Cancer cell culture methods and protocols pdf

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cancer cell culture methods and protocols pdf

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Metrics details. The use of patient-derived primary cell cultures in cancer preclinical assays has increased in recent years. The management of resected tumor tissue remains complex and a number of parameters must be respected to obtain complete sample digestion and optimal vitality yield.

3D Cell Culture: An Introduction

Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice. We have compared protocols for primary cultures, differing in media components and plate coatings. Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses.

Stable cancer cell lines have been considered valuable tools for analysis of cancer biology as well as applicable platforms for preclinical drug testing for many years [ 1 , 2 ]. Being relatively easy to culture basic media requirements, simple culturing protocols and having virtually limitless lifespan, these lines gained recognition among the majority of scientists.

Since cancers are well known for high heterogeneity [ 3 ], stable cancer cell lines may be unable to adequately represent the complexity of these diseases [ 4 , 5 ]. As an example, no stable cell line obtained from the primary prostate tumour site is available [ 6 , 7 ].

Such an approach, however, is associated with some technical hurdles—primary culture may be difficult not only to establish e. Many attempts have been made to overcome problems with epithelial primary cancer cultures, resulting in development of various strategies that employ special enriched media [ 12 ], feeder layer [ 13 , 14 ] or feeder layer and Rho kinase ROCK inhibitor-supplemented medium [ 15 ].

It has to be noted that feeder layer preparation is not only time-consuming, but also difficult to standardize. Mouse fibroblasts, most often employed as feeder cells, tend to transform or undergo senescence, thus they do not provide primary cells with adequate support [ 16 ].

Hence, usage of feeder layer coating is associated with a risk of inconsistencies in culture, affecting test outcomes. In the present study, we propose the protocol for primary epithelial prostate cancer PC and breast cancer BC cell cultures, involving application of extracellular matrix reconstitution along with tissue-specific primary cell medium.

The proposed approach yields results comparable to feeder layer coating, is relatively simple and constitutes an easily validatable testing platform. All procedures were performed in accordance with the protocol approved by the Bioethical Committee of the Regional Medical Chamber in Lodz Approval No.

Patients signed informed consent form and their data were processed and stored according to the principles expressed in the Declaration of Helsinki. Cells were seeded onto attachment factor Life Technologies coated plates at density 1.

Prior to enzymatic dissociation prostate tumour samples were washed thrice in HBSS and mechanically disintegrated into pieces smaller than 1 mm 3 using scalpels. Such tissue pieces were incubated 1. Subsequently, PC cells were seeded at density 0. Cells were either further cultured and passaged using trypsin—EDTA 0. Prior to enzymatic dissociation breast tumour samples were washed thrice in HBSS and mechanically disintegrated into pieces smaller than 1 mm 3 using scalpels.

Further disintegration was performed in two steps: via gentle pipetting in trypsin 0. Subsequently, cells were seeded at density 0. In order to confirm the specificity of amplification signal, gene dissociation curve was considered in each case. Coverslips were mounted with mounting medium—N-propyl gallate 0. The multiplex ligation-dependent probe amplification MLPA reactions were performed as recently described [ 10 ] using commercially available probemixes P and P and kits MRC-Holland following the manufacturer's recommendations.

The comparative analyses were performed using Coffalyzer. Net MRC-Holland. For each gene, the resultant ratio was calculated and interpreted as gain above 1. Senescence detection was performed using Histochemical Staining Kit Sigma—Aldrich according to the manufacturer's protocol. After the incubation, cells were washed thrice with PBS and analysed using light microscope Opta-Tech.

During the experiment course, primary epithelial breast and prostate cancer cells were cultured utilizing protocols differing in terms of media components and plate coatings Figure 1. Errors bars indicate SEM. Selected primary epithelial breast cancer cells PEBCs; 8 cases were also subjected to such analysis. Despite no difference was observed in culture initiation efficiency for breast specimens, the number of adherent cells following first two passages was decreasing with each passage.

Therefore, an attempt to long-term culture on feeder layer was discontinued in case of BC cells. We hypothesized that ROCK inhibitor Y may constitute the key component of primary cancer culture medium. Such enrichment, however, did not enhance the effectiveness of culturing approach based on commercial media.

Although overexpression of MUC1 gene is typical for both normal epithelium and epithelial tumour cells, its isoform Y is considered tumour-specific [ 19 , 20 ].

Prostate and breast tumour samples demonstrated low averaged level of this gene expression 1. Additional analysis of prostate tumour specimens was focused on expression of AMACR —gene encoding an enzyme considered to be overexpressed in PC [ 25 , 26 ], yet with many contradictory data published [ 27 , 28 ], which occurred to be slightly lower in PEPCs than in tissue samples 0.

