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Suspension cells do not attach to the surface of the culture vessels. These cells are also called anchorage independent or non-adherent cells which can be grown floating in the culture medium. Hematopoietic stem cells derived from blood, spleen and bone marrow and tumor cells can be grown in suspension. These cells grow much faster which do not require the frequent replacement of the medium and can be easily maintained. These are of homogeneous types and enzyme treatment is not required for the dissociation of cells; similarly these cultures have short lag period.

After the cells are isolated from the tissue and proliferated under the appropriate conditions, they occupy all of the available substrate i. For a few days it can become too crowded for their container and this can be detrimental to their growth, generally leading to cell death if left for long time.

The cells thus have to be subculture i. Hence subculture keeps cells in healthy and in growing state. A passage number refers specifically to how many times a cell line has been sub-cultured. In contrast with the population doubling level in that the specific number of cells involved is not relevant. It simply gives a general indication of how old the cells may be for various assays. When a primary culture is sub-cultured, it is known as secondary culture or cell line or sub-clone.

The process involves removing the growth media and disassociating the adhered cells usually enzymatically. Sub-culturing of primary cells to different divisions leads to the generation of cell lines. During the passage, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. However, as they are sub-cultured serially, they become different from the original cell. The cell lines which go through a limited number of cell division having a limited life span are known as finite cell lines.

The cells passage several times and then lose their ability to proliferate, which is a genetically determined event known as senescence. Cell lines derived from primary cultures of normal cells are finite cell lines. Cell cultures prepared in this way can be sub-cultured and grown indefinitely as permanent cell lines and are immortal. These cells are less adherent, fast growing, less fastidious in their nutritional requirements, able to grow up to higher cell density and different in phenotypes from the original tissue.

Such cells grow more in suspension. They also have a tendency to grow on top of each other in multilayers on culture-vessel surfaces. Lineage of cells originated from the primary culture is called strain. These are either derived from a primary culture or a cell line by the positive selection or cloning of cells having specific properties or characteristics.

A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line. The culture media used for cell cultures are generally quite complex, and culture condition widely varies for each cell type. However, media generally include amino acids, vitamins, salts maintain osmotic pressure , glucose, a bicarbonate buffer system maintains a pH between 7.

To obtain best growth, addition of a small amount of blood serum is usually necessary, and several antibiotics, like penicillin and streptomycin are added to prevent bacterial contamination. Temperature varies on the type of host cell. Most mammalian cells are maintained at 37 o C for optimal growth, while cells derived from cold- blooded animals tolerate a wider temperature range i. Serum-free media for cultures of primitive and mature hematopoietic cells.

Biotechnol Bioeng. Chromatin three-dimensional interactions mediate genetic effects on gene expression. Autoclaving: a modification in the preparation of tissue culture medium Can J Microbiol. Weller T, Wheeldon S. The cultivation in vitro of cells derived from adult Schistosoma mansoni. Methodology; criteria for evaluation of cultures; and development of media. Am J Trop Med Hyg. Yang H. Zhonghua Yan Ke Za Zhi.

Advances in cell culture

Evaluation of media, time and temperature of incubation, and method of enumeration of several strains fo Clostridium perfringens spores. Specific cell types and their requirements. In: Davis JM, editor. Oxford: Oxford University Press; L1 drives IFN in senescent cells and promotes age-associated inflammation. Chambers S, Swalley S. Designing experiments for high-throughput protein expression. Methods Mol Biol. Keen M, Rapson N. Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line.

Basal medium development for serum-free culture: a historical perspective. Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway. Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Mol Cell.

Dimerization quality control ensures neuronal development and survival. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. The nucleolus functions as a phase-separated protein quality control compartment. Epithelial-type systemic breast carcinoma cells with a restricted mesenchymal transition are a major source of metastasis. Sci Adv. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Identification of small molecule activators of cryptochrome.

Regulation of cyclic 3',5'-adenosine monophosphate phosphodiesterase in Friend erythroleukemia cells. Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes. PLoS Biol. A stem cell-based approach to cartilage repair. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses. Clathrin-independent pathways do not contribute significantly to endocytic flux.

Animal Cell Culture

Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells. The Rho exchange factors Vav2 and Vav3 favor skin tumor initiation and promotion by engaging extracellular signaling loops. Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states. The lac repressor displays facilitated diffusion in living cells. Modulation of the maladaptive stress response to manage diseases of protein folding. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota.

Phosphofructokinase 1 glycosylation regulates cell growth and metabolism. A role for PVRL4-driven cell-cell interactions in tumorigenesis. Information transduction capacity of noisy biochemical signaling networks. Pessina F, Lowndes N. Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells.

