of the treated spheroids showed the emergence of an abnormal giant-cell population, and the progressive clonogenic cell loss that occurred after treatment was thought to be due to oxygen and nutrient deprivation of the remaining viable cells by this doomed cell population. Correction of the growth delay measurements for changes in cell size and clonogenic cell population allowed correlation of the growth delay and cell survival data.
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A hypo-osmolar medium and tissue processing technique is described which is useful for disaggregation of residual human tumor cell clumps persisting after mechanical or enzymatic treatment of solid tumors and malignant effusions. The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors by 47% and in malignant effusions by 67%. In 5 of the 26 solid tumor specimens tested in the human tumor stem cell assay, clonogenic single cells were obtained with the hypo-osmolar procedure, whereas no growth was observed using standard methods. Overall, the success rate for clonogenicity increased from 46% to 65% for the 26 solid tumors, with the major improvement occurring in ovarian cancer. Clonogenicity was obtained in 80% of malignant effusions both by standard methods and the hypo-osmolar techniques. The increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.
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The antiproliferative and cytotoxic effects of purified IFN-alpha and recombinant IFN-gamma were investigated using both direct cell counting and a clonogenic assay on a panel of 5 established human lung cancer cell lines and for 2 of them also on their multidrug-resistant counterparts. There was considerable heterogeneity in the response of the cell lines to the IFNs in terms of growth inhibition. Clonogenic assay of IFN-treated cells indicated that, where a cell line had responded markedly to an IFN, only a small fraction of the cells remaining after IFN treatment were clonogenically viable. When cells were placed into the clonogenic assay in the presence of IFNs, the time course of colony formation was different from that seen in the control cultures for most of the cell lines. The measured "surviving fraction" was greatly dependent upon the time of colony counting. When the effects of IFNs in combination with ADM were studied, conclusions regarding the interaction of the effects of the agents also depended upon the time at which colonies were counted.
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We need practical laboratory methods for predicting the chemosensitivity of human neoplasms. Over the past 20 years, numerous investigators have implied or stated with increasing certitude that clonogenic assays are the most valid (or only valid) approach to predictive chemosensitivity testing. We feel that this point of view may have insufficient scientific validity and may be harmful to progress in this area. In addition to well-known technical problems, there are serious theoretical problems with clonogenic assays. These include the disruption of normal cell-to-cell interactions, the possibility that true tumor stem cells may be largely nondividing (G0) cells, while cells forming colonies are exclusively dividing cells, the possibility that clonogenic cells may largely represent cells which are not true stem cells, and the fact that clonogenic assays have the ability to measure cell kill over a narrow log range, while meaningful clinical responses require a multiple-log cell kill. This latter fact mandates the use of unrealistically low drug concentrations to avoid excessive false-positive results. Neither theoretical concepts, direct experimental data, nor clinical correlations support the alleged superiority of clonogenic assays. Clonogenic assays may not be advantageous compared to other more practical methods of estimating the general chemosensitivity of proliferating cells. In contrast, there is a growing body of literature which indicates that early evidence of cell damage in the entire tumor cell population may accurately predict for a multiple-log stem cell kill and meaningful clinical response. Future studies should continue to develop and test assays based on alternative methods for detecting cell kill in the proliferating and total tumor cell populations.
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Chemosensitivity assay
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Abstract Clonogenic assays are considered to be the “gold standard assay” to test for the effects of drugs and radiation on the growth and proliferative characteristics of cells in vitro. Clonogenic-based assays are considered superior to short-term cell proliferations-based assays as a clonogenic assay is insensitive to p53 status. The clonogenic assay is the measurement of a drug's cytotoxic activity against a single cell population and is the most valid in vitro approach to predict the chemosensitivity of human cancers. The traditional clonogenic assay is very time-consuming and labor-intensive. In addition, the cell disaggregation step necessary for plating in the clonogenic assay results in a loss of cell-cell and cell-substrate interactions which may be critical for the drug response. To overcome the technical challenges and limitations of the traditional clonogenic assay, a 6-well plate clonogenic assay was developed and optimized. Current 6-well clonogenic assays are initiated with a single seeding density, and then surviving cells grow over 10 to 12 days and form colonies. The data generated from this from of the assay only allows the determination of IC50s but not IC90s due to a narrow one log range of cell killing. We have developed an efficient and simple six-well clonogenic assay that yields a wider range of cell killing covering 5 logs. The key is that a series of different cell numbers are seeded in 6 well plates ranging from a density from 100 cells per well to 100,000 cells per well. After a 24 h cell attachment period, cells in each plate were treated with five different concentrations of the test compound. After incubation for the appropriate period of time at 37oC in a 95% air and 5% CO2 atmosphere incubator, drug containing medium was removed and the cells were cultured in fresh medium for additional 10-15 days until adequate colony formation. At the end of incubation, cell colonies were stained with 0.25% w/v crystal violet in 95% Ethanol. After air drying, the plates were scanned with an image-based colony counter (Alpha Innotech) and colonies with more than 50 cells were quantified. To directly compare the experimental results derived from this improved 6-well plate clonogenic assay and the traditional approach to the clonogenic assay, we ran the two forms of the assays simultaneously using cisplatin and SN-38 as the cytotoxic test agents. Cispatin and SN-38 showed comparable IC50, IC90 and IC99 values in both the improved six-well clonogenic assay and the traditional clonogenic assay. In summary, the key advantage of this 6-well clonogenic assay is that it does not involve the trypsinization and replating step after drug treatment and also that is greatly simplifies the traditional clonogenic assay and allows for its use as a moderate-throughput primary drug screening tool. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5497.
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