Anti-CD90 antibody was employed to visualize any remaining fibroblasts results not shown. B MLPA analysis showing tumour-characteristic changes in gene copy number. In case of analysed tissue samples, none or very few alterations were detected depending on a specimen , most probably due to high percentage of normal cells in surgical samples.

Moreover, some new alterations appeared during culture course, probably as an effect of the inevitable molecular dynamics of neoplastic cells Figure 5 B. Primary breast and prostate epithelial cells were not susceptible to the effect of applied immortalizing agents, since the approach did not increase the number of passages Table 3. In the present study, we provide a simplified approach to primary breast and prostate epithelial cancer cells culture, utilizing extracellular matrix reconstitution and tissue-specific primary cell medium.

Feeder layer is associated with numerous technical issues, including incomplete inactivation of fibroblasts as well as their tendency to lose contact inhibition, transform or even become unresponsive to mitomycin C in a long-term culture [ 16 ].

Therefore, there is a risk that feeder cells will not be able to provide epithelial cancer cells with adequate support or even contaminate the culture. Initially, ROCK inhibitor was applied in cultures of stem cells to enable more efficient culturing of dissociated spheres [ 29 ] and keratinocytes to enhance their proliferation and culture time [ 30 ]. Afterwards, this inhibitor was reported to constitute a key factor in long-term culture maintenance of normal and neoplastic cells from various tissues of origin [ 15 ].

Such a difference is consistent with current availability of stable cancer cell lines derived from these tumour types—there is a plenty of BC cell lines, whereas the selection of possible testing platform for PC is severely limited lack of stable cancer cell line from the primary tumour site [ 6 , 7 ]. Each of the very few commercially available PC lines was established from metastatic sites mostly bone metastases or tumour xenografts usually implanted heterotopically.

MGB1 expression was detected at similar level in both BC specimens and PEBCs, which was in line with our expectations, since this marker is specific for mammary gland, both normal and neoplastic tissue [ 21 , 22 ]. On the contrary, there are very few markers that may be considered PC specific. Latest literature reports indicate the important role of AMACR gene, however, it is not clear whether it can be considered tissue or cancer specific marker [ 25 — 28 ].

Our analysis demonstrated that in contrary to other analysed markers, the expression of AMACR observed in tissue samples decreased during culture course Figure 4 B.

Considering the fact that role of this gene in PC is not fully understood, it is not clear why pattern of its expression differs from other analysed markers. Interestingly, when cultured in dedicated commercial media, primary epithelial cancer cells tend to be characterized by earlier onset of senescence than those maintained in tissue-specific primary cell media.

This phenomenon is inseparably associated with alterations in the molecular context of the cell [ 32 , 33 ], especially factors implicated in cell-cycle regulation [ 34 ].

Such a phenomenon, however, is also observed in stable cancer cell lines. Consequently, we have recently reported that even population of cancer cells with almost infinite proliferative potential is characterized by low percentage of senescent cells [ 11 ].

Generally, stable cell lines either stabilize due to spontaneous immortalization very rare event, mostly due to acquisition of new mutations or are obtained via isolation from xenografts or treatment with immortalizing agents of viral, chemical or other origin.

None of the utilized genes significantly prolonged culture time, neither in breast nor in prostate cells. It is worth mentioning that primary cancer cells are less prone to genetic manipulations than normal cells. Scientific reports provide several methods for normal cells immortalization, based on induction of various genes expression, e.

On the contrary, only few successful attempts to immortalize primary cancer cells were described in the literature, including induction of hTERT [ 43 ] or E6 and E7 genes of HPV [ 42 ]. Undoubtedly, such platforms do not provide the representation of the whole spectrum of tumour subpopulations. Therefore, primary cell cultures, reflecting high heterogeneity of cells within a tumour, seem to be an essential tool for reliable studies on cancer treatment, drug development and cancer biology.

Importantly, availability of primary cancer cells derived from a particular patient may facilitate the attempts to personalize anticancer therapy. In the era of molecular diagnostics, testing drugs or their combinations directly on patient tumour material seems a powerful approach towards improvement of anticancer treatment efficiency. All authors read and approved the final version of the manuscript.

This does not alter their adherence to all the Bioscience Reports policies on sharing data and materials. The authors have declared that no other competing interests exist.

National Center for Biotechnology Information , U. Journal List Biosci Rep v. Biosci Rep. Published online Dec 9. Prepublished online Nov 1. Author information Article notes Copyright and License information Disclaimer. This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4.

This article has been cited by other articles in PMC. Keywords: breast cancer, extracellular matrix, primary cancer cell cultures, prostate cancer, Rho kinase ROCK inhibitor. Prostate cancer primary cultures Prior to enzymatic dissociation prostate tumour samples were washed thrice in HBSS and mechanically disintegrated into pieces smaller than 1 mm 3 using scalpels. Breast cancer primary cultures Prior to enzymatic dissociation breast tumour samples were washed thrice in HBSS and mechanically disintegrated into pieces smaller than 1 mm 3 using scalpels.