The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification. The molecular basis of acid insensitivity in the African naked mole-rat. Langerhans cells facilitate epithelial DNA damage and squamous cell carcinoma. Cell Stem Cell. Fbw7 repression by hes5 creates a feedback loop that modulates Notch-mediated intestinal and neural stem cell fate decisions.

In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. A neuron-glia co-culture model system to study neuroinflammation. To prevent cell cultures contamination, the copper CO 2 incubators can be used, due to inhibition the growth of many different microorganisms e. The copper ions disrupt key proteins and proteins essential to microbial life [ 97 ],. Use of routine antibiotics should be avoided, and using antibiotics might cause selection of the resistant microbial strains,.

Regular filtering of culture media using 0. Avoiding chemical contamination by testing all new lots of reagents—media, sera, trypsin, water,. Use of medical grade gases rather than industrial grade gas mixture that may contain toxic impurities, for example, carbon monoxide,. In , Leonard Hayflick and Paul Moorhead defined the finite life span of normal human cells. Hayflick inspired by Carrel's observations started research on the possible viral etiology of human cancer [ 99 , ]. Hayflick thought that he made a mistake in culture medium composition, glassware cleaning or other technical procedures.

A few years later in , when working with the cytogenetist Paul Moorhead, he performed a series of experiments that validated Carrel's theory. In their work, they demonstrated that normal human fibroblasts doubled a finite number of times, stopped dividing and entered the phase III phenomenon. Hayflick divided the time of primary cell culture into three phases [ 99 , ]. Finally, the cell strain enters Phase III. In phase III, the cells stop dividing, and the cell strain is lost after a finite period of time. On the basis of these experiments, Hayflick argued that normal cells have a finite capacity to replicate as opposed to cancer cells e.

Hayflick and Moorhead findings revised Carrel's idea of cellular immortality. Due to evidence of defined life span of normal cells, Dr. Witkowski conducted his own private investigations to find the answer to the phenomenon of Dr. Carrel's immortal cells. The transformed cells usually display changes in morphology and behavior, but Carrel's cells were described as being unchanged in appearance. The second theory concerns cell contamination. Hayflick suggested that embryo extract contained living cells, and those cells grew and gave the impression that the original cells were stimulated by the extract.

The presented theories tried to explain the phenomenon of Carrel's culture. In , Henrietta Lacks was diagnosed with aggressive adenocarcinoma of the cervix by Dr. Jones at Johns Hopkins Hospital in Baltimore. After cervical biopsy, the samples were send to Dr. George Gay — —director of the Tissue Culture Laboratory [ 52 ].

His assistant, Mary Kubicek, first noticed that the cells remained viable in a nutrient solution of chicken plasma [ , ]. This cell line was called HeLa derived from patient's name—Henrietta Lacks , but for years, HeLa cells were also interpreted as originating from Harriet Lane or Helen Larsen [ 71 , , ]. This situation was associated with confidential information about the originator of HeLa cells, and it was until the Obstetrics and Gynecology named Henrietta Lack as the HeLa cell source in [ — ]. In , Dr. It was demonstrated that the cells of HeLa line grew in various media—in chicken plasma medium, bovine embryo extract and human placental cord serum [ , ].

The poliomyelitis virus was successfully propagated in HeLa cell cultures by Dr. Gay [ ]. HeLa cell line was cultured in almost all known culture media and was rapidly distributed to the laboratories in the United States and other countries to scientists who were interested in cancer studies. HeLa cell line was also distributed to pharmaceutical companies, and thus, HeLa cells became the most popular and valuable resource for cancer studies [ 96 , ].

The most famous cell line was studied intensively, in particular the mechanisms that made it so aggressive. Currently, it is known that HeLa cells were infected with human papillomavirus 18 responsible for protein synthesis that degrades the protein of the p53 tumor suppressor gene [ ]. In , HeLa genome was fully sequenced and published without the knowledge of Lack's family later, the family has endorsed restricted access to HeLa genome data [ ].

This result was consistent with previous studies, but additionally, nine putative viral integration sites were found. It was also discovered that four of the HeLa chromosomes had been shattered and reassembled into highly rearranged chromosomes. The presence of chromothripsis was also confirmed especially in chromosome 11 [ , ]. Other rearrangements were observed on chromosomes 5, 19 and X.

The chromothripsis process is also manifested in a high number of CN copy number along the genome switches, high interconnectivity and alternations between a low number 2—3 of CN states [ — ]. The comparison of transcriptomes of HeLa with normalized gene expression levels of 16 tissues from Illumina Human BodyMap 2.

