Establishment of Tumor Cell Lines PDF

61 4 Establishment of Tumor Cell...

61 4 Establishment of Tumor Cell Lines: From Primary Tumor Cells to a Tumor Cell Line Katharina Meditz and Beate Rinner All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law. 4.1 Importance of Primary Tumor Cell Culture: As Close to In Vivo as Possible – 62 4.1.1 Cell Culture Definition – 62 4.2 A Long Way from Tissue to the Desired Cells – 63 4.2.1 Tumor Tissue – 63 4.2.2 Four Steps to Success – 64 4.2.3 Protocol: Tumor Cell Line Establishment for Adherent Growing Tumors – 65 4.2.4 Characterization – 65 4.3 General Tips About the Establishment of Tumor Cell Lines – 66 4.3.1 Hayflick Problem – 66 4.3.2 To Get Rid of Other Cells – 66 4.4 Establishment of Tumor Cell Lines: Three Examples – 67 4.4.1 Establishment of Chordoma Cell Lines – 67 4.4.2 Establishment of Aggressive Growing Melanoma Cell Line: MUG-Mel2 – 69 4.5 Quality Check – 70 4.5.1 Recording and Freezing – 70 4.5.2 Contaminations – 71 References – 72 Copyright 2018. Springer. © Springer International Publishing AG, part of Springer Nature 2018 C. Kasper et al. (eds.), Cell Culture Technology, Learning Materials in Biosciences, https://doi.org/10.1007/978-3-319-74854-2_4 EBSCO Publishing : eBook Collection (EBSCOhost) - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES AN: 2547114 ; Cornelia Kasper, Verena Charwat, Antonina Lavrentieva.; Cell Culture Technology Account: s8999451.main.ehost  62 K. Meditz and B. Rinner What You Will Learn in This Chapter The present chapter will give an overview about handling of tumor tissue, isolation of tumor cells, and finally the establishment of tumor cell lines. Characterization and identifi- cation of established cell lines will be shown, on the basis of three examples: clival and sacral chordomas and NRAS-mutated melanoma. Understanding the culture conditions will help to find the perfect requirements for the culturing of each different cell line. Contamination in the cell culture is a touchy topic; detection and elimination of microbial and cell-cell contamination will be discussed in detail. By adhering some golden rules, suc- 4 cess of cell line establishment will be guaranteed! 4.1 I mportance of Primary Tumor Cell Culture: As Close to In Vivo as Possible George and Margaret Gey spent almost 30 years trying to establish an immortal human cell line, in order to understand the biology and mechanism of tumors. After establishing the Tissue Culture Laboratory, George Gey made his monumental breakthrough – he iso- lated the first tumor cell line from a cervix carcinoma which he named HeLa. George Gey always said: “The key to cancer is right at our fingertips - if we could only reach out and grab it” [12, 26]. Sadly he died from a pancreatic tumor. Since the discovery of cell lines, their importance has continued and has even increased. The reasons are as follows: (i) they are easy to handle, (ii) they offer unlimited self-­ replication, (iii) they have a relatively high degree of homogeneity, and (iv) they are easily replaceable from frozen stocks. There is no positive without some negative because (i) cell lines are prone to genotypic and phenotypic drift during passaging, (ii) cell lines don’t store well over many years, (iii) cell line subpopulations cause phenotypic changes, and (iv) cell lines from different labs show different karyotypes and therefore reproducibility might be lacking. 4.1.1 Cell Culture Definition Primary culture is the initial culture taken directly from the tissue; it mimics the situation most closely to the original tissue and is fundamental for research. Cells can migrate and grow directly from the tissue (explant technology), or cells can be enzymatically or mechanically isolated from the tumor tissue. In relation to their growth behavior, cells can grow in vitro as adherent culture or they can grow in suspension. After the tumor cells grow out and cover the whole cell culture flask, cells must be transferred into a new cell culture flask, the nomenclature of primary tumor cells changes, and it becomes a cell line. There are two classes of cell lines, the continuous cell line with indefinite population expansion or finite cell lines with a defined life span. Currently, there is a wide range of different cell lines in cell banks, which are well char- acterized. However, it is still useful to establish new cell lines, because heterogeneous