Table 1 Primer sequences. Open in a separate window. Multiplex ligation-dependent probe amplification The multiplex ligation-dependent probe amplification MLPA reactions were performed as recently described [ 10 ] using commercially available probemixes P and P and kits MRC-Holland following the manufacturer's recommendations.

Figure 1. Schematic experiment workflow—various attempts to culture primary epithelial breast and prostate cancer cells. Figure 2. Figure 3. Figure 4. Figure 5. Table 3 Passage number of primary cancer epithelial cells transduced with immortalizing genes compared with control cells. Acknowledgments We thank Michal Bienkowski for his valuable comments and suggestions.

References 1. Gillet J.

Cancer Cell Culture

It seems that you're in Germany. We have a dedicated site for Germany. With many recent advances, cancer cell culture research is more important than ever before. This timely edition of Cancer Cell Culture: Methods and Protocols covers the basic concepts of cancer cell biology and culture while expanding upon the recent shift in cell culture methods from the generation of new cell lines to the use of primary cells. There are methods to characterize and authenticate cell lines, to isolate and develop specific types of cancer cells, and to develop new cell line models. Functional assays are provided for the evaluation of clonogenicity, cell proliferation, apoptosis, adhesion, migration, invasion, senescence, angiogenesis, and cell cycle parameters.

Passaging refers to the diluting of cells that have reached high confluence to supplement cells with fresh medium to enable continuous culture propagation. For mammalian cells, passaging should be performed when cells are toward the end of the logarithmic growth phase, so before they reach the stationary phase Figure 3. Passaging of cells at the stationary phase is not recommended because they tend to take longer to begin the logarithmic growth phase upon seeding. Additionally, the build-up of lactic acid in dense cultures may impact cell metabolism. Add detaching agent e. Resuspend cells in fresh medium, pipette thoroughly to obtain single cell suspension. If your medium does not contain serum, you need to inactivate detaching agents, e.

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Two-dimensional 2D culture of cancer cells in vitro does not recapitulate the three-dimensional 3D architecture, heterogeneity and complexity of human tumors.

Advanced Cell Culture Techniques for Cancer Drug Discovery

Your browser does not have JavaScript enabled and some parts of this website will not work without it. For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome. Our Cookie Policy explains how you can opt-out of the cookies we use. The following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.

Click on the following links to view the sample 1 protocol Part 2 Only and reagents list respectively, for conducting a Digesting DNA Reaction. When you have finished viewing the protocol, complete the theoretical lab assessment problem set. Click on the following links to see sample protocols for conducting a restriction enzyme digestion reaction and agarose gel electrophoresis. Protocol Samples

Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice. We have compared protocols for primary cultures, differing in media components and plate coatings. Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses. Stable cancer cell lines have been considered valuable tools for analysis of cancer biology as well as applicable platforms for preclinical drug testing for many years [ 1 , 2 ]. Being relatively easy to culture basic media requirements, simple culturing protocols and having virtually limitless lifespan, these lines gained recognition among the majority of scientists.

1. Introduction

Она не могла поверить, что такой алгоритм может быть создан, но ведь доказательство налицо - у нее перед глазами. ТРАНСТЕКСТ не может с ним справиться. Сьюзан подумала о Стратморе, о том, как мужественно он переносит тяжесть этого испытания, делая все необходимое, сохраняя спокойствие во время крушения. Иногда она видела в нем что-то от Дэвида. У них было много общего: настойчивость, увлеченность своим делом, ум. Иногда ей казалось, что Стратмор без нее пропадет; ее любовь к криптографии помогала коммандеру отвлечься от завихрений политики, напоминая о молодости, отданной взламыванию шифров. Но и она тоже многим была обязана Стратмору: он стал ее защитником в мире рвущихся к власти мужчин, помогал ей делать карьеру, оберегал ее и, как сам часто шутил, делал ее сны явью.

Это файл высочайшей сложности. Я должен был тебя предупредить, но не знал, что сегодня твое дежурство. Сотрудник лаборатории систем безопасности не стал выдавать дежурного. - Я поменялся сменой с новым сотрудником. Согласился подежурить в этот уик-энд. Глаза Стратмора сузились.

 Не стоит благодарности.  - Она улыбнулась и села напротив шефа. Стратмор был крупным кряжистым мужчиной, чье невыразительное лицо скрывало присущие ему решительность, настойчивость и неизменное стремление к совершенству. Серые глаза светились уверенностью, с которой сочеталась профессиональная скрытность, но сегодня в них проглядывали беспокойство и нерешительность. - У вас испуганный вид, - сказала Сьюзан.

Халохот стиснул револьвер в руке, не вынимая из кармана.

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