In the light of the results presented by Landry and his coworkers, the suggestion of the biologist Van Valen made in that HeLa cells have become new species— Helacyton gartleri— as a result of countless cell passages, viral infections or other cell line contaminants seem to take on a new importance [ , , ]. In cell cultures, the transformation may occur spontaneously, and immortal cell populations were observed in many laboratories from the early s to the early s [ ].

Immortal cells arise spontaneously from normal cells, and murine cell cultures are especially prone to that process [ 67 , ].

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Cell cultures can be transformed by oncogenic viruses, for example, SV40 [ , ] or by radiation x irradiation [ , ] and chemical carcinogens, for example, methylocholanthracene [ 64 , ]. Cell culture observations in the fifties brought the conclusions that cells derived from, for example, skin and muscle exhibit contact inhibition of growth. Other findings were made for cells infected with the Rous sarcoma virus.

The dense focus assay was widely used to describe oncogenic activity and indicated that the transformed cells displayed the ability to continue proliferate even after they reached confluence. In studies focused on the transformed cells, it was also noted that those cells were able to form a multilayer on top of normal cells. It was also argued that the loss of contact inhibition was correlated with tumorigenicity.

SV40 was used for transformations of many different animal and human cell cultures, for example, 3T3 cell line was established [ , , ]. The mechanisms that play a crucial role in immortalization and transformation are not very well defined, but several cell lines provide evidence that telomere maintenance, pRB and p53 tumor suppressor protein pathways are important in these processes [ ]. The first hybrid mammalian cells were obtained via viral fusion in human and mouse cells in by Harris and Watkins.

In their work, they demonstrated that fusion of cells of different species was possible [ ]. Using a new technique of UV inactivation, Harris and Watkins obtained heterokaryons from human HeLa cells and Ehrlich ascites tumor cells from mice [ ]. Cell fusions were obtained using mouse myeloma and mouse spleen cells from an immunized donor. Thus, the cell line secreted a myeloma protein—IgG2A.

Cell fusion was performed using an inactivated Sendai virus. Jerne, Georges J. Cells used in the recombinant protein synthesis maintain a recombinant gene with key transcriptional regulatory elements and a selection gene. The most popular genes for selection process are dihydrofolatereductase DHFR , which encodes an enzyme involved in nucleotide metabolism and glutamine synthetase GS responsible for the expression of an enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia [ ].

The production of recombinant proteins in mammalian cells can be performed in two main forms: adherent cell cultures and suspension cell cultures [ ].

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  • The example of adherent cells widely used in protein production is the CHO cell line [ ]. This immortalized cell line was established by Dr. For establishing primary cell cultures, 0. This small difference in diploid character of primary cells is generally rare in primary cells of full diploid karyotype. After some time from establishing the culture, the morphology of cells changed, and it seemed that the cell culture underwent spontaneous immortalization.

    This cell line established by Dr.

    • History of Cell Culture?
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    • Those changes made the cells totally incapable of the reduction of foliate to dihydrofolate DHF. This cell line was a system for the production of human tPA in a roller bottle system. The bottles are slowly rotated to assure oxygen supply and to wet the cells [ , ]. The second form of the production of recombinant proteins in mammalian cells is a suspension culture. Production on a higher scale is possible by using bioreactors.

      The main types of mammalian cell cultures are batch, fed batch, repeated batch, continuous and perfusion cultures [ ]. Finally, mammalian cell culture ensures most often consistent glycosylation patterns and relatively homogeneous in comparison with E. The consistent glycosylation profile maintained between batches is crucial for the recombinant biotherapeutic protein production, but the extent of glycosylation may decrease over time in a batch culture.

      The depletion of nutrients e. Some studied presented that the pattern of protein glycosylation is dependent on the expression of various glycosyltransferase enzymes that occur in the Golgi of the cell and display different relative activity among species. Those differences can account for significant variations in structure. Complete glycosylation process is usually associated with maximization of two processes—galactosylation and sialylation that usually are incomplete and result in glycan structural variation. An alternative approach involves glycoengineering of the proteins in vitro [ ].

      Reprogrammed cells were selected by the presence of Fbx15 gene expression, which is characteristic of early development and embryo stem cells. The induced pluripotent stem cells iPSCs exhibited traits of mouse ES cells but also showed differences in gene expression and chromatin organization in comparison with ES cells [ , ]. After that experiment, another discovery was made by Yamanaka and his coworkers. They introduced mouse retrovirus receptor into human cells to obtain higher transduction frequency by amphotropic retrovirus. Then, the same four genes were introduced into adult human dermal fibroblasts, and the first human iPS was created.