tumors require different cell line systems to investigate the biology of the tumor. Cell lines that have been very long in culture can change their genetic and phenotypic char- acteristics. EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  Establishment of Tumor Cell Lines 63 4 4.2 A Long Way from Tissue to the Desired Cells From tissue to the desired cell can take a lot of time and can also be very tricky; depending on the growth behavior of the cells and the quantity of existing tumor cells in the available tissue. This chapter provides a general overview of tumor tissue, media, and cell isolation. By using contrasting examples in terms of tumor growth, the establishment of a cell line named MUG-Mel2 from a very aggressive NRAS melanoma tumor and the establish- ments of very slow-growing chordoma tumors, named MUG-Chor1 and MUG-CC1, will be explained (. Figs. 4.1 and 4.2). 4.2.1 Tumor Tissue The translation of the Latin word tumor is “swelling.” The distinguished pathologist Wallace H. Clark gave an excellent definition of the word tumor: “A tumor is a population of abnormal cells characterized by temporally unrestricted growth and the ability to grow a b c d..      Fig. 4.1 Establishment of a tumor cell line. The tumor tissue should be obtained immediately after surgery, surrounding tumor tissue is removed, and to avoid contamination an antibiotic bath for 10 min in PBS + 10% PS should be done a. After the antibiotic bath, transfer the tumor tissue into PBS or media, and the tissue is cut into very small pieces (1–2 mm3) by scissors or scalpels b. After disaggregation of the tumor tissue into small pieces c, the suspension is collected, and tumor pieces in PBS or media will be centrifuged. If provided in the protocol, vortex the minced tumor tissue d, and different fractions should be set in suitable media Tumor Tissue Morphology Verification that the Individual tumor Identity Growth rate cells are tumor cells: markers DNA Profiling (MTS, MTT, WST, Cell cycle (flow Surface markers (STR-analysis) BrdU) cytometry) (flow cytometry, Karyotypic analysis Western blot) Colony forming unit Immunohistochemistry assay (CFU) Genetic profile Tumorigenicity in immunodeficient mice..      Fig. 4.2 Overview of a characterization of a new established tumor cell line EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  64 K. Meditz and B. Rinner in at least three different tissue compartments - the original compartment; the mesen- chyme of the primary site (tumor invasion); and a distant mesenchyme (tumor metasta- sis)” [5, 7]. Solid tumors are structured as parenchyma (neoplastic cells) and stroma cells (surrounding tumor cells). Tumors with epithelial origin can show a separation of these compartments with a basal lamina. That means tumors do not exist only of malignant cells, but many other cells as well. Also essential is the tumor microenvironment (TME). Depending on tumor entity, the TME can consist of cells of the immune system, vascular and lymphatics cells, fibroblasts, pericytes, and adipocytes in vastly different amounts. 4 Some cells are easy to distinguish by typical morphology, like the epithelial fraction, pre- dominantly single cells with classical cobblestone morphology, and stromal fraction con- sisting of fibroblast cells, cells with a bipolar spindle shape typical for fibroblasts; immune cells present spheroid cells above the adherent cells or mast cells/macrophages. 4.2.2 Four Steps to Success In the first step, the condition of the tumor tissue is of great importance. Short ischemia times are an advantage, but you can isolate cells from a tissue 24 h after surgery, as long as the tissue is kept cold (4 °C) in media. The transport of tissue from surgery to the lab should be in media without any additives, such as sera (FBS) or antibiotics, to prevent it from drying out. Being FBS-free is important to inhibit cell growth during transport. Antibiotic-free, because in the lab each tissue will be incubated for 10 min in a 10 time antibiotic bath to avoid contamination and if you have antibiotics in the transport media you cannot be sure of the antibiotic concentration and incubation time. Living cells can only be isolated from fresh viable tumor tissue. The second step in creating cell lines is to know what kind of cells you want to isolate and then to keep track of them! A good instinct is necessary for the successful establish- ment of cell lines. In the third step, each tumor requires its own isolation method which, as well as medium, can be found in most literature. A very widespread method is the mechanical dissociation or cutting the tissue with scissors or sharp blades into very small pieces – usu- ally 1–2 mm3, with the option of homogenization by filtration through a nylon filter (50– 100 μm opening), vortexing and repeating those steps to get rid of dead cells, debris, and various different cell types. One must be aware that during isolation of tumor cells, there can be a varying amount of non-neoplastic cells that will complicate tumor cell line establishment. Therefore it is important that a histological section of the tumor is examined by a pathologist to deter- mine the proportion of tumor cells present, as this will affect the success of cell line growth. Enzymatic dissociation is also a way to separate single cells: there are different enzymes that are suitable for dissociating cells from solid tumor and to digest minced tissue into single cells. The concentration, temperature, and incubation time of the enzyme to pre- serve the cell viability and integrity are important. There are various enzymes available including collagenase, trypsin, papain, and elastase. Each enzyme with distinct target specifies with an affinity for different tumors. In the fourth step, each cell needs its own media; therefore a thorough research of all literature is a prerequisite before starting any cell line establishment. There is a wide range of available basic media, which can be adapted by different additives to the needs of the cells. However, in the production of specific media, care must be taken that the osmolality EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  Establishment of Tumor Cell Lines 65 4 is maintained. An increase of FBS, for example, from 10% to 30%, can strongly influence the osmolality of the medium. By comparison, the osmotic value of human blood is 300 mOsmolkg-1, and the osmotic value in most of the basic media is 280–340 mOsmolkg−1. There are different ways to facilitate the adherence or growth of cells with the help of additives in the media, for example, ITS, a mixture of recombinant human insulin, human transferrin, and sodium selenite. Adherence of cells can also be reinforced with coating of cell culture flasks with various enzymes, like fibronectin or Matrigel. 4.2.3  rotocol: Tumor Cell Line Establishment for P Adherent Growing Tumors 55 Preparation of tumor tissue: removal of non-tumor tissue (fatty tissue, blood clots, and connective tissue) by sterile scissors or scalpels. 55 To reduce contamination risk, an antibiotic bath, 10× PS solution, with incubation for approximately 10 min is recommended. 55 Transfer the tumor tissue from antibiotic bath to a PBS or media. 55 Mechanical and/or enzymatic disaggregation with sterile forceps and scissors or scalpels of the tumor in very small pieces to get more surface for outgrowing of the tumor cells. 55 Optional: Vortexing of the tumor pieces. 55 Centrifuge and remove the supernatant. 55 Resuspend the pieces with appropriate media in different cell culture flasks (due to the filter of the flasks, cells might have a better outgrowth than in, e.g., 6-well plates) 55 Incubation of the cells in a humidified incubator at 37 °C in an atmosphere of CO2 (amount of CO2 depends on the used media). 55 Regularly morphological observation of the tumor cells. 55 When the whole cell culture flask is overgrown and the media is consumed, subculti- vation (splitting) is required. 55 Subcultivation: detachment of the adherent monolayer by the use of proteolytic enzyme like trypsin or more gentle with Accutase. Cell-cell contact is broken and cells are in suspension; after centrifugation cells can be seeded into new flasks with appropriate cell amount and new fresh media to continue cell growth. 