      The selection was based on morphology and growth characteristics of these cells. It was also found that each clone of iPS carried from three to six retroviral integrations for each of four factors. It has also been found that KIf4 interacts with p histone acetyltransferase and plays a role in gene transcription regulation via histone acetylation. Effective reprogramming was also evaluated in primary, genetically unmodified human fibroblasts—IMR The cells had normal karyotype and were verified by the presence of cell surface markers and genes typical for ESC.

      The induced cells were able to differentiate into embryoid bodies and teratomas [ ]. Firstly noticed that the differences between iPSC and adult human cells were morphology and growth characteristic. They reported differences in hundreds of genes expression. Deng et al. Induced pluripotent stem cells IPSCs are genetically identical to the mature body cells from which they were derived. It was noticed that the same genes are chemically altered in stem cells derived from adult cells, when cells undergo differentiation, and also when the normal cells become cancer cells. The difference between adult and iPSCs is subtle.

      The study that focused on fibroblasts and the pluripotent stem cells into which they were reprogrammed shows that difference was classified as epigenetic it was described as—what gets copied when the cell divides, although it is not the part of the DNA sequence. It is due chemical change—methylation that is associated with silencing genes. During that study, differentially methylated regions DMRs of genes whose expression was changed in the process of being reprogrammed from a parent cell to a stem cell were identified.

      The process of reprogramming an adult cell to a stem cell involves DMRs and genes. Studies based on cancer cell showed that differently methylated sites were located in cancer cells which matching up with many of the methylated areas that had been implicated during differentiation processes of normal tissues [ ].

      It was stated that there is the high degree of overlap between the differently methylated regions and genes that are involved in reprogramming fibroblasts into stem cells and also reprogramming a normal cells into a cancer cells [ ]. Guordon for the discoveries that proved reversible nature of cell specialization and Shinya Yamanaka for reprogramming mature mouse cells to immature cells [ , ]. Both discoveries are of great importance in many areas of medicine, for example, oncology and regenerative medicine.

      It was reported that ESC were successfully used in cartilage repair, peripheral nerve repair or cardiac regenerative therapy. Moreover, MSC were used in certain types of therapies, for example, autologous transplantations or hematopoietic disease therapies [ ].

      Introduction and history of cell culture, Primary Culture, Secondary Cell Culture and cell lines

      The culture of multicellular tumor spheroid MCTS for anticancer drug screening was developed. The advantages are as follows: More representative in vitro model that exhibits biochemical and morphological features specific for the in vivo state,. More accurate for drug and cancer biology experiments [ 56 ],. Some existing systems fail to mimic the biomechanical characteristics of tissue in vivo represent a static condition [ ],. Difficulties in encapsulated cells recovering e. In the cartridge, the ink was replaced with a biological material, and the paper with a stage with controlled elevator to control of the xyz axis.

      Now, inkjet printers for bioprinting applications use thermal or acoustic methods to eject drops of bioink onto substrate. The function of the microextrusion printers is to control extrusion of small bead of material, which is deposited onto substrate. The extrusion of material can be controlled by pneumatic or mechanical dispensing systems.

      Materials should be characterized by good printability, high biocompatibility, known degradation kinetics and byproducts, material biomimicry and proper structural and mechanical attributes [ ]. The successful bioprinting process depends on cells selection for tissue or organ printing. Printing organs or tissues requires multiple cell types, for example, the primary functional cells, embryonic and induced pluripotent stem cells.

      Introduction to Cell Culture | Thermo Fisher Scientific - US

      The cells chosen for bioprinting must be robust to survive the printing process, and thus, in many studies, cell lines are used. For example, fibroblast or transformed cell lines are robust enough to shear stress and pressure [ , ]. The organ and tissue printing will not only solve problem of organ transplantation but will give possibilities to use those construct in drug discovery, chemical, biological or toxicological analysis, and cancer research [ , ].

      The future of animal cell technology will enlarge its applications, for example, use of viral vectors for gene therapy, vaccine technology, recombinant protein production for therapeutic purposes. Moreover, human cell cultures can also be used for personal therapies—gene therapies, tissue engineering, transplantation of organs. In the future, more human diseases will be treated by new form of therapies based on organ and tissue cultures [ 74 ]. Since HeLa established, immoral cancer cell lines are intensively studied as a biological models to investigate cancers biology e.

      Although many important investigations were performed using cancer cell lines, the results give limited information and present low clinical correlation. The genetic aberrations of cancer cell lines that are related with increasing passage numbers are one of the reasons why this type of study does not fully represent clinical situation.