4.2.4 Characterization There are a large number of different cells in the tumor tissue, so it is very important to characterize the outgrowing cells as quickly as possible. One characteristic of cancer cells is replicative immortality, which means that cancer cells can divide many times more than normal cells. Very often only fibroblasts develop and grow over the tumor cells. Fibroblasts also have the ability to remain for long in culture, up to 50 passages in culture. The prob- lem with fibroblasts is that they are not always recognizable by their morphology and fibroblasts are often mistakenly confused with tumor cells. For those reasons the tumor potential has to be tested as soon as possible. In most cases only few tumor cells are avail- able at the beginning, and the characterization method must be adapted to the number of cells. During the first growth phase, only morphological observation is possible. The established cell line should have the same characteristic as the origin tissue, whereas, for EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  66 K. Meditz and B. Rinner example, mesenchymal origin can be detected with vimentin, epithelial characteristic with cytokeratin. To check the tumor potential of the cells, a colony-forming unit assay can be done; this assay requires only a small number of cells and indicates the tumor potential of a cell through clone growth. Tumor cells are indicated by aneuploidy and chromosome instability, cell cycle analyses by flow cytometry can be done to reveal the ploidy state of the cells, whereas karyotype analyses reveal the chromosome instability of the tumor cells. Tumor marker and genetic profiles should remain constant expressions during cultivation; however, some markers can be lost during cultivation. To confirm that 4 the established cell line has been developed from the tissue of origin, a DNA profiling by STR analyses to prove the identity of the tumor is highly recommended. 4.3 General Tips About the Establishment of Tumor Cell Lines In general, to generate tumor cell lines, cells should (i) be regularly cryopreserved, (ii) be passaged more than 100 times and retained in culture for more than 12 months, and (iii) mainly keep their genetic phenotype and morphology during cultivation. 4.3.1 Hayflick Problem One problem during cultivation is the so-called Hayflick limit. Leonard Hayflick and Paul Moorhead discovered that human cells derived from embryonic tissues could only divide a finite number of times in culture. Stages of cell culture were divided into three phases. Outgrowth of cells from an explant or tissue was named phase I. Division of cells represents phase II. The cells then proliferate in culture, depending on the cell type, and after a certain time period, cells start to divide more slowly and can also stop dividing, and this is defined as phase III. Hayflick and Moorhead described the phenomenon of growth arrest as the Hayflick limit or replicative senescence. If cells decrease or stop dividing, they should be transferred to a new flask with a higher cell density, FBS concentration can be increased for a short time, and it is highly recommended that only 50% of the media is changed, in order to keep autologous growth factors in culture. It’s much easier to cultivate fast-growing cells than slow-growing cells; however, cells can stop dividing without any conceivable reason. 4.3.2 To Get Rid of Other Cells Mentioned in 7 4.2.1 a lot of different cells are in a tumor tissue. To get rid of non-­ tumorigenic cells, selective adhesion and detachment techniques can be used [10, 23]. Fibroblast and tumor cells present a different density and size; after trypsinization, cells can be transferred to a new cell culture flask. After a time period (e.g., 10 min–2 h), certain cells will settle to the bottom, remaining media should be moved into a new flask, and the processes can be repeated (. Fig. 4.3). It is also very useful to take different fractions of cells isolated from a tumor, as in the example, for the establishment of MUG-Chor1. Keep all fractions in culture and hope that in one fraction tumor cells will start to grow! The fibroblast separation described above together with the fractions taken can mean more than 30 flasks in the incubator at one EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  Establishment of Tumor Cell Lines 67 4 10 min..      Fig. 4.3 Easy separation of different cell types due to different size and density time during cell line establishment. Therefore a complete description of each flask needs to be recorded. The most important point of primary culture is do not discard anything during cultivation and be patient! In the next passage, the establishment of three tumor cell lines will be discussed. 4.4 Establishment of Tumor Cell Lines: Three Examples 4.4.1 Establishment of Chordoma Cell Lines Chordomas are rare malignant tumors that develop from embryonic remnants of the notochord and arise only in the midline from the clivus to the sacrum. Surgery followed by radiotherapy is the standard treatment. As chordomas are resistant to standard chemo- therapy, the establishment of more chordoma cell lines is urgently needed to find alterna- tive treatment options [6, 8, 9, 13]. Chordomas and resulting cell lines are characterized according to their morphology, expressed by a variety of lysosomes, vacuoles, and typical physaliferous bodies. Immunohistochemistry shows brachyury and cytokeratin 8 positiv- ity and reveals a slow growth kinetic. It is very tricky to establish chordoma cell lines, due to tumor cells which are very slow growing and being surrounded by other fast-growing cells, such as stroma and immune cells. To get rid of fibroblasts, selective adhesion and detachment techniques can be used [10, 23]. 4.4.1.1 Establishment of Sacral Chordoma Cell Line MUG-Chor1 To establish a chordoma cell line, tumor tissue was obtained immediately after surgery. Following mechanical disaggregation of the tumor tissue into approximately 1–2 mm3 pieces, the mixture of cultures was carried out in fractions. Fraction I contained mechanically dis- sociated tumor cells. Fraction II comprised a cell fraction after the tumor pieces were treated enzymatically, in detail with 200 μL collagenase for 30 min at 37    C ̊ , and fraction III included only the tumor pieces, pressed down with a cover glass (. Fig. 4.4), to facilitate the growth of the cells to the cell culture flask. All fractions were cultured in Iscove/RPMI 4:1 containing 10% fetal bovine serum, 1% insulin-transferrin-sodium selenite (ITS), 2 mM glutamine, and 1% penicillin/streptomycin. Incubation was carried out at 37 °C in a humidified atmosphere of 5% CO2. The chordoma cells grow at a pH of 7.4. Following a culture period of 4 months and a passage number of 5, the cells underwent a crisis. FBS concentration from 10% to 20% and the ITS concentration from 1% to 5% were increased. After applying these conditions for 1 week, the initial medium was used again. After that crisis, the cells grew consistently and showed a doubling time of approximately 7–10 days. Culture medium was changed twice a week, and splitting of the cell culture was performed every 10 days at a confluence of 70–80%. All cell cultures were periodically checked for mycoplasma by PCR. EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  68 K. Meditz and B. Rinner..      Fig. 4.4 The sterile cover glass (blue arrow) is pressed down on the tumor tissue with a forceps 4 4.4.1.2 Establishment of Clival Chordoma Cell Line MUG-CC1 A further problem in the establishment of tumor cell lines is to maintain the vitality of cells during tumor surgery. Various surgery techniques, for example, resectoscopy, can have an influence on the viability of the cells; also clival chordoma cells are very difficult to establish due to their slow growth and the location of the tumor. Therefore a particu- larly careful surgery method was chosen. The interdisciplinary skull base unit of the Medical University of Graz has contributed substantially to the development of four- handed transnasal transsphenoidal purely endoscopic approaches. The focus lies in care- ful tissue preservation and procurement of sufficient cell material as a prerequisite for optimal cell culturing. After appropriate access to the clival region and tumor exposure, preliminary tumor debulking was performed. The specific advantage of this surgical approach lies in the direct visualization of the lesion and the opportunity to obtain non- contaminated (by blood, mucus, mucosa, etc.) and structurally intact tumor tissue from the vital tumor mass. The primary cells for the establishment of the cell line were obtained from a 72-year-old male patient who was referred to the Department of Neurosurgery because of a right-sided abducent nerve palsy causing double vision, ver- tigo, and cephalea. After mechanical dissociation, the cells were in vital conditions but expressed a slow growth. If cells of interest show a very slow growth behavior, there is a risk that other cells will consume the media and displace the tumor cells. To separate the cells immediately from the culture start is also tricky, because they require growth factors and cytokine from sur- rounding cells. During the establishment of the cell line MUG-CC1, there was an over- growth of B-cells, and tumor cells began to die, either because of medium consumption of fast-growing B-cells or of disharmony of both cell lines (. Fig. 4.5). Suspension cells are easy to separate from adherent cells. Nevertheless the right sepa- ration time point is important; otherwise the tumor culture will be lost. At that time point, when there are suspension and adherent cells together in culture, a fatal error can happen; in some cases it has been wrongly thought that the spontaneously immortalized suspen- sions cells might be tumor cells. Therefore the characterization of the suspension cells is very important. EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  Establishment of Tumor Cell Lines 69 4 a b c d..      Fig. 4.5 Establishment of MUG-CC1 and outgrowth of tumor cells and surrounding cells from a tumor piece a, morphological typical chordoma cells with large vacuoles and physaliferous bodies sepa- rated from all other cells b, chordoma cells and suspension cells (brown cells on the top of the tumor cells) c, overgrowth of lymphoblastoid cells and displacement of tumor cells d In the characterization of lymphoblastoid cell lines (LCL) during the establishment of MUG-CC1 by morphology, lymphoblastoid cells grew in a typically rosette morphology in suspension clusters , along with single cells. Flow cytometry analyses were positive for B-cell markers (CD19), while markers for T cells (CD3) and natural killer cells (CD56) were absent. DNA content of the cells can also be useful for characterization, while the tumor cells showed mostly an aneuploid DNA content and lymphoblastoid cells a diploid DNA profile. Copy number profiling showed a balanced profile, suggesting a non-­ tumorigenic cell line. Strong positivity for the EB2 (BMLf1) gene of EBV (TC 70 and TC 72) was detected in MUG-CC1-LCL, indicates an EBV transfection, and explains the immortal state. 4.4.2  stablishment of Aggressive Growing Melanoma E Cell Line: MUG-Mel2 The cell line MUG-Mel2 was obtained from a fresh specimen of a cutaneous metastasis of a 48-year-old male patient with a cutaneous primary, ulcerated melanoma (8.5 mm thickness). The genetic analysis of the primary tumor and cutaneous metastasis revealed a mutation in NRAS Q61R; no mutations in BRAF or c-kit were detected. The tumor EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  70 K. Meditz and B. Rinner a b c 4..      Fig. 4.6 Establishment of a fast-growing tumor cell line. Outgrowth of melanoma tumor cells sur- rounded by stroma cells a; MUG-Mel2 cell line after 10 days of cultivation, magnification ×10 b; MUG- Mel2 cell line in a higher magnification ×20 c and the established cell line expressed melanoma markers such as Melan-A, HMB-45, S100, and tyrosinase. The tumor tissue (cutaneous metastasis) was obtained immedi- ately after surgery; after mechanical disaggregation of the tumor tissue into approxi- mately 1–2 mm3 pieces, cells were cultured in RPMI (Life Technologies, Carlsbad, CA) containing 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. Melanoma cells grew at a pH of 7.4. Cells were grown to 80% confluence and detached from the flasks with Accutase. Especially during culture start, cells should be kept in a very high density to encourage cell-cell contact and cell growth. Incubation of all cells was carried out at 37 °C in a humidified atmosphere of 5% CO2. All cells were periodically checked for mycoplasma by PCR.. Figure 4.6 shows prominent cells with triangular dendritic morphology as typically seen in melanoma and surrounding fibroblast-like cells, from culture start. Due to the rapid growth of the melanoma cells, other cells like fibroblasts were displaced. After 10 days, exclusively melanoma cells were further cultured (. Fig. 4.6b, c), and all melanoma markers mentioned above were able to be kept in culture. 4.5 Quality Check 4.5.1 Recording and Freezing The most important task during cell line establishment is to keep a complete documenta- tion of the cell culture work. Each lab should create their suitable labeling modality. For each cell line, a separate aliquoted medium flask should be used. Inscription is very important; also expiry dates have to be respected. Morphological observation and pictures during culturing start is a must, as well as cryopreservation in regular intervals, to have a frozen stock of different passages available. For cryopreservation a mixture of 90% FBS and 10% DMSO is recommended. Don’t freeze to less cells; if you have a small amount of cells, use a smaller cryo-vial with a lower volume. EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  Establishment of Tumor Cell Lines 71 4 4.5.2 Contaminations There is a variety of contaminations in cell culture, divided into two main areas: microbial organisms and cell-cell contaminations. The more you work with a culture, the greater the risk of contamination. Handle only one cell line at one time, especially when working with primary cells. Aseptic techniques are required to prevent microbial contaminations. 4.5.2.1 Microbial Contaminations Major contaminations in cell culture are bacteria, mycoplasma, fungi, yeasts, and viruses. Contamination source can be poor aseptic techniques, media additives, or the tumor tissue (e.g., mycoplasma). It is essential to monitor and test for contamination. Easy to detect are bacteria (increased turbidity or cloudiness, fine granules, movement), yeasts (small oval- shaped organism in short chains), and fungi (thin filamentous mycelia), whereas myco- plasma and viruses are more difficult to detect. Mycoplasmas are small prokaryotes and their presence cannot be observed by microscopy. The consequence of mycoplasma con- tamination can include growth rate effect , chromosome alterations [1, 18] nucleic acid and amino acid synthesis, as well as membrane alterations [17, 22, 27, 28]. A lot of different mycoplasma detections have been described with advantage and disadvantage in respect to cost, time, reliability, specificity, and sensitivity. PCR and ELISA technologies present a very sensitive, specific, and rapid option to detect mycoplasma while covering a broad mycoplasma panel. The most difficult contamination to detect is virus contamination, but unless they are cytopathic, they may have little effect on their host cells. 4.5.2.2 Cell-Cell Contamination “False” cell lines – cross contaminations of cells in culture was highlighted more than 30 years ago by demonstration that cell lines are contaminated by HeLa cells. Especially when working with primary cells, cell-cell contaminations can easily occur. Causes of cell misidentifications can be human errors (mislabeling, unsterile working conditions, cross contamination) or an undesired result of the used techniques (use of feeder layers or xenografting). The more you work with a culture, the greater the risk of contamination. Handle only one cell line at one time and make sure that each cell line has its own media, trypsin, and PBS. Especially adherent cells can be kept apart due to their ­morphology, but the morphology of primary cells is still unclear. To be sure to work with the right cells, DNA profiling should be done. STR DNA profiling technology is used for routine identification (authentication) of human cell lines, stem cells, and tissues. STR analyses refer to short tandem repeat DNA, to examine individual areas in DNA. Difference for certain DNA regions can be used to distinguish between individuals. However, a human STR analysis is limited to the identification of species. This means that contamina- tion with cell lines from another species, such as mice or rats, cannot be identified. The presence of nonhuman DNA remains undetected in human-based STR analyses. Animal species can be detected by isoenzyme analysis. Even during the establishment of cell lines, the cross contamination risk is very high! Therefore never treat primary cell culture and continuous cell lines at the same time. False cell lines cost time and money and not to be underestimated can cause incorrect publications. EBSCOhost - printed on 8/15/2023 10:47 PM via ALBANY COLLEGE OF PHARMACY AND HEALTH SCIENCES. All use subject to https://www.ebsco.com/terms-of-use  72 K. Meditz and B. Rinner Take-Home Message Ten Golden Rules to Be Successful in the Establishment of Tumor Cell Lines 1. Well-preserved tumor tissue with high amounts of tumor cells. 2. Do not discard anything during cultivation. 3. Choose and track carefully cells of interest. 4. Regarding of media osmolality. 5. Tumor cells which grow adherent in the body will keep this ability and will grow 4 adherent in cell culture! 6. Don’t forget to check quality regularly. 7. Handle only one primary cell or cell line at one time. 8. Carefully and correctly labeling. 9. 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